**Role of the Biofilms in Wastewater Treatment**

Shama Sehar and Iffat Naz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63499

#### **Abstract**

Biological wastewater treatment systems play an important role in improving water quality and human health. This chapter thus briefly discusses different biological methods, specially biofilm technologies, the development of biofilms on different filter media, factors affecting their development as well as their structure and function. It also tackles various conventional and modern molecular techniques for detailed explora‐ tion of the composition, diversity and dynamics of biofilms. These data are crucial to improve the performance, robustness and stability of biofilm-based wastewater treatment technologies. media, factors affecting their development as well as their structure and function. It wastewater biofilm molecular water crises are due to a rapid increase in population, climatic variation, environmental pollution,

**Keywords:** biofilm, wastewater treatment, biofilm technologies, molecular methods, biofilter media

### **1. Introduction**

Water is a basic necessity, but its availability for human use is hardly about 1%. Current global watercrisesareduetoarapidincreaseinpopulation,climaticvariation,environmentalpollution, urbanization, industrialization and contamination of existing water reservoirs. The quality of freshwater in rivers and streams is affected because much of the wastage is discharged without prior treatment from industries, municipal sewers and agricultural areas. The quality of groundwater is declining due to unprocessed sewage containing domestic waste along with human and animal excretion products, leading to worldwide deaths and other environmental factors, including biodiversity reduction and an increasing number of water-related infec‐ tions, among others. According to WHO, approximately 30% of all diseases and 40% of deaths throughout the world are due to polluted water [1].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Wastewater* is a broad term comprising effluents or discharge from household seepage, agriculture, industries and storm water [2]. The organic material present in wastewater includes detergents, pesticides, fats and oils. In addition, many types of microorganisms, including bacteria, viruses, protozoa and helminths, can be present in wastewater. Basic nutrients (nitrogen, phosphorous and ammonia, etc.) as well as metals and inorganic materi‐ als (mercury, lead, cadmium, nickel and hydrogen sulfide, etc.) are also present in wastewa‐ ter. By keeping the hazardous effects of wastewater and its usage for daily lives, wastewater treatment plants have become a focal path in securing our future water supply.

by being resuspended. In dispersed growth systems, the density of dispersed biomass is close to the sewage and moves in the same direction and velocity thereof. Thus, biomass is ex‐ posed to the same fraction of liquid for a larger interval with less substrate concentration in the neighboring cell, leading to low bacterial activity and substrate removal rate. The hydraul‐ ic retention time (average time water molecules stay in the system) has to be greater than the doubling time of microorganisms (time required to generate new cells) to increase bacterial activity and population size. Bacteria can easily be "washed out" of the system if the hydraul‐ ic retention time is shorterthan the bacterial doubling time [4]. This is the main hurdle in sizing biological reactors, as reactor volume and retention time are directly related to each other. Some of the commonly used dispersed growth systems are described in the following

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Activated sludge systems comprise a multichamber reactor unit in which aerobic microor‐ ganisms are used to degrade organic components of wastewater to produce a high-quality effluent. Constant supply of oxygen is required to maintain aerobic conditions in an aeration tank. Besides aerobic bacteria, anaerobic and/or nitrifying bacteria along with higher organ‐ isms can be present. These microorganisms oxidize the organic carbon present in wastewa‐ ter to produce carbon dioxide, water and new cells that form small clusters or flocs during the aeration and mixing process. After aeration, the mixture is transferred to a secondary clarifier for settling of floc particles and the effluent moves on for further treatment or discharge. The sludge is then recycled back to the aeration tank, where the process is repeated. A schematic of the entire process is shown in **Figure 2**. Activated sludge technology is most commonly used

subsections.

*2.1.1. Activated sludge technology*

**Figure 2.** Schematic of a typical activated sludge system [6].

### **2. Types of biological wastewater treatment systems**

There are a number of wastewater treatment processes based on the physical and chemical removal of contaminants. These processes offer varying degrees of effectiveness in addition to presenting environmental and economic disadvantages. However, biological wastewater treatment technologies have been gaining much attention in recent years. They offer low operational costs, provide easy handling and have comparatively less harmful effects on the corresponding environment. On the basis of structural configuration of biomass, biological wastewater treatment processes can be divided into two basic configurations: dispersed growth system and attached growth system.

#### **2.1. Dispersed growth system**

In dispersed/suspended growth systems, biomass grows in suspended or dispersed form in liquid medium without any attachment to the surface (**Figure 1**).

**Figure 1.** Typical examples of biomass growth [3].

Microorganisms in biomass absorb organic matter and nutrients in their vicinity, which allows them to grow and reproduce to form microcolonies. These microcolonies settle as sludge, which is then either removed or treated in a sludge treatment process or reused in the process

by being resuspended. In dispersed growth systems, the density of dispersed biomass is close to the sewage and moves in the same direction and velocity thereof. Thus, biomass is ex‐ posed to the same fraction of liquid for a larger interval with less substrate concentration in the neighboring cell, leading to low bacterial activity and substrate removal rate. The hydraul‐ ic retention time (average time water molecules stay in the system) has to be greater than the doubling time of microorganisms (time required to generate new cells) to increase bacterial activity and population size. Bacteria can easily be "washed out" of the system if the hydraul‐ ic retention time is shorterthan the bacterial doubling time [4]. This is the main hurdle in sizing biological reactors, as reactor volume and retention time are directly related to each other. Some of the commonly used dispersed growth systems are described in the following subsections.

#### *2.1.1. Activated sludge technology*

*Wastewater* is a broad term comprising effluents or discharge from household seepage, agriculture, industries and storm water [2]. The organic material present in wastewater includes detergents, pesticides, fats and oils. In addition, many types of microorganisms, including bacteria, viruses, protozoa and helminths, can be present in wastewater. Basic nutrients (nitrogen, phosphorous and ammonia, etc.) as well as metals and inorganic materi‐ als (mercury, lead, cadmium, nickel and hydrogen sulfide, etc.) are also present in wastewa‐ ter. By keeping the hazardous effects of wastewater and its usage for daily lives, wastewater

There are a number of wastewater treatment processes based on the physical and chemical removal of contaminants. These processes offer varying degrees of effectiveness in addition to presenting environmental and economic disadvantages. However, biological wastewater treatment technologies have been gaining much attention in recent years. They offer low operational costs, provide easy handling and have comparatively less harmful effects on the corresponding environment. On the basis of structural configuration of biomass, biological wastewater treatment processes can be divided into two basic configurations: dispersed

In dispersed/suspended growth systems, biomass grows in suspended or dispersed form in

Microorganisms in biomass absorb organic matter and nutrients in their vicinity, which allows them to grow and reproduce to form microcolonies. These microcolonies settle as sludge, which is then either removed or treated in a sludge treatment process or reused in the process

treatment plants have become a focal path in securing our future water supply.

**2. Types of biological wastewater treatment systems**

liquid medium without any attachment to the surface (**Figure 1**).

growth system and attached growth system.

**Figure 1.** Typical examples of biomass growth [3].

**2.1. Dispersed growth system**

122 Microbial Biofilms - Importance and Applications

Activated sludge systems comprise a multichamber reactor unit in which aerobic microor‐ ganisms are used to degrade organic components of wastewater to produce a high-quality effluent. Constant supply of oxygen is required to maintain aerobic conditions in an aeration tank. Besides aerobic bacteria, anaerobic and/or nitrifying bacteria along with higher organ‐ isms can be present. These microorganisms oxidize the organic carbon present in wastewa‐ ter to produce carbon dioxide, water and new cells that form small clusters or flocs during the aeration and mixing process. After aeration, the mixture is transferred to a secondary clarifier for settling of floc particles and the effluent moves on for further treatment or discharge. The sludge is then recycled back to the aeration tank, where the process is repeated. A schematic of the entire process is shown in **Figure 2**. Activated sludge technology is most commonly used

**Figure 2.** Schematic of a typical activated sludge system [6].

in industrialized countries for the removal of biological solids by sedimentation. Poor settling of these solid pollutants can lead to increased solid treatment costs, increased effluent solid concentrations, decreased disinfection efficiencies, washout/low biomass concentration and increased risks to downstream ecosystems and public health [5].

the microbes that absorb dissolved organic matter for their growth and reproduction as the wastewater cascades randomly through the voids between the media [8]. A schematic of the entire process is shown in **Figure 3**. TFs are suitable for small- to medium-sized communi‐ ties with a high filterloading rate and marked by their ease of operation, self-cleaning capacity and efficient removal of ammonia. However, additional treatment may be needed for the effluent to meet strict discharge standards as it generates large amounts of sludge and a

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The rotating biological contactor (RBC) is an efficient attached growth system that purifies wastewater from different industries, namely food and beverage, refinery and petrochemi‐ cal, pulp and paper industries. In addition, it is efficient in purifying municipal wastewater, landfill leachate and lagoon effluent. The system consists of biomass media, usually plastic (polyethylene, polyvinyl chloride [PVC] and expanded polystyrene), that are partially immersed in wastewater. As it slowly rotates, it lifts a film of wastewater into the air. The wastewater trickles down across the media and absorbs oxygen from the air provided by the rotating action. A living biomass (biofilm) attached to the discs assimilates the organic materials and nutrients in the wastewater. Any excess biomass that sloughs off the discs by shearing forces exerted with disc rotation and gravitational force is then removed from clear water through a conventional clarification process. A schematic of the entire process is shown in **Figure 4**. The RBC system has an edge over suspended growth systems in terms of reduced life cycle costs, less sludge production, less space requirement, ease of operation and high

relatively high incidence of clogging [9].

**Figure 3.** Schematic of a typical trickling filter system [10].

*2.2.2. Rotating biological contactor (RBC) system*

### *2.1.2. Extended aeration system*

The extended aeration system is one of the modifications of the activated sludge process. It is a complete mixed system that provides biological treatment for the removal of biodegrada‐ ble organic waste under aerobic conditions. Air may be supplied by mechanical or diffused aeration means. The raw sewage directly flows into the aerobic digestion chamber where all the solids are digested by aerobic bacteria. This is possible because the sewage is aerated for a minimum of 24 h, giving vastly increased time for almost complete digestion of all solids. Since there is complete stabilization in the aeration tank, there is no need for a separate sludge digester. Furthermore, there is no need for a primary settling tank as organic solids are allowed to settle in the aeration tank due to theirlong detention time. The major advantages of extended aeration include ease of construction as well as operation, high oxygen transfer efficiency, absence of odor, less sludge yield and exceptional mixing energy from a controlled aeration chain environment. However, extended aeration plants do not achieve denitrification and phosphorus removal without additional unit processes.

### **2.2. Attached growth system**

In attached growth systems, the biomass grows attached to a support medium to create a biofilm. Attachment to the support medium is influenced by composition of the media used, cell-cell interactions and the presence of polymer molecules on the surface [7]. The support medium can be immersed in the liquid medium or receive continuous or intermittent discharges. The support medium can be of any nature, such as solid natural (rocks, stones, gravels, sand and soil), artificial (rubber, plastic) or agglomerates of the biomass itself (granules). These biofilms grow on support media by feeding off the organic matter and nutrients in the wastewater that flows over them. In attached growth systems, there is a difference in the density gradient of the support medium together with biomass and the density of the liquid inside the reactorthat allows the velocity gradient between the liquid and the external layer of biofilm. Therefore, bacterial cells being continually exposed to new substrates tend to increase their activity. Some of the commonly used attached growth systems are described in the following subsections.

#### *2.2.1. Trickling filters*

Wastewater treatment through trickling filters (TFs) is among the oldest and most well characterized treatment technologies. TFs generally comprise a vessel packed with inert media (rocks, coke, lava, slag, gravel, polyurethane foam, ceramic, sphagnum peat moss or plastic media). The distribution system is used to sprinkle wastewater over filter media, and the wastewater trickles through the filter media supporting biomass under the influence of gravitational force. A biological slime layer grows on the media, and treatment is provided by

the microbes that absorb dissolved organic matter for their growth and reproduction as the wastewater cascades randomly through the voids between the media [8]. A schematic of the entire process is shown in **Figure 3**. TFs are suitable for small- to medium-sized communi‐ ties with a high filterloading rate and marked by their ease of operation, self-cleaning capacity and efficient removal of ammonia. However, additional treatment may be needed for the effluent to meet strict discharge standards as it generates large amounts of sludge and a relatively high incidence of clogging [9].

**Figure 3.** Schematic of a typical trickling filter system [10].

in industrialized countries for the removal of biological solids by sedimentation. Poor settling of these solid pollutants can lead to increased solid treatment costs, increased effluent solid concentrations, decreased disinfection efficiencies, washout/low biomass concentration and

The extended aeration system is one of the modifications of the activated sludge process. It is a complete mixed system that provides biological treatment for the removal of biodegrada‐ ble organic waste under aerobic conditions. Air may be supplied by mechanical or diffused aeration means. The raw sewage directly flows into the aerobic digestion chamber where all the solids are digested by aerobic bacteria. This is possible because the sewage is aerated for a minimum of 24 h, giving vastly increased time for almost complete digestion of all solids. Since there is complete stabilization in the aeration tank, there is no need for a separate sludge digester. Furthermore, there is no need for a primary settling tank as organic solids are allowed to settle in the aeration tank due to theirlong detention time. The major advantages of extended aeration include ease of construction as well as operation, high oxygen transfer efficiency, absence of odor, less sludge yield and exceptional mixing energy from a controlled aeration chain environment. However, extended aeration plants do not achieve denitrification and

In attached growth systems, the biomass grows attached to a support medium to create a biofilm. Attachment to the support medium is influenced by composition of the media used, cell-cell interactions and the presence of polymer molecules on the surface [7]. The support medium can be immersed in the liquid medium or receive continuous or intermittent discharges. The support medium can be of any nature, such as solid natural (rocks, stones, gravels, sand and soil), artificial (rubber, plastic) or agglomerates of the biomass itself (granules). These biofilms grow on support media by feeding off the organic matter and nutrients in the wastewater that flows over them. In attached growth systems, there is a difference in the density gradient of the support medium together with biomass and the density of the liquid inside the reactorthat allows the velocity gradient between the liquid and the external layer of biofilm. Therefore, bacterial cells being continually exposed to new substrates tend to increase their activity. Some of the commonly used attached growth systems

Wastewater treatment through trickling filters (TFs) is among the oldest and most well characterized treatment technologies. TFs generally comprise a vessel packed with inert media (rocks, coke, lava, slag, gravel, polyurethane foam, ceramic, sphagnum peat moss or plastic media). The distribution system is used to sprinkle wastewater over filter media, and the wastewater trickles through the filter media supporting biomass under the influence of gravitational force. A biological slime layer grows on the media, and treatment is provided by

increased risks to downstream ecosystems and public health [5].

phosphorus removal without additional unit processes.

*2.1.2. Extended aeration system*

124 Microbial Biofilms - Importance and Applications

**2.2. Attached growth system**

are described in the following subsections.

*2.2.1. Trickling filters*

#### *2.2.2. Rotating biological contactor (RBC) system*

The rotating biological contactor (RBC) is an efficient attached growth system that purifies wastewater from different industries, namely food and beverage, refinery and petrochemi‐ cal, pulp and paper industries. In addition, it is efficient in purifying municipal wastewater, landfill leachate and lagoon effluent. The system consists of biomass media, usually plastic (polyethylene, polyvinyl chloride [PVC] and expanded polystyrene), that are partially immersed in wastewater. As it slowly rotates, it lifts a film of wastewater into the air. The wastewater trickles down across the media and absorbs oxygen from the air provided by the rotating action. A living biomass (biofilm) attached to the discs assimilates the organic materials and nutrients in the wastewater. Any excess biomass that sloughs off the discs by shearing forces exerted with disc rotation and gravitational force is then removed from clear water through a conventional clarification process. A schematic of the entire process is shown in **Figure 4**. The RBC system has an edge over suspended growth systems in terms of reduced life cycle costs, less sludge production, less space requirement, ease of operation and high

process stability with load variations as well as high effluent quality with regard to both biological oxygen demand (BOD) and nutrients. However, RBC system optimization and adaptability under different environmental conditions and influent characteristics still pose challenges for the efficient design and use of this technology.

efficiency of CWs, including hydraulic retention time, temperature, macrophytes, composi‐ tion of substrate orfill media and microorganisms [14]. In CWs, the role of macrophytes is very important for the removal of nutrients from wastewater, and they also speed up the purifica‐ tion process by increasing the chemical and biological reactions in the rhizosphere. CWs require low operational and maintenance costs, less energy consumption and a reduced

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Membrane bioreactors (MBRs), which are used for municipal/industrial wastewater treat‐ ment, are a combination of a suspended growth treatment method with membrane filtration equipped with low-pressure microfiltration (MF) or ultrafiltration (UF) membranes. A membrane is simply a two-dimensional material used to separate components offluids usually on the basis of their relative size or electrical charge. MBRs are generally categorized into the following: (i) vacuum or gravity-driven systems, immersed and normally employing hollow fiber or flat sheet membranes installed in bioreactors or a subsequent membrane tank and (ii) pressure-driven systems or pipe cartridge systems located external to the bioreactor. A

An MBR system is often composed of 10 or 11 subsystems and includes fine screening, the membrane zone and, in most cases, some type of post-disinfection process. The initial step in a biological process occurs in membrane zones where microbes are used to degrade pollu‐ tants that are then filtered by a series of submerged membranes. The individual membranes are housed in units known as modules, cassettes or racks, and a combined series of these modules is referred to as a working membrane unit. Air is introduced through integral diffusers to continually scour membrane surfaces during filtration, facilitate mixing and, in some cases, contribute oxygen to the biological process. The major advantage of MBRs is that they allow high concentrations of mixed liquor suspended solids (MLSSs) with low sludge production, increased removal efficiencies of BOD and COD, water reclamation, reduced footprints and no further polishing requirement for disinfection/clarification. However, membrane surface fouling is a major obstacle to the wide application of MBRs. Additionally,

amount of sludge, and they are environmentally friendly [15].

**Figure 5.** Schematic of a constructed wetland system for wastewater treatment [14].

*2.2.4. Membrane bioreactors (MBRs)*

schematic of MBRs is depicted in **Figure 6**.

**Figure 4.** Schematic of a typical rotating biological contactor (RBC) [11].

### *2.2.3. Constructed wetland system*

Constructed wetlands (CWs) are engineered attached growth or fixed film systems compris‐ ing beds loaded with inadequately sapped graded medium (sand, soil, gravel, etc.) and planted with suitable vegetation and their microbial inhabitants to treat contaminants in surface water, groundwater or waste streams. CWs generally may be categorized into two major groups: surface flow and subsurface flow. In the case of surface flow, the water runs over the surface, while for subsurface flow, itruns beneath the surface to overcome the issues of odor. In surface flow, the bacteria and substrate contact angle with water is lower than that in subsurface flow, resulting in the much enhanced treatment efficiency of subsurface flow systems [12]. Further subsurface flow systems are categorized into horizontal and vertical subsurface flow wet‐ lands depending on the flow path. All these systems are efficient in removing contaminants and pathogens from wastewater; however, the evaporation rate of CWs in general is much higher than that of ponds or lagoons, thus posing a low potential for irrigation. The configu‐ ration of hybrid CWs (combination of vertical and horizontal flows) is considered to be an appropriate choice that has minimum water loss to overcome this flaw (**Figure 5**). Hence, the discharge of nitrified and partly denitrified effluents is possible with lower total N contents [13].

Generally, water purification in constructed wetlands involves a series of physical, chemical and biological processes, such as adsorption, filtration, sedimentation, chemical precipita‐ tion, microbial activities and macrophyte uptake. Various factors contribute to the removal

efficiency of CWs, including hydraulic retention time, temperature, macrophytes, composi‐ tion of substrate orfill media and microorganisms [14]. In CWs, the role of macrophytes is very important for the removal of nutrients from wastewater, and they also speed up the purifica‐ tion process by increasing the chemical and biological reactions in the rhizosphere. CWs require low operational and maintenance costs, less energy consumption and a reduced amount of sludge, and they are environmentally friendly [15].

**Figure 5.** Schematic of a constructed wetland system for wastewater treatment [14].

#### *2.2.4. Membrane bioreactors (MBRs)*

process stability with load variations as well as high effluent quality with regard to both biological oxygen demand (BOD) and nutrients. However, RBC system optimization and adaptability under different environmental conditions and influent characteristics still pose

Constructed wetlands (CWs) are engineered attached growth or fixed film systems compris‐ ing beds loaded with inadequately sapped graded medium (sand, soil, gravel, etc.) and planted with suitable vegetation and their microbial inhabitants to treat contaminants in surface water, groundwater or waste streams. CWs generally may be categorized into two major groups: surface flow and subsurface flow. In the case of surface flow, the water runs over the surface, while for subsurface flow, itruns beneath the surface to overcome the issues of odor. In surface flow, the bacteria and substrate contact angle with water is lower than that in subsurface flow, resulting in the much enhanced treatment efficiency of subsurface flow systems [12]. Further subsurface flow systems are categorized into horizontal and vertical subsurface flow wet‐ lands depending on the flow path. All these systems are efficient in removing contaminants and pathogens from wastewater; however, the evaporation rate of CWs in general is much higher than that of ponds or lagoons, thus posing a low potential for irrigation. The configu‐ ration of hybrid CWs (combination of vertical and horizontal flows) is considered to be an appropriate choice that has minimum water loss to overcome this flaw (**Figure 5**). Hence, the discharge of nitrified and partly denitrified effluents is possible with lower total N contents

Generally, water purification in constructed wetlands involves a series of physical, chemical and biological processes, such as adsorption, filtration, sedimentation, chemical precipita‐ tion, microbial activities and macrophyte uptake. Various factors contribute to the removal

dischargenitrifiedpossiblecontentsfiltration, and

on

lagoons,

challenges for the efficient design and use of this technology.

**Figure 4.** Schematic of a typical rotating biological contactor (RBC) [11].

*2.2.3. Constructed wetland system*

126 Microbial Biofilms - Importance and Applications

[13].

Membrane bioreactors (MBRs), which are used for municipal/industrial wastewater treat‐ ment, are a combination of a suspended growth treatment method with membrane filtration equipped with low-pressure microfiltration (MF) or ultrafiltration (UF) membranes. A membrane is simply a two-dimensional material used to separate components offluids usually on the basis of their relative size or electrical charge. MBRs are generally categorized into the following: (i) vacuum or gravity-driven systems, immersed and normally employing hollow fiber or flat sheet membranes installed in bioreactors or a subsequent membrane tank and (ii) pressure-driven systems or pipe cartridge systems located external to the bioreactor. A schematic of MBRs is depicted in **Figure 6**.

An MBR system is often composed of 10 or 11 subsystems and includes fine screening, the membrane zone and, in most cases, some type of post-disinfection process. The initial step in a biological process occurs in membrane zones where microbes are used to degrade pollu‐ tants that are then filtered by a series of submerged membranes. The individual membranes are housed in units known as modules, cassettes or racks, and a combined series of these modules is referred to as a working membrane unit. Air is introduced through integral diffusers to continually scour membrane surfaces during filtration, facilitate mixing and, in some cases, contribute oxygen to the biological process. The major advantage of MBRs is that they allow high concentrations of mixed liquor suspended solids (MLSSs) with low sludge production, increased removal efficiencies of BOD and COD, water reclamation, reduced footprints and no further polishing requirement for disinfection/clarification. However, membrane surface fouling is a major obstacle to the wide application of MBRs. Additionally, membrane channel clogging and process complexity are the main cause of increased capital as well as running costs of the entire system [16].

medium; (2) irreversible adhesion upon the production of microorganism-mediated EPSs as polyhydroxyl groups in EPSs colonize bacteria to the surface via hydrogen bonding [18]; (3) formation of monolayer microcolonies on the fixed surface due to replication of early colonizers; (4) maturation of biofilm into a three-dimensional arrangement by attaching debris from the adjacent environment and by employing new planktonic bacteria and (5) disper‐ sion or expansion by active and passive processes in which sessile, matrix-encased biofilm cells convert to freely swimming planktonic bacteria through quorum sensing (QS) or a cell-to-cell

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The following subsections discuss the factors that help in promoting the process of biofilm

Biofilm formation varies under diverse nutrient conditions ranging from high to almost nondetectable. However, they are more abundant and dense in a nutrient-rich environment as it promotes the transition of bacterial cells from planktonic to biofilm state, while depletion of these nutrients causes detachment of biofilm cells from surfaces. There are different means by which bacterial biofilms obtain nutrients: (i) concentrating trace organics on surfaces through extracellular polymer, (ii) using the waste products from secondary colonizers and (iii) pooling the biochemical resources with the help of different enzymes to break down food supplies.

Any change in pH greatly affects the growth and development of bacterial and biofilm formation as it can overwhelm different mechanisms and have negative or killing effects on the microorganisms. In response to internal or external changes in pH, bacteria quickly adjust the activity and synthesis of proteins that are associated with different cellular processes. However, some of the cellular processes, including excretion of exopolymeric substances or polysaccharides, do not adapt to pH variations so easily. The optimum pH for polysacchar‐ ide production varies among different species, but for the majority of bacteria, it is around 7 [21]. Microbial activities are very sensitive to change in temperature. Optimum temperature results in healthy growth of bacterial populations, whereas a slight variation may reduce bacterial growth efficiency. The reason forthis is a reduction in bacterial enzyme reaction rates. For many bacteria found in cooling water systems, the optimum temperature for maximum

Surface topography greatly influences the ability of bacteria to adhere to a surface. During the initial steps of colonization, surface roughness at nanoscale and microscale levels enhances the adhesion of bacteria to substrates by providing more surface area for cell attachment. Surface roughness reduces the shearforce on bacterial cells and communities presentin flowing liquids at high flow rates, such as water pipes in industrial plants. A material surface exposed in an aqueous medium will inevitably become conditioned or coated by polymers from the medium,

signaling mechanism [19].

growth is about 40°C [22].

*3.1.2. Surface topography*

formation.

**3.1. Factors effecting biofilm formation**

*3.1.1. Effects of nutrients, pH and temperature*

**Figure 6.** Typical schematic for a membrane bioreactor [17].

### **3. Biofilm development: structure and function**

An assemblage of microbial cells enclosed in a matrix of bacterial self-generated extracellu‐ lar polymeric substances (EPSs) irreversibly associated with a surface is termed a *biofilm*. Generally, the development of biofilms is composed of five main stages (**Figure 7**): (1) initial attachment of planktonic microorganisms with the exposure of a surface to an aqueous

**Figure 7.** Stages of biofilm development [20]: (1) initial attachment; (2) irreversible attachment; (3) replication; (4) matu‐ ration and (5) dispersion.

medium; (2) irreversible adhesion upon the production of microorganism-mediated EPSs as polyhydroxyl groups in EPSs colonize bacteria to the surface via hydrogen bonding [18]; (3) formation of monolayer microcolonies on the fixed surface due to replication of early colonizers; (4) maturation of biofilm into a three-dimensional arrangement by attaching debris from the adjacent environment and by employing new planktonic bacteria and (5) disper‐ sion or expansion by active and passive processes in which sessile, matrix-encased biofilm cells convert to freely swimming planktonic bacteria through quorum sensing (QS) or a cell-to-cell signaling mechanism [19].

### **3.1. Factors effecting biofilm formation**

membrane channel clogging and process complexity are the main cause of increased capital

An assemblage of microbial cells enclosed in a matrix of bacterial self-generated extracellu‐ lar polymeric substances (EPSs) irreversibly associated with a surface is termed a *biofilm*. Generally, the development of biofilms is composed of five main stages (**Figure 7**): (1) initial attachment of planktonic microorganisms with the exposure of a surface to an aqueous

**Figure 7.** Stages of biofilm development [20]: (1) initial attachment; (2) irreversible attachment; (3) replication; (4) matu‐

as well as running costs of the entire system [16].

128 Microbial Biofilms - Importance and Applications

**Figure 6.** Typical schematic for a membrane bioreactor [17].

ration and (5) dispersion.

**3. Biofilm development: structure and function**

The following subsections discuss the factors that help in promoting the process of biofilm formation.

### *3.1.1. Effects of nutrients, pH and temperature*

Biofilm formation varies under diverse nutrient conditions ranging from high to almost nondetectable. However, they are more abundant and dense in a nutrient-rich environment as it promotes the transition of bacterial cells from planktonic to biofilm state, while depletion of these nutrients causes detachment of biofilm cells from surfaces. There are different means by which bacterial biofilms obtain nutrients: (i) concentrating trace organics on surfaces through extracellular polymer, (ii) using the waste products from secondary colonizers and (iii) pooling the biochemical resources with the help of different enzymes to break down food supplies.

Any change in pH greatly affects the growth and development of bacterial and biofilm formation as it can overwhelm different mechanisms and have negative or killing effects on the microorganisms. In response to internal or external changes in pH, bacteria quickly adjust the activity and synthesis of proteins that are associated with different cellular processes. However, some of the cellular processes, including excretion of exopolymeric substances or polysaccharides, do not adapt to pH variations so easily. The optimum pH for polysacchar‐ ide production varies among different species, but for the majority of bacteria, it is around 7 [21]. Microbial activities are very sensitive to change in temperature. Optimum temperature results in healthy growth of bacterial populations, whereas a slight variation may reduce bacterial growth efficiency. The reason forthis is a reduction in bacterial enzyme reaction rates. For many bacteria found in cooling water systems, the optimum temperature for maximum growth is about 40°C [22].

### *3.1.2. Surface topography*

Surface topography greatly influences the ability of bacteria to adhere to a surface. During the initial steps of colonization, surface roughness at nanoscale and microscale levels enhances the adhesion of bacteria to substrates by providing more surface area for cell attachment. Surface roughness reduces the shearforce on bacterial cells and communities presentin flowing liquids at high flow rates, such as water pipes in industrial plants. A material surface exposed in an aqueous medium will inevitably become conditioned or coated by polymers from the medium,

and the resulting chemical modification will affect the rate and extent of microbial attach‐ ment. Moreover, other factors such as charge, hydrophobicity and elasticity are also influen‐ tial in microbial attachment [23].

surroundings [29]. Different biofilms produce different amounts of EPSs, and the amount of

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131

Extracellular DNA (eDNA) has been reported to be a major constituent of various single and multispecies biofilms. eDNA or naked DNA is a central part of bacterial self-produced extracellular polymeric substances (EPSs) and has similarity to chromosomal DNA in its primary sequence [31]. Its role is very important in various stages of biofilm formation, such as initial bacterial adhesion, aggregation and microcolony formation that favors wastewater treatment. eDNA also helps strengthen biofilms, provides protection to biofilms from physical stress, antibiotics and detergents as well as serves as an excellent source of nutrients for biofilm growth [32]. In addition, eDNA can be utilized in engineering of biofilms for beneficial purposes, such as remediation of environmental pollutants and electricity or fuel production

Divalent cations such as Ca2+ are abundant in terrestrial and aquatic environments; therefore, calcium may be one of the factors that bacteria sense during biofilm-associated growth. Recent studies showed that eDNA chelates divalent cations that help in the modification of bacteri‐ al cell surface properties and thus favor resistance of biofilms to detergents and antimicrobi‐ al agents [33]. Divalent cations, such as those of calcium, play a critical role in the initial attachment of microbial aggregates of activated sludge flocs, anaerobic sludge granules and biofilms by bridging negatively charged sites on extracellular polymers [34]. Recent studies have shown that the thickness of a biofilm can be enhanced by introducing more divalent cations, as a result of which the biofilm becomes denser and mechanically more stable [35]. Calcium has been found to not only act as a cofactor for certain proteins but also act in cell signaling, biofilm virulence, cellular and extracellular product formation and alginate

Biofilm system is a well-developed technology in which solid media are added to suspend‐ ed growth reactors to provide attachment surfaces for biofilms, so as to increase the microbi‐ al concentration as well as rates of contaminant degradation biofilms to take advantage of a number ofremoval mechanisms, including biodegradation, bioaccumulation, biosorption and biomineralization [8]. The microbial communities in the biofilm break down different nutrients, such as phosphorous and nitrogen-containing compounds, carbonaceous materi‐ als as well as trapped pathogens from the wastewater. Once pollutants are removed, treated water of a biofilter is either released to the environment or used for agriculture and other recreational purposes. Removal of the pollutants from wastewater by biofilm on the filter

EPSs increases with the age of biofilms [30].

in bioelectrochemical systems or bioreactors.

**4. Biofilm in wastewater treatment**

media is schematically represented in **Figure 8**.

*3.1.6. Extracellular DNA (eDNA)*

*3.1.7. Divalent cations*

regulation [36].

### *3.1.3. Velocity, turbulence and hydrodynamics*

The area from the surface where no turbulent flow is experienced is known as the boundary layer. Within this area, the flow velocity has been shown to be insufficient to remove bio‐ films. The area outside this layer is characterized by high levels of turbulent flow and has an influence on the attachment of cells to the surface. The size of the boundary layer is depend‐ ent on the flow velocity of water. At high velocities, the boundary layer decreases in size and the cells are exposed to a high turbulence level. Hydrodynamic conditions can influence the formation, structure, EPS production, thickness, mass and metabolic activities of biofilms [24].

### *3.1.4. Gene regulation and quorum sensing (QS)*

Studies have shown that up-regulation and down-regulation of a number of genes are involved in the initial attachment of cells with the substratum. Approximately 22% of genes were upregulated and 16% were down-regulated in the biofilm formation of *Pseudomonas aeruginosa* [25]. In addition, *algD*, *algU*, *rpoS* and genes controlling polyphosphokinase synthesis were also up-regulated in the biofilm formation of *P. aeruginosa* [23]. Biofilms of *Staphylococcus aureus* were up-regulated for genes encoding enzymes involved in glycolysis or fermentation, such as phosphoglycerate mutase, triphosphate and alcohol dehydrogenase [26]. Cell-to-cell signaling, also termed *QS*, has recently been proven to play a significant role in cell attach‐ ment and detachment from biofilms. Growth and development of biofilms on different surfaces are mediated by a density-dependent chemical signal released by bacterial cells densely packed with an EPS matrix. QS makes use of a transcriptional activator protein that acts in concert with small autoinducers (AIs) signaling molecules to stimulate expression of target genes, resulting in changes in chemical behavior. After accumulation of sufficient AIs, this form of intercellular communication serves to coordinate gene expression, morphologi‐ cal differentiation and the development responses of bacterial cells [27].

### *3.1.5. Production of extracellular polymeric substances (EPSs)*

Extracellular polymeric substances (EPSs) are a complex mixture of high-molecular-weight polymer (*M*w = 10,000) excreted by microorganisms, products from lysis and hydrolysis as well as adsorbed organic matters from wastewater. Generally, EPSs have been shown to be a rich matrix of polymers, including polysaccharides, proteins, glycoproteins, DNA oligomers, phospholipids and humic acids [28]. EPSs are also highly hydrated because they can incorpo‐ rate large amounts of water into their structure by hydrogen bonding. EPSs are typically reported to aid in the formation of a gel-like network that keeps bacteria together in biofilms due to bridging with multivalent cations and hydrophobic interactions. In addition, EPSs also cause the adherence of biofilms to surfaces, flocculation and granulation, protect bacteria against noxious environmental conditions and enable bacteria to capture nutrients from the surroundings [29]. Different biofilms produce different amounts of EPSs, and the amount of EPSs increases with the age of biofilms [30].

#### *3.1.6. Extracellular DNA (eDNA)*

and the resulting chemical modification will affect the rate and extent of microbial attach‐ ment. Moreover, other factors such as charge, hydrophobicity and elasticity are also influen‐

The area from the surface where no turbulent flow is experienced is known as the boundary layer. Within this area, the flow velocity has been shown to be insufficient to remove bio‐ films. The area outside this layer is characterized by high levels of turbulent flow and has an influence on the attachment of cells to the surface. The size of the boundary layer is depend‐ ent on the flow velocity of water. At high velocities, the boundary layer decreases in size and the cells are exposed to a high turbulence level. Hydrodynamic conditions can influence the formation, structure, EPS production, thickness, mass and metabolic activities of biofilms

Studies have shown that up-regulation and down-regulation of a number of genes are involved in the initial attachment of cells with the substratum. Approximately 22% of genes were upregulated and 16% were down-regulated in the biofilm formation of *Pseudomonas aeruginosa* [25]. In addition, *algD*, *algU*, *rpoS* and genes controlling polyphosphokinase synthesis were also up-regulated in the biofilm formation of *P. aeruginosa* [23]. Biofilms of *Staphylococcus aureus* were up-regulated for genes encoding enzymes involved in glycolysis or fermentation, such as phosphoglycerate mutase, triphosphate and alcohol dehydrogenase [26]. Cell-to-cell signaling, also termed *QS*, has recently been proven to play a significant role in cell attach‐ ment and detachment from biofilms. Growth and development of biofilms on different surfaces are mediated by a density-dependent chemical signal released by bacterial cells densely packed with an EPS matrix. QS makes use of a transcriptional activator protein that acts in concert with small autoinducers (AIs) signaling molecules to stimulate expression of target genes, resulting in changes in chemical behavior. After accumulation of sufficient AIs, this form of intercellular communication serves to coordinate gene expression, morphologi‐

Extracellular polymeric substances (EPSs) are a complex mixture of high-molecular-weight polymer (*M*w = 10,000) excreted by microorganisms, products from lysis and hydrolysis as well as adsorbed organic matters from wastewater. Generally, EPSs have been shown to be a rich matrix of polymers, including polysaccharides, proteins, glycoproteins, DNA oligomers, phospholipids and humic acids [28]. EPSs are also highly hydrated because they can incorpo‐ rate large amounts of water into their structure by hydrogen bonding. EPSs are typically reported to aid in the formation of a gel-like network that keeps bacteria together in biofilms due to bridging with multivalent cations and hydrophobic interactions. In addition, EPSs also cause the adherence of biofilms to surfaces, flocculation and granulation, protect bacteria against noxious environmental conditions and enable bacteria to capture nutrients from the

playGrowthmakes stimulate

cal differentiation and the development responses of bacterial cells [27].

*3.1.5. Production of extracellular polymeric substances (EPSs)*

tial in microbial attachment [23].

130 Microbial Biofilms - Importance and Applications

[24].

*3.1.3. Velocity, turbulence and hydrodynamics*

*3.1.4. Gene regulation and quorum sensing (QS)*

Extracellular DNA (eDNA) has been reported to be a major constituent of various single and multispecies biofilms. eDNA or naked DNA is a central part of bacterial self-produced extracellular polymeric substances (EPSs) and has similarity to chromosomal DNA in its primary sequence [31]. Its role is very important in various stages of biofilm formation, such as initial bacterial adhesion, aggregation and microcolony formation that favors wastewater treatment. eDNA also helps strengthen biofilms, provides protection to biofilms from physical stress, antibiotics and detergents as well as serves as an excellent source of nutrients for biofilm growth [32]. In addition, eDNA can be utilized in engineering of biofilms for beneficial purposes, such as remediation of environmental pollutants and electricity or fuel production in bioelectrochemical systems or bioreactors. that ofas boundaryturbulent formation,

#### *3.1.7. Divalent cations*

Divalent cations such as Ca2+ are abundant in terrestrial and aquatic environments; therefore, calcium may be one of the factors that bacteria sense during biofilm-associated growth. Recent studies showed that eDNA chelates divalent cations that help in the modification of bacteri‐ al cell surface properties and thus favor resistance of biofilms to detergents and antimicrobi‐ al agents [33]. Divalent cations, such as those of calcium, play a critical role in the initial attachment of microbial aggregates of activated sludge flocs, anaerobic sludge granules and biofilms by bridging negatively charged sites on extracellular polymers [34]. Recent studies have shown that the thickness of a biofilm can be enhanced by introducing more divalent cations, as a result of which the biofilm becomes denser and mechanically more stable [35]. Calcium has been found to not only act as a cofactor for certain proteins but also act in cell signaling, biofilm virulence, cellular and extracellular product formation and alginate regulation [36].

### **4. Biofilm in wastewater treatment**

Biofilm system is a well-developed technology in which solid media are added to suspend‐ ed growth reactors to provide attachment surfaces for biofilms, so as to increase the microbi‐ al concentration as well as rates of contaminant degradation biofilms to take advantage of a number ofremoval mechanisms, including biodegradation, bioaccumulation, biosorption and biomineralization [8]. The microbial communities in the biofilm break down different nutrients, such as phosphorous and nitrogen-containing compounds, carbonaceous materi‐ als as well as trapped pathogens from the wastewater. Once pollutants are removed, treated water of a biofilter is either released to the environment or used for agriculture and other recreational purposes. Removal of the pollutants from wastewater by biofilm on the filter media is schematically represented in **Figure 8**.

Wastewater treatment with biofilm systems has several advantages, including operational flexibility, low space requirements, reduced hydraulic retention time, resilience to changes in the environment, increased biomass residence time, high active biomass concentration, enhanced ability to degrade recalcitrant compounds as well as a slower microbial growth rate, resulting in lower sludge production.

analyze the surface chemistry of a material. It measures the elemental composition at the parts per thousand range, empirical formulas, chemical state and electronic state of the elements that exist within a material [46]. On the other hand, EDS is a useful technique applied for the

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The following subsections discuss various biofilm community characterization approaches.

Biofilm weight can be determined in terms of dry weight and wet weight by using a digital weighing balance. The wet weight of the biofilm is measured after soft rinsing with distilled water. However, the dry weight of the biofilm is estimated by allowing it to dry under aseptic conditions in laminar flow until the attainment of the constant weight of polypropylene and polystyrene filter media [41, 42]. On the other hand, natural filter media, such as rock, granite or stone media, should be dried in the oven at 60°C to constant weight [44]. The weight of the biofilm is then calculated from the difference between the weight of medium with biofilm and

The biofilm is also measured by the OD method. The filter media supporting biofilms are first rinsed with sterilized waterto ensure the removal of any material on their surface. The biofilm is then removed from the filter media in 0.9% saline by sonication for 15 min. Finally, the spectrophotometric absorbance of dissolved biofilms is recorded at 550 nm wavelength

The HPC concentration (HPC/mL) of biofilms on filter media is determined by the conven‐ tional serial dilution method. The biofilm dissolved in 0.9% saline is serially diluted (up to 10−5) and then spread on the selective growth media plates and incubated at 37°C for a specific period (24–48 h). The microbial growth appearing on specific media is enumerated in terms of HPC/mL (pathogen indicators). Pure cultures from these plates are further identified by

Non-invasive microscopic techniques provide a more accurate way of visualizing biofilms without disturbing their structure. The traditional microscopic techniques involve light microscopy (LM) and electron microscopy (SM), used for imaging analysis of biofilm sam‐

elemental analysis/chemical characterization of filter media [47].

**6. Biofilm community characterization approaches**

*6.1.1. Determination of biofilm weight (wet weight and dry weight)*

**6.1. Traditional methods**

that of medium without biofilm.

(OD550) using saline as blank [41, 42, 44].

*6.1.4. Microscopic analysis of biofilms*

*6.1.2. Determination of the biofilm optical density (OD)*

*6.1.3. Determination of heterotrophic plate count (HPC)*

colony morphology as well as microscopic and biochemical tests.

**Figure 8.** Removal of the pollutants from wastewater by biofilm on the filter media [37].

### **5. Biofilm development on different filter media**

Packing or filter medium is the basic unit of attached growth wastewater treating technolo‐ gies. It provides a surface for the growth of the biofilm. The filter medium needs to be durable, insoluble and resistant to chemicals. Its selection is based on size, porosity, density as well as resistance to erosion and chemicals [38]. The ideal medium provides a high specific surface area, low cost andporosity high enough to avoidclogging andpromote ventilation. The surface area and geometry of the support materials affect the hydrodynamic conditions in the reactor and thus affect biofilm formation, which in turn affects wastewater treatment [39, 40]. Presently, different synthetic and natural materials have been employed. Various research‐ ers have used polystyrene [41], polypropylene [42], tire-derived rubber [43] and pebbles [44, 45] as bio-filter media in fixed biofilm reactors for wastewater treatment. The chemical composition of the filter media is very critical, with respect to its compatibility with the developing biofilms; its elemental composition should be evaluated. For the detection and quantification of the elements in a filter medium, different spectroscopic techniques can be applied, such as X-ray photoelectron spectroscopy (XPS) and energy-dispersive X-ray spectroscopy (EDS or EDX or XEDS). XPS is a surface chemical analysis technique used to analyze the surface chemistry of a material. It measures the elemental composition at the parts per thousand range, empirical formulas, chemical state and electronic state of the elements that exist within a material [46]. On the other hand, EDS is a useful technique applied for the elemental analysis/chemical characterization of filter media [47].

### **6. Biofilm community characterization approaches**

The following subsections discuss various biofilm community characterization approaches.

### **6.1. Traditional methods**

Wastewater treatment with biofilm systems has several advantages, including operational flexibility, low space requirements, reduced hydraulic retention time, resilience to changes in the environment, increased biomass residence time, high active biomass concentration, enhanced ability to degrade recalcitrant compounds as well as a slower microbial growth rate,

**Figure 8.** Removal of the pollutants from wastewater by biofilm on the filter media [37].

Packing or filter medium is the basic unit of attached growth wastewater treating technolo‐ gies. It provides a surface for the growth of the biofilm. The filter medium needs to be durable, insoluble and resistant to chemicals. Its selection is based on size, porosity, density as well as resistance to erosion and chemicals [38]. The ideal medium provides a high specific surface area, low cost andporosity high enough to avoidclogging andpromote ventilation. The surface area and geometry of the support materials affect the hydrodynamic conditions in the reactor and thus affect biofilm formation, which in turn affects wastewater treatment [39, 40]. Presently, different synthetic and natural materials have been employed. Various research‐ ers have used polystyrene [41], polypropylene [42], tire-derived rubber [43] and pebbles [44, 45] as bio-filter media in fixed biofilm reactors for wastewater treatment. The chemical composition of the filter media is very critical, with respect to its compatibility with the developing biofilms; its elemental composition should be evaluated. For the detection and quantification of the elements in a filter medium, different spectroscopic techniques can be applied, such as X-ray photoelectron spectroscopy (XPS) and energy-dispersive X-ray spectroscopy (EDS or EDX or XEDS). XPS is a surface chemical analysis technique used to

**5. Biofilm development on different filter media**

resulting in lower sludge production.

132 Microbial Biofilms - Importance and Applications

### *6.1.1. Determination of biofilm weight (wet weight and dry weight)*

Biofilm weight can be determined in terms of dry weight and wet weight by using a digital weighing balance. The wet weight of the biofilm is measured after soft rinsing with distilled water. However, the dry weight of the biofilm is estimated by allowing it to dry under aseptic conditions in laminar flow until the attainment of the constant weight of polypropylene and polystyrene filter media [41, 42]. On the other hand, natural filter media, such as rock, granite or stone media, should be dried in the oven at 60°C to constant weight [44]. The weight of the biofilm is then calculated from the difference between the weight of medium with biofilm and that of medium without biofilm.

### *6.1.2. Determination of the biofilm optical density (OD)*

The biofilm is also measured by the OD method. The filter media supporting biofilms are first rinsed with sterilized waterto ensure the removal of any material on their surface. The biofilm is then removed from the filter media in 0.9% saline by sonication for 15 min. Finally, the spectrophotometric absorbance of dissolved biofilms is recorded at 550 nm wavelength (OD550) using saline as blank [41, 42, 44].

### *6.1.3. Determination of heterotrophic plate count (HPC)*

The HPC concentration (HPC/mL) of biofilms on filter media is determined by the conven‐ tional serial dilution method. The biofilm dissolved in 0.9% saline is serially diluted (up to 10−5) and then spread on the selective growth media plates and incubated at 37°C for a specific period (24–48 h). The microbial growth appearing on specific media is enumerated in terms of HPC/mL (pathogen indicators). Pure cultures from these plates are further identified by colony morphology as well as microscopic and biochemical tests.

#### *6.1.4. Microscopic analysis of biofilms*

Non-invasive microscopic techniques provide a more accurate way of visualizing biofilms without disturbing their structure. The traditional microscopic techniques involve light microscopy (LM) and electron microscopy (SM), used for imaging analysis of biofilm sam‐

ples. However, scanning electron microscopy (SEM) is a well-established fundamental technique to examine the morphology of bacteria and the topography of the material surface, and it is even capable of demonstrating the relation of biofilms to surfaces. On the other hand, other new advanced techniques have been established, including laser scanning microscopy (LSM), confocal laser scanning microscopy (CLSM), magnetic resonance imaging (MRI) and scanning transmission X-ray microscopy (STXM). These new techniques allow *in situ* analy‐ sis of the structure, composition, processes and dynamics of microbial communities. These techniques represent powerful tools for the examination of mixed microbial communities, those usually in the form of aggregates and biofilms [48].

library construction have been applied in combination with other advanced techniques in

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Microbial fingerprinting methods provide the overall profile of a biofilm community by making a distinction between microorganisms and groups of microorganisms on the basis of their distinctive characteristics of a universal component/section of a biomolecule, such as phospholipids, DNA or RNA [52, 53]. These methods include phospholipid fatty acid analysis (PLFA), denaturing gradient gel electrophoresis (DGGE) and terminal restriction

Phospholipids are a structural component of all cell membranes, but their type and propor‐ tion are distinctive to different microorganisms and break down rapidly upon cell death. Thus, the mass of PLFAs in a biofilm sample is directly proportional to viable biomass. Some groups of organisms have unique or "signature" types of PLFA [54]. PLFA analysis of the biofilm involves (1) extraction of phospholipids from the biofilm sample; (2) separation by gas chromatography with flame ionization detection; and, if required, (3) confirmation and identification by mass spectroscopy. It is not a good choice as a stand-alone method and can be combined with stable isotope probing (SIP). The SIP technique includes (1) incorporation of the stable isotope label (typically 13C) into biomass, (2) incubation of microorganisms to metabolize for a specific time, (3) extraction of biomolecules from the incubated biofilm sample, (4) quantification of the extracted biomolecules by 13C-PLFA using GC/IRMS and separation of unlabeled nucleic acids by density gradient ultracentrifugation and (5) identifi‐

DGGE is a nucleic acid-based technique employed to generate a genetic fingerprint of a complex microbial community [51]. It encompasses the following steps: (1) extraction of the DNA or RNA from the biofilm sample; (2) amplification of the extracted nucleic acids by PCR, generating a multitude of copies of a variable region within a target gene usually with universal primers to give a mixture of DNA fragments, all of the same length and each representing a species present in the original sample; (3) separation of the DNA mixture by denaturant gradient electrophoresis on an acrylamide gel with an increasing urea/formamide gradient, with the DNA molecules migrating toward the positive pole and halting on the gel upon reaching their corresponding denaturant force (*T*m), depending on the DNA sequence, with every band on the gel corresponding to a different microorganism in the sample; (4) visuali‐ zation of these bands and (5) sequence identification by excision of the individual "bands" from the gel and its comparison with the 16S rDNA database for the phylogenetic affiliation

cation of the genes/microorganisms by PCR or fingerprinting or sequencing.

wastewater treatment for the exploration of biofilm communities.

*6.2.2. Microbial fingerprinting methods*

fragment length polymorphism (T-RFLP).

*6.2.2.1. Phospholipid ester-linked fatty acid analysis (PLFA)*

*6.2.2.2. Denaturing gradient gel electrophoresis (DGGE)*

of the microorganism.

### *6.1.5. Determination of biofilm activity*

The metabolic activity of the microorganisms constituting biofilms can be estimated by considering the rate of the conversion of the specific substrate after inoculation with the seed of the biomass. For example, the physiological activity of *Nitrosomonas* spp. can be deter‐ mined by measuring the strength of the nitrites (NO2-N) formed in the growth medium from the known concentration of (NH4)2SO4 after a specific period [49]. Similarly, the removal of carbonaceous (COD and BOD) and nitrogenous (NH4─N) pollutants by biofilms can be estimated.

#### **6.2. Advanced methods**

#### *6.2.1. Clone library technique*

Cloning and sequencing of the 16S rRNA gene have been extensively and successfully employed for the study of microbial biofilms since the beginning of the 1990s, and this technique is still most widely used [50]. The cloning methodology for studying a biofilm community involves (1) extraction of the nucleic acid from the biofilm sample; (2) amplifica‐ tion ofthe 16S rRNA gene by polymerase chain reaction (PCR), usually using universal primers for bacteria or archaea, for obtaining a mixture of rDNA copies of the microorganisms; (3) cloning of the PCR products into an appropriately high number of copies of plasmid and then transformation of competent *Escherichia coli* cells with this vector; (4) selection of the trans‐ formed clones on the basis of an indicator contained in the plasmid; (5) extraction of the plasmid DNA from the colons; (6) creating a cone library by sequencing of the cloned gene and finally (7) identification and affiliation of the isolated cloned sequence with the aid of phylogenetic software and various dedicated computer programs (ARB, Seqlab, PAUP, PHYLIP).

These illustrate that the clone library method allows complete 16S rRNA sequencing and identification with very precise taxonomic studies of both cultured and non-cultured micro‐ organisms in biofilms, design of primers for PCR and probes for fluorescence *in situ* hybridi‐ zation (FISH) [51]. However, cloning is a time-consuming method, impractical for a high sample throughput and non-quantitative; in addition, extraction of a DNA pool from a microbial community can be difficult and the PCR steps are also biased. Furthermore, this technique needs specialized personnel and equipment [52]. In general, cloning and rRNA gene library construction have been applied in combination with other advanced techniques in wastewater treatment for the exploration of biofilm communities.

### *6.2.2. Microbial fingerprinting methods*

ples. However, scanning electron microscopy (SEM) is a well-established fundamental technique to examine the morphology of bacteria and the topography of the material surface, and it is even capable of demonstrating the relation of biofilms to surfaces. On the other hand, other new advanced techniques have been established, including laser scanning microscopy (LSM), confocal laser scanning microscopy (CLSM), magnetic resonance imaging (MRI) and scanning transmission X-ray microscopy (STXM). These new techniques allow *in situ* analy‐ sis of the structure, composition, processes and dynamics of microbial communities. These techniques represent powerful tools for the examination of mixed microbial communities,

The metabolic activity of the microorganisms constituting biofilms can be estimated by considering the rate of the conversion of the specific substrate after inoculation with the seed of the biomass. For example, the physiological activity of *Nitrosomonas* spp. can be deter‐ mined by measuring the strength of the nitrites (NO2-N) formed in the growth medium from the known concentration of (NH4)2SO4 after a specific period [49]. Similarly, the removal of carbonaceous (COD and BOD) and nitrogenous (NH4─N) pollutants by biofilms can be

Cloning and sequencing of the 16S rRNA gene have been extensively and successfully employed for the study of microbial biofilms since the beginning of the 1990s, and this technique is still most widely used [50]. The cloning methodology for studying a biofilm community involves (1) extraction of the nucleic acid from the biofilm sample; (2) amplifica‐ tion ofthe 16S rRNA gene by polymerase chain reaction (PCR), usually using universal primers for bacteria or archaea, for obtaining a mixture of rDNA copies of the microorganisms; (3) cloning of the PCR products into an appropriately high number of copies of plasmid and then transformation of competent *Escherichia coli* cells with this vector; (4) selection of the trans‐ formed clones on the basis of an indicator contained in the plasmid; (5) extraction of the plasmid DNA from the colons; (6) creating a cone library by sequencing of the cloned gene and finally (7) identification and affiliation of the isolated cloned sequence with the aid of phylogenetic software and various dedicated computer programs (ARB, Seqlab, PAUP,

These illustrate that the clone library method allows complete 16S rRNA sequencing and identification with very precise taxonomic studies of both cultured and non-cultured micro‐ organisms in biofilms, design of primers for PCR and probes for fluorescence *in situ* hybridi‐ zation (FISH) [51]. However, cloning is a time-consuming method, impractical for a high sample throughput and non-quantitative; in addition, extraction of a DNA pool from a microbial community can be difficult and the PCR steps are also biased. Furthermore, this technique needs specialized personnel and equipment [52]. In general, cloning and rRNA gene

those usually in the form of aggregates and biofilms [48].

*6.1.5. Determination of biofilm activity*

134 Microbial Biofilms - Importance and Applications

estimated.

PHYLIP).

**6.2. Advanced methods**

*6.2.1. Clone library technique*

Microbial fingerprinting methods provide the overall profile of a biofilm community by making a distinction between microorganisms and groups of microorganisms on the basis of their distinctive characteristics of a universal component/section of a biomolecule, such as phospholipids, DNA or RNA [52, 53]. These methods include phospholipid fatty acid analysis (PLFA), denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP).

#### *6.2.2.1. Phospholipid ester-linked fatty acid analysis (PLFA)*

Phospholipids are a structural component of all cell membranes, but their type and propor‐ tion are distinctive to different microorganisms and break down rapidly upon cell death. Thus, the mass of PLFAs in a biofilm sample is directly proportional to viable biomass. Some groups of organisms have unique or "signature" types of PLFA [54]. PLFA analysis of the biofilm involves (1) extraction of phospholipids from the biofilm sample; (2) separation by gas chromatography with flame ionization detection; and, if required, (3) confirmation and identification by mass spectroscopy. It is not a good choice as a stand-alone method and can be combined with stable isotope probing (SIP). The SIP technique includes (1) incorporation of the stable isotope label (typically 13C) into biomass, (2) incubation of microorganisms to metabolize for a specific time, (3) extraction of biomolecules from the incubated biofilm sample, (4) quantification of the extracted biomolecules by 13C-PLFA using GC/IRMS and separation of unlabeled nucleic acids by density gradient ultracentrifugation and (5) identifi‐ cation of the genes/microorganisms by PCR or fingerprinting or sequencing.

#### *6.2.2.2. Denaturing gradient gel electrophoresis (DGGE)*

DGGE is a nucleic acid-based technique employed to generate a genetic fingerprint of a complex microbial community [51]. It encompasses the following steps: (1) extraction of the DNA or RNA from the biofilm sample; (2) amplification of the extracted nucleic acids by PCR, generating a multitude of copies of a variable region within a target gene usually with universal primers to give a mixture of DNA fragments, all of the same length and each representing a species present in the original sample; (3) separation of the DNA mixture by denaturant gradient electrophoresis on an acrylamide gel with an increasing urea/formamide gradient, with the DNA molecules migrating toward the positive pole and halting on the gel upon reaching their corresponding denaturant force (*T*m), depending on the DNA sequence, with every band on the gel corresponding to a different microorganism in the sample; (4) visuali‐ zation of these bands and (5) sequence identification by excision of the individual "bands" from the gel and its comparison with the 16S rDNA database for the phylogenetic affiliation of the microorganism.

DGGE is the fastest and most economical way of comparing large numbers of samples without culturing on expensive media, isolations and analysis, and it permits rapid/simple monitor‐ ing of the spatial-temporal distribution of microbial populations by only considering band. However, depending on the nature of the sample, extraction and amplification of representa‐ tive genomic DNA can be difficult. The DNA copy number, proportional to the abundance of a particular microorganism, can be very different after amplification by PCR, and thus the intensity of the bands on a DGGE gel is not quantitative. Furthermore, the sequences of the bands obtained from a gel correspond to short DNA fragments (200–600 bp), and so phyloge‐ netic relations are less reliably established and short sequences are less useful for designing new specific primers (for PCR) and probes (for FISH).

results are carried out with a conventional epifluorescence microscope for multicolor FISH. However, a charged coupled device (CCD) camera and appropriate image analysis software can be used for the digitalization/manipulation of images, enumeration of microorganisms and measurement of the activity of single cells in biofilms by quantification of their rRNA

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On the other hand, CLSM is used with FISH analysis for thick samples with a high back‐ ground (sludge flocs, biofilms) and for obtaining three-dimensional images. Different software

FISH is an easy and fast technique, and, if required, probes are available for direct visualiza‐ tion and quantification of microorganisms. This technique is apt for routine analyses, highly trained/specialized personnel are not necessary, and only basic knowledge of microscopy and laboratory experience is required. However, prior knowledge of the microbial habitat/ environment conditions and the target microorganisms to be detected is necessary. The rRNA sequence for a particular microorganism to be detected and quantified must also be known

FISH is a widely applied technique and can be combined with other techniques to increase its sensitivity and upgrade it to overcome some of its pitfalls. FISH-based methods have revolu‐ tionized investigations into the morphology and microbial composition of biofilms and enable bacteria to be mapped [58]. These methods include FISH-MAR (FISH with micro-autoradiog‐ raphy, CARD-FISH (FISH with catalyzed reporter deposition), Clone-FISH (FISH preceded by generating the expression of the 16S or 18S rRNA targeted gene), CLASI (combinatorial labeling and spectral imaging with FISH), DOPE-FISH (double labeling of oligonucleotide probes with FISH), RING-FISH (recognition of individual genes with FISH), DVC-FISH (FISH with direct viable count) and RCA-FISH (FISH with rolling circle amplification). In the FISH-BrdU method, identification of the microbes is carried out by using 5-bromo-2′-deoxy‐ uridine (BrdU) without any need for paraformaldehyde for cellfixation orformamide for DNA denaturation. In a technique called Spike-FISH, quantification based on an internal standard (*E. coli*) is introduced by spiking the biofilm samples with known amounts of *E. coli* cells. In RAMAN-FISH, Raman microspectroscopy is combined with FISH. NanoSIMS is based on the visualization of oligonucleotide probe-conferred hybridization signals in single microbial cells

DNA microarray technology detects hundreds or even thousands of DNA sequences simul‐ taneously and rapidly [59]. It involves (1) extraction of DNA from the sample, (2) amplifica‐ tion by PCR, (3) direct hybridization of the amplified PCR products from total DNA to known molecular probes attached on the microarrays and (4) scoring of positive signals using CLSM after hybridization of the fluorescently labeled PCR amplicons to the probes. Generally, the hybridization signal intensity on microarrays is directly proportional to the abundance of the target organism. The main pitfalls of this technique are cross-hybridization and that it is not useful in identifying and detecting novel prokaryotic taxa. Moreover, if the genus does not

and isotopic measurement using high-resolution ion microprobes [58].

content.

[51].

packages are also available.

*6.2.4. DNA microarray technology*

### *6.2.2.3. Terminal restriction fragment length polymorphism (T-RFLP)*

T-RFLP is a nucleic acid-based method and provides the profile of a microbial community, which is used to identify specific microbial populations [55]. It has four steps: (1) total community DNA or RNA extraction from a sample; (2) PCR amplification with a fluorescent PCR primer to make multiple copies of a target gene; (3) enzymatic digestion of the PCR products with restriction enzymes to cut the DNA molecule at known sequences, indicative of a specific microorganism and finally (4) fragment identification by electrophoretically separating the amplified gene sequences of different sizes.

Furthermore, it is also possible to sequence and identify the generated sequences by compar‐ ison with a sequence database. The strength of the fluorescent signal yields additional information regarding the abundance of the different species, similar to the band intensity in the patterns of a DGGE gel. T-RFLP offers more sensitivity than DGGE, and it may detect the lower number sequences in a sample and is commercially available. However, sometimes, the heterogeneous size of fragments makes phylogenetic analysis less confident [56].

### *6.2.3. Fluorescence in situ hybridization (FISH)*

For FISH, the most commonly used target molecules are 16S rRNA, 18S rRNA, 23S rRNA and mRNA. FISH is an excellent method for the identification, localization, visualization and quantification of non-cultured microorganisms in their microcosm. The specificity of the florescent probe enables detection/identification on any desired taxonomic level, from domain down to a resolution suitable for differentiating between individual species [57].

FISH is carried out in a few steps: (1) the specimen is fixed by precipitating agents (ethanol or methanol), cross-linking agents (aldehydes) or a mixture depending on the target organism and the type of sample; (2) the sample is prepared, with the process including specific pretreatment steps. For better attachment of specimens to glass slides, their surfaces should be treated with coating (gelatin, poly-L lysine) or silanising agents; (3) hybridization is directly carried out on the fixed sample by addition of a mixture of salts, formamide, detergents and fluorescent probe in a dark humid chamber, usually at temperatures between 37°C and 50°C. Its time varies between 30 min and several hours; (4) slides are rinsed with distilled water to remove unbound probe, dried and mounted and (5) visualization and documentation of

results are carried out with a conventional epifluorescence microscope for multicolor FISH. However, a charged coupled device (CCD) camera and appropriate image analysis software can be used for the digitalization/manipulation of images, enumeration of microorganisms and measurement of the activity of single cells in biofilms by quantification of their rRNA content.

On the other hand, CLSM is used with FISH analysis for thick samples with a high back‐ ground (sludge flocs, biofilms) and for obtaining three-dimensional images. Different software packages are also available.

FISH is an easy and fast technique, and, if required, probes are available for direct visualiza‐ tion and quantification of microorganisms. This technique is apt for routine analyses, highly trained/specialized personnel are not necessary, and only basic knowledge of microscopy and laboratory experience is required. However, prior knowledge of the microbial habitat/ environment conditions and the target microorganisms to be detected is necessary. The rRNA sequence for a particular microorganism to be detected and quantified must also be known [51].

FISH is a widely applied technique and can be combined with other techniques to increase its sensitivity and upgrade it to overcome some of its pitfalls. FISH-based methods have revolu‐ tionized investigations into the morphology and microbial composition of biofilms and enable bacteria to be mapped [58]. These methods include FISH-MAR (FISH with micro-autoradiog‐ raphy, CARD-FISH (FISH with catalyzed reporter deposition), Clone-FISH (FISH preceded by generating the expression of the 16S or 18S rRNA targeted gene), CLASI (combinatorial labeling and spectral imaging with FISH), DOPE-FISH (double labeling of oligonucleotide probes with FISH), RING-FISH (recognition of individual genes with FISH), DVC-FISH (FISH with direct viable count) and RCA-FISH (FISH with rolling circle amplification). In the FISH-BrdU method, identification of the microbes is carried out by using 5-bromo-2′-deoxy‐ uridine (BrdU) without any need for paraformaldehyde for cellfixation orformamide for DNA denaturation. In a technique called Spike-FISH, quantification based on an internal standard (*E. coli*) is introduced by spiking the biofilm samples with known amounts of *E. coli* cells. In RAMAN-FISH, Raman microspectroscopy is combined with FISH. NanoSIMS is based on the visualization of oligonucleotide probe-conferred hybridization signals in single microbial cells and isotopic measurement using high-resolution ion microprobes [58]. internal

#### *6.2.4. DNA microarray technology*

DGGE is the fastest and most economical way of comparing large numbers of samples without culturing on expensive media, isolations and analysis, and it permits rapid/simple monitor‐ ing of the spatial-temporal distribution of microbial populations by only considering band. However, depending on the nature of the sample, extraction and amplification of representa‐ tive genomic DNA can be difficult. The DNA copy number, proportional to the abundance of a particular microorganism, can be very different after amplification by PCR, and thus the intensity of the bands on a DGGE gel is not quantitative. Furthermore, the sequences of the bands obtained from a gel correspond to short DNA fragments (200–600 bp), and so phyloge‐ netic relations are less reliably established and short sequences are less useful for designing

T-RFLP is a nucleic acid-based method and provides the profile of a microbial community, which is used to identify specific microbial populations [55]. It has four steps: (1) total community DNA or RNA extraction from a sample; (2) PCR amplification with a fluorescent PCR primer to make multiple copies of a target gene; (3) enzymatic digestion of the PCR products with restriction enzymes to cut the DNA molecule at known sequences, indicative of a specific microorganism and finally (4) fragment identification by electrophoretically

Furthermore, it is also possible to sequence and identify the generated sequences by compar‐ ison with a sequence database. The strength of the fluorescent signal yields additional information regarding the abundance of the different species, similar to the band intensity in the patterns of a DGGE gel. T-RFLP offers more sensitivity than DGGE, and it may detect the lower number sequences in a sample and is commercially available. However, sometimes, the

For FISH, the most commonly used target molecules are 16S rRNA, 18S rRNA, 23S rRNA and mRNA. FISH is an excellent method for the identification, localization, visualization and quantification of non-cultured microorganisms in their microcosm. The specificity of the florescent probe enables detection/identification on any desired taxonomic level, from domain

FISH is carried out in a few steps: (1) the specimen is fixed by precipitating agents (ethanol or methanol), cross-linking agents (aldehydes) or a mixture depending on the target organism and the type of sample; (2) the sample is prepared, with the process including specific pretreatment steps. For better attachment of specimens to glass slides, their surfaces should be treated with coating (gelatin, poly-L lysine) or silanising agents; (3) hybridization is directly carried out on the fixed sample by addition of a mixture of salts, formamide, detergents and fluorescent probe in a dark humid chamber, usually at temperatures between 37°C and 50°C. Its time varies between 30 min and several hours; (4) slides are rinsed with distilled water to remove unbound probe, dried and mounted and (5) visualization and documentation of

heterogeneous size of fragments makes phylogenetic analysis less confident [56].

down to a resolution suitable for differentiating between individual species [57].

new specific primers (for PCR) and probes (for FISH).

136 Microbial Biofilms - Importance and Applications

*6.2.2.3. Terminal restriction fragment length polymorphism (T-RFLP)*

separating the amplified gene sequences of different sizes.

*6.2.3. Fluorescence in situ hybridization (FISH)*

out

DNA microarray technology detects hundreds or even thousands of DNA sequences simul‐ taneously and rapidly [59]. It involves (1) extraction of DNA from the sample, (2) amplifica‐ tion by PCR, (3) direct hybridization of the amplified PCR products from total DNA to known molecular probes attached on the microarrays and (4) scoring of positive signals using CLSM after hybridization of the fluorescently labeled PCR amplicons to the probes. Generally, the hybridization signal intensity on microarrays is directly proportional to the abundance of the target organism. The main pitfalls of this technique are cross-hybridization and that it is not useful in identifying and detecting novel prokaryotic taxa. Moreover, if the genus does not have a corresponding probe on the microarray, then the biological significance of a genus could be totally missed. The application of this technique is comparatively less in the study of wastewater treating biofilms.

about the composition of filter media and developing biofilms is highly necessary. The composition and quantification of different elements in filter media can be determined using spectroscopic techniques, such as X-ray photoelectron spectroscopy (XPS) and energydispersive X-ray spectroscopy (EDS). The traditional methods used for the study of biofilms include analyses of their gravimetric weight spectroscopic absorbance, substrate utilization activity and viable plate count as well as microscopic techniques. Complete biofilm commun‐ ity profiling is carried out by advanced techniques such as microbial sequencing, clone library generation, genetic fingerprinting, DNA microarray, denaturant gradient electrophoresis (DGGE) and next-generation sequencing (NGS), on the basis of their availability, to increase

Role of the Biofilms in Wastewater Treatment

http://dx.doi.org/10.5772/63499

139

1 Centre for Marine Bio-Innovation (CMB), School of Biological, Earth and Environmental

[1] Kantawanichkul S, Kladprasert S, Brix H. Treatment of high-strength wastewater in tropical vertical flow constructed wetlands planted with *Typha angustifolia* and *Cyperus*

[2] Olutiola PO. Relationship between bacterial density and chemical composition of a tropical sewage oxidation pond. African Journal of Environmental Science and

[3] von Sperling M. Basic Principles of Wastewater Treatment [Biological Wastewater Treatment Series (Volume 2)]. IWA Publishing: London, UK. 2007. 15 pp. eISBN:

[4] Lubberding HJ. Applied Anaerobic Digestion. In: International Course on Anaerobic Treatment. Wageningen Agricultural University/IHE Delft: Wageningen, The

2 Department of Biochemistry, Deanship of Educational Services, Qassim University,

the performance, stability and robustness of biofilm reactors.

\*Address all correspondence to: iffatkhattak@yahoo.com

Sciences (BEES), University of New South Wales, Sydney, Australia

*involucratus*. Ecological Engineering. 2009; 35(2): 238–247.

and Iffat Naz2\*

Buraidah, Kingdom of Saudi Arabia

Technology. 2010; 4(9): 595–602.

9781780402093.

Netherlands. 1995.

**Author details**

Shama Sehar1

**References**

#### *6.2.5. Next-generation sequencing (NGS) technology*

NGS, such as pyrosequencing, is a novel DNA sequencing technology developed at the Royal Institute of Technology (KTH) based on the sequencing-by-synthesis principle [60] and on the detection of released pyrophosphate (PPi) during DNA synthesis [61]. This technology transforms microbial ecology, explores deeper layers of microbial communities and is vital in presenting an unbiased view of the composition and diversity of communities [62]. NGS platforms such as Roche/454, Illumina/Solexa, Life/APG and HeliScope/Helicos BioSciences are much faster and less expensive than the first-generation Sanger sequencing technology [63].

The steps in pyrosequencing techniques include the following:(1) extraction of the DNA from the biofilm samples; (2) quantification and detection of the purity of the extracted DNA using a NanoDrop spectrophotometer; (3) amplification of the sample 16S rRNA gene by using universal PCR primers (28F and 519R) and incorporation of different barcodes between the 454 adaptor and the forward primer, with the duplicate PCR products pooled and purified using the QIAquick Gel Extraction Kit; (4) use of the purified PCR products for pyrosequenc‐ ing and then ligation of short adaptors onto both ends for the segregation of the sequences; (5) attachment of the modified products to DNA capture beads, followed by emulsion-based clonal amplification, with the beads set into the wells of a PicoTiterPlate device, with appro‐ priate chemicals, four enzymes (DNA polymerase, ATP sulfurylase, luciferase, apyrase), adenosine 5′-phosphosulfate (APS) and luciferin, and then inserted into the Genome Sequencer according to the manufacturer's directions to record programs; (6) preprocessing of all partial 16S rRNA gene sequences using the pyrosequencing pipeline at the Ribosomal Database Project (RDP) to trim barcodes, remove primers from the partialribotags and discard low-quality and short (<250 bp long) sequences; (7) denoising and assemblage of the sequen‐ ces into clusters using the precluster command, thus generating the FASTA file data sets (\*.fna and \*.qual files) and (8) further analysis of these sequences through MOTHUR, with MOTHUR analysis pipeline and R-Scripts used to start sequencing the taxonomy and analyze the data

The technique of pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing and easy automation. It has no need for labeled primers, labeled nucleotides and gel electrophoresis. It has been successful for both confirmatory sequencing and *de novo* sequencing [61].

### **7. Conclusions**

Of the different wastewater treatment technologies, biofilm-based systems have potential advantages. For better designing of these biofilm wastewater treatment systems, knowledge about the composition of filter media and developing biofilms is highly necessary. The composition and quantification of different elements in filter media can be determined using spectroscopic techniques, such as X-ray photoelectron spectroscopy (XPS) and energydispersive X-ray spectroscopy (EDS). The traditional methods used for the study of biofilms include analyses of their gravimetric weight spectroscopic absorbance, substrate utilization activity and viable plate count as well as microscopic techniques. Complete biofilm commun‐ ity profiling is carried out by advanced techniques such as microbial sequencing, clone library generation, genetic fingerprinting, DNA microarray, denaturant gradient electrophoresis (DGGE) and next-generation sequencing (NGS), on the basis of their availability, to increase the performance, stability and robustness of biofilm reactors.

### **Author details**

have a corresponding probe on the microarray, then the biological significance of a genus could be totally missed. The application of this technique is comparatively less in the study of

NGS, such as pyrosequencing, is a novel DNA sequencing technology developed at the Royal Institute of Technology (KTH) based on the sequencing-by-synthesis principle [60] and on the detection of released pyrophosphate (PPi) during DNA synthesis [61]. This technology transforms microbial ecology, explores deeper layers of microbial communities and is vital in presenting an unbiased view of the composition and diversity of communities [62]. NGS platforms such as Roche/454, Illumina/Solexa, Life/APG and HeliScope/Helicos BioSciences are much faster and less expensive than the first-generation Sanger sequencing technology

The steps in pyrosequencing techniques include the following:(1) extraction of the DNA from the biofilm samples; (2) quantification and detection of the purity of the extracted DNA using a NanoDrop spectrophotometer; (3) amplification of the sample 16S rRNA gene by using universal PCR primers (28F and 519R) and incorporation of different barcodes between the 454 adaptor and the forward primer, with the duplicate PCR products pooled and purified using the QIAquick Gel Extraction Kit; (4) use of the purified PCR products for pyrosequenc‐ ing and then ligation of short adaptors onto both ends for the segregation of the sequences; (5) attachment of the modified products to DNA capture beads, followed by emulsion-based clonal amplification, with the beads set into the wells of a PicoTiterPlate device, with appro‐ priate chemicals, four enzymes (DNA polymerase, ATP sulfurylase, luciferase, apyrase), adenosine 5′-phosphosulfate (APS) and luciferin, and then inserted into the Genome Sequencer according to the manufacturer's directions to record programs; (6) preprocessing of all partial 16S rRNA gene sequences using the pyrosequencing pipeline at the Ribosomal Database Project (RDP) to trim barcodes, remove primers from the partialribotags and discard low-quality and short (<250 bp long) sequences; (7) denoising and assemblage of the sequen‐ ces into clusters using the precluster command, thus generating the FASTA file data sets (\*.fna and \*.qual files) and (8) further analysis of these sequences through MOTHUR, with MOTHUR analysis pipeline and R-Scripts used to start sequencing the taxonomy and analyze

The technique of pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing and easy automation. It has no need for labeled primers, labeled nucleotides and gel electrophoresis. It has been successful for both confirmatory sequencing and *de novo*

Of the different wastewater treatment technologies, biofilm-based systems have potential advantages. For better designing of these biofilm wastewater treatment systems, knowledge

wastewater treating biofilms.

138 Microbial Biofilms - Importance and Applications

[63].

the data

sequencing [61].

**7. Conclusions**

*6.2.5. Next-generation sequencing (NGS) technology*

Shama Sehar1 and Iffat Naz2\*

\*Address all correspondence to: iffatkhattak@yahoo.com

1 Centre for Marine Bio-Innovation (CMB), School of Biological, Earth and Environmental Sciences (BEES), University of New South Wales, Sydney, Australia

2 Department of Biochemistry, Deanship of Educational Services, Qassim University, Buraidah, Kingdom of Saudi Arabia

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**Chapter 8**

**Role of Biofilm in Rainwater Tank**

Additional information is available at the end of the chapter

In order to establish the role of biofilm in rainwater tank, it was investigated the phylogenetic distribution of the bacteria present in an operating rainwater tank. Most of the bacteria were closely related to fresh water, soil, and biofilm bacteria found in natural environments. The high proportion of proteobacteria indicates the generally clean oligotrophic nature of the tank water. To better understand the environmental conditions in rainwater tanks and the development of biofilms therein, the changes in biofilm cells and the bacterial community were investigated during biofilm develop‐ ment. We confirmed that the biofilm development process takes place in three stages: an initial stage characterized by the colonization of different populations, an intermedi‐ ate stage characterized by a limited number of dominant populations utilizing similar resources, and a late/mature stage characterized by mature biofilms of a complex spatial structure. It was investigated microbial behaviour after inoculation of the bacterium, *Pseudomonas aeruginosa*, in pilot and full-scale rainwater tanks with different surface-tovolume (S/V) ratios. Ninety-nine percentage of the inoculated *P. aeruginosa* had been removed from the water phase. The faster removal rate in pilot and full-scale tank was due to its higher S/V ratio. From the results, several recommendations for tank design

**Keywords:** bacterial composition, bacterial community, biofilm, biofilm development, CLSM, DGGE, microbial quality, *P. aeruginosa*, rainwater tank, surface-to-volume ra‐

Almost about one billion people in developing country suffer from water problem. According‐ ly, rainwater harvesting is becoming now one of the major alternatives to tackle water scarci‐ tyandspreadingtonotonlyindevelopingcountrybutalsourbanandremoterural communities

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Mikyeong Kim and Mooyoung Han

and management were suggested.

http://dx.doi.org/10.5772/63373

**Abstract**

tio

**1. Introduction**


**Chapter 8**

## **Role of Biofilm in Rainwater Tank**

Mikyeong Kim and Mooyoung Han

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63373

#### **Abstract**

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In order to establish the role of biofilm in rainwater tank, it was investigated the phylogenetic distribution of the bacteria present in an operating rainwater tank. Most of the bacteria were closely related to fresh water, soil, and biofilm bacteria found in natural environments. The high proportion of proteobacteria indicates the generally clean oligotrophic nature of the tank water. To better understand the environmental conditions in rainwater tanks and the development of biofilms therein, the changes in biofilm cells and the bacterial community were investigated during biofilm develop‐ ment. We confirmed that the biofilm development process takes place in three stages: an initial stage characterized by the colonization of different populations, an intermedi‐ ate stage characterized by a limited number of dominant populations utilizing similar resources, and a late/mature stage characterized by mature biofilms of a complex spatial structure. It was investigated microbial behaviour after inoculation of the bacterium, *Pseudomonas aeruginosa*, in pilot and full-scale rainwater tanks with different surface-tovolume (S/V) ratios. Ninety-nine percentage of the inoculated *P. aeruginosa* had been removed from the water phase. The faster removal rate in pilot and full-scale tank was due to its higher S/V ratio. From the results, several recommendations for tank design and management were suggested. an initial stage characterized by the colonization of different populations, an resources, and a late/mature stage characterized by mature biofilms of a complex *Pseudomonas* , in pilot and full-scale rainwater tanks with different microbial rainwater surface-to-volume ty and spreading to not only in developing country but also urban and remote rural Quantitative phylogenetic assessment of microbial communities in diverse Amann Binder Olson Chisholm Devereux Stahl 16S rRNA-targeted oligonucleotide probes with ow cytometry for analyzing Moter Göbel Fluorescence *in*  for direct Gilbride Lee Beaudette Molecular techniques in ing microbial detecting and real-time process Fakruddin Chowdhury Abhijit Hossain Mannan Mazumda Pyrosequencing-principles and International Journal of Life Sciences Metzker Sequencing the next Nature

**Keywords:** bacterial composition, bacterial community, biofilm, biofilm development, CLSM, DGGE, microbial quality, *P. aeruginosa*, rainwater tank, surface-to-volume ra‐ tio

### **1. Introduction**

Almost about one billion people in developing country suffer from water problem. According‐ ly, rainwater harvesting is becoming now one of the major alternatives to tackle water scarci‐ tyandspreadingtonotonlyindevelopingcountrybutalsourbanandremoterural communities

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in developed country in the world. Rainwater management system has an advantages such as simple technology, low cost and low-energy consuming, but rainwater use is limited by uncertainty about rainwater quality, and especially its microbial quality [1, 2].

rainwater tank, the choice of sites for sample collection was made mainly with regard to the

The system 1 was built in November 2003 and consists of a 200 m3 concrete storage tank located undergroundanda 2098 m2 roof catchment area. The harvestedrainwater supplies to the toilets of 167 households and a garden [10]. In this system, the study about microbial community was

is a concrete roof surface with a total area of 2828 and 824 m2 terrace. Rainwater collected from

When the water in the extra tank reaches 1.2 m in depth, it is pumped into the main tank. About 1000 full-time staff and students occupy the building, and the amount of water used

System 3 and system 4 were installed at Buddle-gol, Seoul National University in October 2007, which collects rainwater from the valley of Mount Gwanak. Tank 3 was designed a concrete

main storage tank,

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 147

supply tank located underground. The catchment area

) and the aforementioned terrace flows into the extra tank.

) flows into the main tank and that from the roof of

. Tank 4 was assembled from polypropylene units

. In this system, study about biofilm

. In these systems, study about microbial

The system 2, which was constructed in October 2005, comprises a 250 m3

availability of the rainwater facility.

smaller extra tank, and a 4 m3

each day to flush the toilet is approximately 60–90 m3

development process in rainwater tank was performed.

with 95% pore space and has a storage volume of 20 m3

**Figure 1.** Schematic diagram of the four study sites in SNU campus.

behaviour by full-scale spike test described was performed.

the roof of one part of the building (960 m2

square with a storage volume of 20 m3

another part of the building (1868 m2

performed.

a 27 m3

Krampitz and Holländer [1] concluded that tank cleaning was contra-productive and Deutsches Institut für Normung (DIN; in English, the German Institute for Standardization) recommend that people do not clean the rainwater tank <10 years. About 13% of all Australian households use rainwater tanks as a source of drinking water [3]. This study was motivated by those questions why the water quality was poorer after cleaning tank and why they are safe in spite of using untreated rainwater.

In the case of roof-harvested water, contamination could mainly occur on the roof collection system orin the storage facility [4]. The contaminant input is limited only from catchment area, and its management is very important for water quality in rainwater tank. Most of the contaminants come into the rainwatertank which is removed by sedimentation [5] and sludge generated thereby is able to lower the water quality by resuspension. Application of simple design such as sludge drain, calm inlet, intermediate wall, and baffle can control the contam‐ inant in rainwater tank to the certain level [6].

Biofilm is one of the factors influencing the rainwater quality in tank. Many researches showed that presence of biofilm includes negative effects, such as biofouling in filter and biocorro‐ sion and biocontamination in drinking water distribution networks, but also positive effects such as biofilm reactors for the degradation or production of chemical substances in waste‐ water treatment process [7–9]. It has been suggested that biofilm may have a function of selfcleaning ofthe tank and regulation ofthe microbial quality in rainwater [1, 2]. Although biofilm might have a positive impact on stored rainwater quality, only few studies investigated bacterial composition and distribution, its development and role in this particular environ‐ ment. Through the research on these characteristics of biofilm in rainwater tank, it is possi‐ bly suggested a betterinformation to improve the rainwater system in management and design perspectives

In this chapter, to establish the role of biofilm in rainwater tank, (1) it was investigated the kinds of bacteria that inhabit rainwater tanks, (2) the changes in the biofilm cells and the bacterial community during biofilm development, (3) the microbiological characteristics of rainwater in two tanks with different S/V ratios to identify how the internal design features of storage tanks affect the microbial quality of rainwater, and then (4) suggested design and maintenance guideline for rainwater tank.

### **2. Method and materials**

#### **2.1. Study sites**

This study was carried out at Seoul National University in Seoul, Korea (**Figure 1**). In order to investigate the microbial community and how biofilms are developed and in operating rainwater tank, the choice of sites for sample collection was made mainly with regard to the availability of the rainwater facility.

in developed country in the world. Rainwater management system has an advantages such as simple technology, low cost and low-energy consuming, but rainwater use is limited by

Krampitz and Holländer [1] concluded that tank cleaning was contra-productive and Deutsches Institut für Normung (DIN; in English, the German Institute for Standardization) recommend that people do not clean the rainwater tank <10 years. About 13% of all Australian households use rainwater tanks as a source of drinking water [3]. This study was motivated by those questions why the water quality was poorer after cleaning tank and why they are safe

In the case of roof-harvested water, contamination could mainly occur on the roof collection system orin the storage facility [4]. The contaminant input is limited only from catchment area, and its management is very important for water quality in rainwater tank. Most of the contaminants come into the rainwatertank which is removed by sedimentation [5] and sludge generated thereby is able to lower the water quality by resuspension. Application of simple design such as sludge drain, calm inlet, intermediate wall, and baffle can control the contam‐

Biofilm is one of the factors influencing the rainwater quality in tank. Many researches showed that presence of biofilm includes negative effects, such as biofouling in filter and biocorro‐ sion and biocontamination in drinking water distribution networks, but also positive effects such as biofilm reactors for the degradation or production of chemical substances in waste‐ water treatment process [7–9]. It has been suggested that biofilm may have a function of selfcleaning ofthe tank and regulation ofthe microbial quality in rainwater [1, 2]. Although biofilm might have a positive impact on stored rainwater quality, only few studies investigated bacterial composition and distribution, its development and role in this particular environ‐ ment. Through the research on these characteristics of biofilm in rainwater tank, it is possi‐ bly suggested a betterinformation to improve the rainwater system in management and design

In this chapter, to establish the role of biofilm in rainwater tank, (1) it was investigated the kinds of bacteria that inhabit rainwater tanks, (2) the changes in the biofilm cells and the bacterial community during biofilm development, (3) the microbiological characteristics of rainwater in two tanks with different S/V ratios to identify how the internal design features of storage tanks affect the microbial quality of rainwater, and then (4) suggested design and

This study was carried out at Seoul National University in Seoul, Korea (**Figure 1**). In order to investigate the microbial community and how biofilms are developed and in operating

uncertainty about rainwater quality, and especially its microbial quality [1, 2].

in spite of using untreated rainwater.

146 Microbial Biofilms - Importance and Applications

inant in rainwater tank to the certain level [6].

maintenance guideline for rainwater tank.

**2. Method and materials**

**2.1. Study sites**

perspectives

The system 1 was built in November 2003 and consists of a 200 m3 concrete storage tank located undergroundanda 2098 m2 roof catchment area. The harvestedrainwater supplies to the toilets of 167 households and a garden [10]. In this system, the study about microbial community was performed.

The system 2, which was constructed in October 2005, comprises a 250 m3 main storage tank, a 27 m3 smaller extra tank, and a 4 m3 supply tank located underground. The catchment area is a concrete roof surface with a total area of 2828 and 824 m2 terrace. Rainwater collected from the roof of one part of the building (960 m2 ) flows into the main tank and that from the roof of another part of the building (1868 m2 ) and the aforementioned terrace flows into the extra tank. When the water in the extra tank reaches 1.2 m in depth, it is pumped into the main tank. About 1000 full-time staff and students occupy the building, and the amount of water used each day to flush the toilet is approximately 60–90 m3 . In this system, study about biofilm development process in rainwater tank was performed.

System 3 and system 4 were installed at Buddle-gol, Seoul National University in October 2007, which collects rainwater from the valley of Mount Gwanak. Tank 3 was designed a concrete square with a storage volume of 20 m3 . Tank 4 was assembled from polypropylene units with 95% pore space and has a storage volume of 20 m3 . In these systems, study about microbial behaviour by full-scale spike test described was performed.

**Figure 1.** Schematic diagram of the four study sites in SNU campus.

### **2.2. Sampling sketches**

### *2.2.1. Sampling and sample preparation for PCR-DGGE*

In the system 1, the rainwater from the roof flows through a filter (VF6 type with a mesh size of 0.65 mm and a capacity of 70.5 L/s) at first, and then enters the main tank through a calm inlet. Inside the tank, the W × L × H ratio changes from 7.4 × 15.4 × 2 to 3.7 × 30.8 × 2 due to the installation of a baffle.

The sampling points were indicated in **Figure 2**. Rainwater of 1.5 L was sampled at a depth of 50 cm in the tank and was carried directly to the lab in a sterile water bottle. Biofilm was collected from 0.04 m2 of the wall surface in the tank and placed in a sterile tube containing 20 ml of distilled water.

**Figure 3.** Schematic diagram of system 3 and system 4 used in full-scale experiments.

To study the biofilm formation on the surface, 3 × 8 × 0.5 cm acrylic coupons were prepared (**Figure 4**). These coupons were immersed at the inlet and outlet of the system 2 tied to an acrylic support placed in the middle of the tank at a depth of 2 m from the bottom (**Figure 5**).

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 149

**Figure 5** shows the plans and cross-sectional views of the tank, and sampling points in system 2. To minimize the influence of flow velocity during the experiment period, no

To evaluate biofilm growth, several tests were carried out with quantify the biofilm develop‐

Water samples were collected from the inlet and outlet portions of the tank once a month in two 1 L sterile screw-cap containers. The samples were taken at a depth of 2 m from the bottom of the tank to collect data on the coupons' environment. The samples were transported to the

*2.2.2.1. Characteristics of biofilm development on the surface*

rainwater was supplied, but overflow was permitted.

**Figure 4.** Picture for coupon prepared and set up in the tank.

laboratory and analysed within 30 min of collection.

ment on the surface, following the schedule presented in **Table 1**.

*2.2.2. Coupon preparation and sampling*

**Figure 2.** Schematic diagram and description of the sampling points in system 1.

Water samples in system 3 and system 4 were collected for physicochemical monitoring and after spiking test at a depth of 1.3 m from the bottom, around the point of supply in each tank (**Figure 3**). Three replicate samples were taken on four different occasions between May and August 2010.

**Figure 3.** Schematic diagram of system 3 and system 4 used in full-scale experiments.

### *2.2.2. Coupon preparation and sampling*

**2.2. Sampling sketches**

148 Microbial Biofilms - Importance and Applications

installation of a baffle.

collected from 0.04 m2

ml of distilled water.

and August 2010.

*2.2.1. Sampling and sample preparation for PCR-DGGE*

**Figure 2.** Schematic diagram and description of the sampling points in system 1.

Water samples in system 3 and system 4 were collected for physicochemical monitoring and after spiking test at a depth of 1.3 m from the bottom, around the point of supply in each tank (**Figure 3**). Three replicate samples were taken on four different occasions between May

In the system 1, the rainwater from the roof flows through a filter (VF6 type with a mesh size of 0.65 mm and a capacity of 70.5 L/s) at first, and then enters the main tank through a calm inlet. Inside the tank, the W × L × H ratio changes from 7.4 × 15.4 × 2 to 3.7 × 30.8 × 2 due to the

The sampling points were indicated in **Figure 2**. Rainwater of 1.5 L was sampled at a depth of 50 cm in the tank and was carried directly to the lab in a sterile water bottle. Biofilm was

of the wall surface in the tank and placed in a sterile tube containing 20

### *2.2.2.1. Characteristics of biofilm development on the surface*

To study the biofilm formation on the surface, 3 × 8 × 0.5 cm acrylic coupons were prepared (**Figure 4**). These coupons were immersed at the inlet and outlet of the system 2 tied to an acrylic support placed in the middle of the tank at a depth of 2 m from the bottom (**Figure 5**).

**Figure 5** shows the plans and cross-sectional views of the tank, and sampling points in system 2. To minimize the influence of flow velocity during the experiment period, no rainwater was supplied, but overflow was permitted.

To evaluate biofilm growth, several tests were carried out with quantify the biofilm develop‐ ment on the surface, following the schedule presented in **Table 1**.

**Figure 4.** Picture for coupon prepared and set up in the tank.

Water samples were collected from the inlet and outlet portions of the tank once a month in two 1 L sterile screw-cap containers. The samples were taken at a depth of 2 m from the bottom of the tank to collect data on the coupons' environment. The samples were transported to the laboratory and analysed within 30 min of collection.

**2.4. Enumeration of bacteria**

**2.5. PCR-DGGE analysis**

*2.5.1. DNA extraction*

EUB 341F-GC, PRUN518R EUB 341F, PRUN518R

photographs of them.

**Table 2.** Reaction conditions for the PCR.

Standard method 9222D (APHA, 1998).

*2.5.2. Polymerase chain reaction (PCR)*

94°C, 15 min

*2.5.3. Denaturing gradient gel electrophoresis (DGGE)*

*2.5.4. Re-amplification of the DGGE bands and sequencing*

The heterotrophic bacteria were quantified using the conventional microbiological culture method. Faecal coliform tests were carried out through membrane filtration procedure,

The sample of rainwater and the detached biofilm sample in the PBS were separately passed through a filter, and genomic DNA was isolated with a water RNA/DNA purification kit

The EUB 341F-GC and PRUN518R primer pair, comprising universal primers specific to bacteria, was used [11] for PCR, which was performed with a thermal cycler (GeneAmp PCR

**Primer set Operation temperature and thermal cycler time (Temp., Time) Cycles**

DGGE analysis was performed using a D-Code system (Bio-Rad, USA). The 8% polyacryla‐ mide gel contained a series of denaturant concentrations ranging from 30 to 60% (forma‐ mide and urea). The gels were run at 70 V for 11 h in a 1 × TAE buffer at 60°C. After electrophoresis, the gels were stained with ethidium bromide in a 1 × TAE buffer for 15 min and then destained in DDW(Deionized distiled water) for 20 min. The DGGE gels were visualized with a UV transilluminator (302 nm) mounted with a digital camera to capture

The DNA bands on the DGGE gels were excised under UV transillumination using sterile scalpels and then soaked overnight in 50 μL of sterile DDW at 4°C. Two μL of DNA solution was used for re-amplification with the same primer pair without a GC clamp. The reaction conditions for the PCR were the same as those described in **Table 2**. The PCR products were purified using a kit (AccuPrep PCR purification kit, Bioneer, Korea) and then sequenced using EUB341F (for bacteria) and F984 (for actinomycetes) in an automatic DNA sequencer (ABI

94°C 45 s

**Initial denaturation Denaturation Annealing Elongation Final extension**

55°C 45 s

72°C 45 s

72°C 7 min

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 151

33

(Norgen, Canada) according to the manufacturer's instructions.

System 9700, Perkin Elmer). The PCR conditions are described in **Table 2**.

**Figure 5.** Schematic diagram and description of the sampling points in system 2.


CLSM, confocal laser scanning microscopy; EC, electric conductivity; HPC, heterotrophic plate count; SS: suspended solids.

**Table 1.** Experimental schedule for coupons and water sampling in system 2.

#### **2.3. Physicochemical characteristics**

The various physicochemical parameters of the rainwater, such as temperature (Sension 1, Hatch, Japan), pH (Sension 1, Hatch, Japan), dissolved oxygen (DO) (ProODO, YSI, USA), electric conductivity (EC) (Sension 378, Hatch, Japan), turbidity (2100P, Hatch, Japan), suspended solid (SS) total nitrogen (TN) (HS-TN-L kit, Humas, Korea), total phosphate (TP) (HS-TP-L kit, Humas, Korea), and total organic carbon (TOC) (V CPH kit, Shimadzu, Japan) were measured.

### **2.4. Enumeration of bacteria**

The heterotrophic bacteria were quantified using the conventional microbiological culture method. Faecal coliform tests were carried out through membrane filtration procedure, Standard method 9222D (APHA, 1998).

### **2.5. PCR-DGGE analysis**

### *2.5.1. DNA extraction*

**Figure 5.** Schematic diagram and description of the sampling points in system 2.

**Table 1.** Experimental schedule for coupons and water sampling in system 2.

Water samples for pH, turbidity, EC, DO, Temp., SS, TN, TP, TOC, HPC

150 Microbial Biofilms - Importance and Applications

**2.3. Physicochemical characteristics**

solids.

were measured.

**Weeks (after immersion in the tank) 0 1 4 5 8 9 12 15 16** Coupon samples for HPC ✔ ✔ ✔ ✔ Coupon samples for CLSM ✔ ✔ ✔ ✔ Coupon samples for PCR-DGGE ✔ ✔ ✔ ✔

Water samples for PCR-DGGE ✔ CLSM, confocal laser scanning microscopy; EC, electric conductivity; HPC, heterotrophic plate count; SS: suspended

The various physicochemical parameters of the rainwater, such as temperature (Sension 1, Hatch, Japan), pH (Sension 1, Hatch, Japan), dissolved oxygen (DO) (ProODO, YSI, USA), electric conductivity (EC) (Sension 378, Hatch, Japan), turbidity (2100P, Hatch, Japan), suspended solid (SS) total nitrogen (TN) (HS-TN-L kit, Humas, Korea), total phosphate (TP) (HS-TP-L kit, Humas, Korea), and total organic carbon (TOC) (V CPH kit, Shimadzu, Japan)

✔ ✔ ✔ ✔ ✔

The sample of rainwater and the detached biofilm sample in the PBS were separately passed through a filter, and genomic DNA was isolated with a water RNA/DNA purification kit (Norgen, Canada) according to the manufacturer's instructions.

#### *2.5.2. Polymerase chain reaction (PCR)*

The EUB 341F-GC and PRUN518R primer pair, comprising universal primers specific to bacteria, was used [11] for PCR, which was performed with a thermal cycler (GeneAmp PCR System 9700, Perkin Elmer). The PCR conditions are described in **Table 2**.


**Table 2.** Reaction conditions for the PCR.

#### *2.5.3. Denaturing gradient gel electrophoresis (DGGE)*

DGGE analysis was performed using a D-Code system (Bio-Rad, USA). The 8% polyacryla‐ mide gel contained a series of denaturant concentrations ranging from 30 to 60% (forma‐ mide and urea). The gels were run at 70 V for 11 h in a 1 × TAE buffer at 60°C. After electrophoresis, the gels were stained with ethidium bromide in a 1 × TAE buffer for 15 min and then destained in DDW(Deionized distiled water) for 20 min. The DGGE gels were visualized with a UV transilluminator (302 nm) mounted with a digital camera to capture photographs of them.

#### *2.5.4. Re-amplification of the DGGE bands and sequencing*

The DNA bands on the DGGE gels were excised under UV transillumination using sterile scalpels and then soaked overnight in 50 μL of sterile DDW at 4°C. Two μL of DNA solution was used for re-amplification with the same primer pair without a GC clamp. The reaction conditions for the PCR were the same as those described in **Table 2**. The PCR products were purified using a kit (AccuPrep PCR purification kit, Bioneer, Korea) and then sequenced using EUB341F (for bacteria) and F984 (for actinomycetes) in an automatic DNA sequencer (ABI Prism 3730 XL DNA Analyzer, PE Applied Biosystems). The DGGE band sequences were compared with 16S rDNA sequences obtained through a BLAST search from the database of the DNA Data Bank of Japan (http://blast.ddbj.nig.ac.jp/top-e.html).

the spike test, the tanks were filled with 100 L of rainwater and stored for 4 weeks. Ten litres of rainwater per day were then replaced, and the retention time was controlled at 10 days. The

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 153

Two full-scale rainwater tanks of system 3 and system 4 were employed to investigate the behaviour of the microbial populations in spike tests carried out with different S/V ratios (**Figure 3**). The S/V ratio was 2 m−1 in system 3 and 15 m−1 in system 4. The retention time was 10 days. The difference of tank material between two tanks was assumed to be negligible because material for biofilm formation primarily affects in the initial steps [13] and the two rainwater

*Pseudomonas aeruginosa* (KCTC #1636), a ubiquitous environmental bacterium that forms biofilms on wet surfaces such as those of rocks and soil, was used in the spike tests. The *P. aeruginosa* were grown to an exponential phase (OD600 = 1.2, containing approximately 5 ×

 CFU/mL) in Luria-Bertani (LB) broths and washed twice with phosphate-buffered saline (PBS) (centrifuge at 8000 rpm, 4°C for 10 min). *P. aeruginosa* was put into the tanks at a final concentration in rainwater of about 5 × 105 CFU/mL for the pilot tests and 1.3 × 104 CFU/mL

Fifty microlitres of water samples were taken in duplicate from the bottom and middle sections of the two pilot tanks, and coupons were tested randomly every day for 8 days (**Figure 6**). In the full-scale test, 1 L rainwater samples were taken in duplicate from the two tanks every day for 10 days. The pH value in both pilot tanks was 7.1 ± 0.1; the DO was 7.9 ± 0.5 mg/L in Pilot

**3.1. Composition and distribution of bacteria in an operating rainwater harvesting tank**

The turbidity, EC, SS and VSS were lower at the outlet than at inlet, and the DO was slightly lower at the inlet than at the outlet. The TN and total phosphorous were 4.9 ± 0.4 and 0.08 ± 0.04 mg/L at the inlet but decreased at the outlet to 4.4 ± 0.2 and 0.05 ± 0.01 mg/L. The COD was 1.9 ± 1.12 and 0.9 ± 0.01 mg/L, and the TOC was 0.78 ± 0.03 and 0.26 ± 0.15 mg/L at the inlet and outlet, respectively. The values of the parameters were better at the outlet than at the inlet. Because the rainwater tank under study is installed underground, the lack of sunlight and the average water temperature of as low as 19°C led to the absence of photosynthetic microbes

*3.1.1. Physicochemical conditions in rainwater tanks create a distinct microbial habitat*

water was stored in the dark at room temperature (20°C).

tanks used in this study had been in operation for 3 years.

*2.7.3. Bacteria preparation and inoculation*

Tank 1 and 6.7 ± 0.4 mg/L in Pilot Tank 2.

**3. Results and discussions**

*2.7.2. Full-scale tanks*

for the full-scale tests.

*2.7.4. Sampling*

107

### **2.6. CLSM analysis**

To observe the thickness of the biofilm via CLSM, two coupons from each part of the tank were sampled in sterile Petri dishes, and a BacLight Live/Dead bacterial viability kit (L-7012, Molecular Probes, USA) was employed to stain the live and dead cells. Photographs of two random locations on each coupon were taken with a CarlZeiss LSM 510 microscope. The CLSM images were analysed with an Image Structure Analyzer (ISA) [12].

#### **2.7. Spike test**

### *2.7.1. Pilot-batch tanks*

To investigate the behaviour of microbial populations in spike tests in pilot-scale batch tanks with different S/V ratios, 200-liter (L) polyethylene (PE) tanks were filled with 100 L of rainwater. The S/V ratios were set to 10 and 50 m−1 by installing acrylic plates (50 × 20 × 0.2 cm) (**Figure 6**). To ensure that a sufficient amount of biofilm attached to the tank walls before

**Figure 6.** Schematic diagram for the pilot-scale batch experiments.

the spike test, the tanks were filled with 100 L of rainwater and stored for 4 weeks. Ten litres of rainwater per day were then replaced, and the retention time was controlled at 10 days. The water was stored in the dark at room temperature (20°C).

### *2.7.2. Full-scale tanks*

Prism 3730 XL DNA Analyzer, PE Applied Biosystems). The DGGE band sequences were compared with 16S rDNA sequences obtained through a BLAST search from the database of

To observe the thickness of the biofilm via CLSM, two coupons from each part of the tank were sampled in sterile Petri dishes, and a BacLight Live/Dead bacterial viability kit (L-7012, Molecular Probes, USA) was employed to stain the live and dead cells. Photographs of two random locations on each coupon were taken with a CarlZeiss LSM 510 microscope. The CLSM

To investigate the behaviour of microbial populations in spike tests in pilot-scale batch tanks with different S/V ratios, 200-liter (L) polyethylene (PE) tanks were filled with 100 L of rainwater. The S/V ratios were set to 10 and 50 m−1 by installing acrylic plates (50 × 20 × 0.2 cm) (**Figure 6**). To ensure that a sufficient amount of biofilm attached to the tank walls before

the DNA Data Bank of Japan (http://blast.ddbj.nig.ac.jp/top-e.html).

images were analysed with an Image Structure Analyzer (ISA) [12].

**Figure 6.** Schematic diagram for the pilot-scale batch experiments.

**2.6. CLSM analysis**

152 Microbial Biofilms - Importance and Applications

**2.7. Spike test**

*2.7.1. Pilot-batch tanks*

Two full-scale rainwater tanks of system 3 and system 4 were employed to investigate the behaviour of the microbial populations in spike tests carried out with different S/V ratios (**Figure 3**). The S/V ratio was 2 m−1 in system 3 and 15 m−1 in system 4. The retention time was 10 days. The difference of tank material between two tanks was assumed to be negligible because material for biofilm formation primarily affects in the initial steps [13] and the two rainwater tanks used in this study had been in operation for 3 years.

### *2.7.3. Bacteria preparation and inoculation*

*Pseudomonas aeruginosa* (KCTC #1636), a ubiquitous environmental bacterium that forms biofilms on wet surfaces such as those of rocks and soil, was used in the spike tests. The *P. aeruginosa* were grown to an exponential phase (OD600 = 1.2, containing approximately 5 × 107 CFU/mL) in Luria-Bertani (LB) broths and washed twice with phosphate-buffered saline (PBS) (centrifuge at 8000 rpm, 4°C for 10 min). *P. aeruginosa* was put into the tanks at a final concentration in rainwater of about 5 × 105 CFU/mL for the pilot tests and 1.3 × 104 CFU/mL for the full-scale tests. thetests. ×

### *2.7.4. Sampling*

Fifty microlitres of water samples were taken in duplicate from the bottom and middle sections of the two pilot tanks, and coupons were tested randomly every day for 8 days (**Figure 6**). In the full-scale test, 1 L rainwater samples were taken in duplicate from the two tanks every day for 10 days. The pH value in both pilot tanks was 7.1 ± 0.1; the DO was 7.9 ± 0.5 mg/L in Pilot Tank 1 and 6.7 ± 0.4 mg/L in Pilot Tank 2.

### **3. Results and discussions**

### **3.1. Composition and distribution of bacteria in an operating rainwater harvesting tank**

### *3.1.1. Physicochemical conditions in rainwater tanks create a distinct microbial habitat*

The turbidity, EC, SS and VSS were lower at the outlet than at inlet, and the DO was slightly lower at the inlet than at the outlet. The TN and total phosphorous were 4.9 ± 0.4 and 0.08 ± 0.04 mg/L at the inlet but decreased at the outlet to 4.4 ± 0.2 and 0.05 ± 0.01 mg/L. The COD was 1.9 ± 1.12 and 0.9 ± 0.01 mg/L, and the TOC was 0.78 ± 0.03 and 0.26 ± 0.15 mg/L at the inlet and outlet, respectively. The values of the parameters were better at the outlet than at the inlet.

Because the rainwater tank under study is installed underground, the lack of sunlight and the average water temperature of as low as 19°C led to the absence of photosynthetic microbes

such as algae. The nutrient input depended on rainfall. Rainwater tanks indicated an oligo‐ trophic environment, as the concentration of dissolved organic matter in these habitats is commonly <10 mg/L [14]. The inflow and outflow of rainwater in such tanks change accord‐ ing to the precipitation and rainwater usage. Thus, rainwater tanks constitute a unique habitat for microbes.

such as *Sphingopyxis* sp., *Sphingomonas* sp., *Novosphingobium resinovorum* and *Sphingobium*

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 155

The bacterial composition in the biofilm differed according to the location. *Sphingopyxis* sp. (Band No. 7) and *Blastochloris sulfoviridis* (Band No. 9) were detected in the inlet samples, whereas *Ralstonia insidiosa*(Band No. 4), *Novosphingobium resinovorum* (Band No. 6), *Sphingo‐ monas* sp. (Band No. 8), *Sphingobium* sp. (Band No. 15) were found only in the outlet samples. *Sphingobium yanoikuyae* (Band No. 12), *Bacillus* sp. (Band No. 13), *Sphingomonas* sp. (Band No. 14), and *Beijerinckiaceae bacterium* (Band No. 17) were detected in both locations. Similar

The samples contained mostly nonpathogenic proteobacteria. Many of the bacteria identi‐ fied were closely related to fresh water, soil and biofilm bacteria found in natural environ‐ ments [15–20]. Eighty-eight percentage of the identified bacteria were proteobacteria. It have been reported that proteobacteria are consistently more abundant at pristine sites, whereas Firmicutes and Actionobacteria are dominant at polluted sites [21]. Though estimates were made in terms of detection ratio only in this study and the species were not quantified, the

The bacterial composition in the biofilm was different from that in the rainwater. It is known that biofilm formation provides an advantage for bacteria that exist in oligotrophic environ‐ ments [22]. Some of the species identified in the biofilm in this study, such as *Bacillus* sp., *Sphingomonas* sp. and *Sphingobium* sp., have been demonstrated to degrade certain contami‐ nants and to act as bio-control agents [17, 18, 23]. These species may be relatively sensitive to nutrients in oligotrophic conditions and thus tend to develop a biofilm to survive. Therefore, in oligotrophic rainwater tanks, microbial species possibly remain constant in rainwater tank

*3.1.3. Self-purification possibility of rainwater tanks and implications for rainwater quality*

Bacterial communities in nature play a key role in the production and degradation of organ‐ ic matter and many types of environmental contamination, and the cycling of nitrogen, sulphur, and many metals [24]. In addition, the sorptive capacity of biofilm for dissolved organic matter and metals has been widely demonstrated in sewage and marine systems [25, 26]. Thus, biofilm formation in rainwater tanks seems not only to promote the survival of bacteria, but also serves as a natural filter by removing contaminants and bacteria from

The temperature of the stored rainwater ranged from 16 to 22°C, and the pH was around 7. At the inlet and outlet of the tank, the turbidity was 2.9 ± 1.6 and 2.1 ± 1.0 NTUs, respectively; the SS count was 3.2 ± 1.8 and 1.3 ± 0.8 mg/L, respectively; the TOC was 1.56 ± 0.54 and 0.91 ± 0.97 mg/L, respectively; and the TP count was 0.07 ± 0.04 and 0.04 ± 0.01 mg/L, respectively. Thus,

*yanoikuyae* were found in the both rainwater and biofilm samples.

bacterial composition indicated at the inlet and outlet rainwater samples.

results still indicate the clean oligotrophic nature of the tank water.

**3.2. Characteristics of biofilm development in rainwater tank**

*3.2.1. Physicochemical and microbial conditions in rainwater*

through biofilm formation.

rainwater.

### *3.1.2. Bacterial composition and distribution*

The bacterial composition in the rainwater and biofilm samples showed different tendencies. Seventeen species were identified from the selected DGGE bands (**Figure 7**). According to the standard phylogenetic classification of prokaryotes, the species belonged to 13 genera, 10 families, 8 orders, 5 classes and 3 phyla. Proteobacteria accounted for 88% of the species identified, with the remainder being Bacteroidetes and Firmicutes.


**Figure 7.** DGGE profiles at each sampling point and closest identified phylogenetic relatives found in the DGGE bands.

The DGGE profiles showed a clear difference between the planktonic bacterial community and the community in the biofilm (**Figure 7**). The bacterial composition tended to differ across the biofilm samples, but was similar across the rainwater samples. *Rubrivivax gelatinosus*, *Roseivirga ehrenbergii*, *Limnohabitans* sp., *Aquaspirillum*sp. and*Rhodobacter gluconicum* were identified only in the rainwater, whereas *Sphingomonas* sp., *Sphingobium* sp., *Ralstonia insidiosa*, *Blastochloris sulfoviridis*, *Bacillus* sp. and *Beijerinckiaceae bacterium* were found in the biofilm. Some species, such as *Sphingopyxis* sp., *Sphingomonas* sp., *Novosphingobium resinovorum* and *Sphingobium yanoikuyae* were found in the both rainwater and biofilm samples.

such as algae. The nutrient input depended on rainfall. Rainwater tanks indicated an oligo‐ trophic environment, as the concentration of dissolved organic matter in these habitats is commonly <10 mg/L [14]. The inflow and outflow of rainwater in such tanks change accord‐ ing to the precipitation and rainwater usage. Thus, rainwater tanks constitute a unique habitat

The bacterial composition in the rainwater and biofilm samples showed different tendencies. Seventeen species were identified from the selected DGGE bands (**Figure 7**). According to the standard phylogenetic classification of prokaryotes, the species belonged to 13 genera, 10 families, 8 orders, 5 classes and 3 phyla. Proteobacteria accounted for 88% of the species

**Figure 7.** DGGE profiles at each sampling point and closest identified phylogenetic relatives found in the DGGE

The DGGE profiles showed a clear difference between the planktonic bacterial community and the community in the biofilm (**Figure 7**). The bacterial composition tended to differ across the biofilm samples, but was similar across the rainwater samples. *Rubrivivax gelatinosus*, *Roseivirga ehrenbergii*, *Limnohabitans* sp., *Aquaspirillum*sp. and*Rhodobacter gluconicum* were identified only in the rainwater, whereas *Sphingomonas* sp., *Sphingobium* sp., *Ralstonia insidiosa*, *Blastochloris sulfoviridis*, *Bacillus* sp. and *Beijerinckiaceae bacterium* were found in the biofilm. Some species,

identified, with the remainder being Bacteroidetes and Firmicutes.

for microbes.

bands.

*3.1.2. Bacterial composition and distribution*

154 Microbial Biofilms - Importance and Applications

The bacterial composition in the biofilm differed according to the location. *Sphingopyxis* sp. (Band No. 7) and *Blastochloris sulfoviridis* (Band No. 9) were detected in the inlet samples, whereas *Ralstonia insidiosa*(Band No. 4), *Novosphingobium resinovorum* (Band No. 6), *Sphingo‐ monas* sp. (Band No. 8), *Sphingobium* sp. (Band No. 15) were found only in the outlet samples. *Sphingobium yanoikuyae* (Band No. 12), *Bacillus* sp. (Band No. 13), *Sphingomonas* sp. (Band No. 14), and *Beijerinckiaceae bacterium* (Band No. 17) were detected in both locations. Similar bacterial composition indicated at the inlet and outlet rainwater samples. outlet

The samples contained mostly nonpathogenic proteobacteria. Many of the bacteria identi‐ fied were closely related to fresh water, soil and biofilm bacteria found in natural environ‐ ments [15–20]. Eighty-eight percentage of the identified bacteria were proteobacteria. It have been reported that proteobacteria are consistently more abundant at pristine sites, whereas Firmicutes and Actionobacteria are dominant at polluted sites [21]. Though estimates were made in terms of detection ratio only in this study and the species were not quantified, the results still indicate the clean oligotrophic nature of the tank water.

The bacterial composition in the biofilm was different from that in the rainwater. It is known that biofilm formation provides an advantage for bacteria that exist in oligotrophic environ‐ ments [22]. Some of the species identified in the biofilm in this study, such as *Bacillus* sp., *Sphingomonas* sp. and *Sphingobium* sp., have been demonstrated to degrade certain contami‐ nants and to act as bio-control agents [17, 18, 23]. These species may be relatively sensitive to nutrients in oligotrophic conditions and thus tend to develop a biofilm to survive. Therefore, in oligotrophic rainwater tanks, microbial species possibly remain constant in rainwater tank through biofilm formation.

#### *3.1.3. Self-purification possibility of rainwater tanks and implications for rainwater quality*

Bacterial communities in nature play a key role in the production and degradation of organ‐ ic matter and many types of environmental contamination, and the cycling of nitrogen, sulphur, and many metals [24]. In addition, the sorptive capacity of biofilm for dissolved organic matter and metals has been widely demonstrated in sewage and marine systems [25, 26]. Thus, biofilm formation in rainwater tanks seems not only to promote the survival of bacteria, but also serves as a natural filter by removing contaminants and bacteria from rainwater.

#### **3.2. Characteristics of biofilm development in rainwater tank**

#### *3.2.1. Physicochemical and microbial conditions in rainwater*

The temperature of the stored rainwater ranged from 16 to 22°C, and the pH was around 7. At the inlet and outlet of the tank, the turbidity was 2.9 ± 1.6 and 2.1 ± 1.0 NTUs, respectively; the SS count was 3.2 ± 1.8 and 1.3 ± 0.8 mg/L, respectively; the TOC was 1.56 ± 0.54 and 0.91 ± 0.97 mg/L, respectively; and the TP count was 0.07 ± 0.04 and 0.04 ± 0.01 mg/L, respectively. Thus, the particle and nutrient parameters of the rainwater, namely turbidity, SS, TOC, and TP, at the outlet of the tank were slightly lower than those at the inlet.

suggesting there was greater biofilm growth at the former. After 15 weeks, the correspond‐

at the outlet, and the difference in

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 157

at the inlet and 3.0 CFU/cm2

cell numbers between the two sites had been reduced to 1.2 times.

**Figure 9.** Number of viable cells on the coupons immersed at the tank inlet and outlet for 15 weeks.

it appears that biofilms develop within 1 week in this environment.

Mature biofilm development may take anywhere from several hours to several weeks, depending on the system [27]. Biofilm formation is one possible survival strategy for bacte‐ ria, and one of the advantages of bacterial adherence is the greater availability of nutrients attached to the surface [28]. Geesey et al. [29] reported high rates of biofilm development in oligotrophic environments. In the current study, biofilm formation was observed on the coupons after 1 week of immersion. Considering the oligotrophic nature of rainwater tanks [2],

In this study, cell number in outlet site would be smaller than that in inlet part because of attachment to the existing biofilms on the wall in inlet part and sedimentation with small particles. The difference in biofilm formation between inlet and outlet part would be results of nutrient concentration and planktonic cell number in rain water flowing from inlet part. The influence of flow velocity and the substratum effect was most likely excluded in this study because, during the experiment period, a coupon of identical material was placed in the middle of the tank and to minimize the effect of water flow, the water flow was controlled without

The CLSM images of the biofilm thickness on the coupons exhibited similar viable cell patterns (**Figure 10**). At the tank inlet, the thickness was 4.5 ± 0.1 μm at the end of the first week, increasing to 48.4 ± 1.3 μm at week 9 and then decreasing to 25.0 ± 2.8 μm at week 15.

ing figures were 3.6 CFU/cm2

supply by overflowing.

*3.2.3. Biofilm thickness*

**Figure 8** shows the number of viable cells at the inlet and outlet of the tank during the experimental period. The difference between the two sites was significant (P < 0.05): the number of viable cells at the inlet was triple that at the outlet (3 × 105 versus 1 × 105 CFU/mL, respectively).

**Figure 8.** Comparison of the viable cell quantities at the tank inlet and outlet. The difference between them is statisti‐ cally significant (Student's t-test; P < 0.05, n = 12).

The rainwater tank used in this study was designed with an internal wall in the inlet section and a baffle in the middle to improve sediment efficiency. Ryu [6] reported that such design factors as inlet barrier and baffles can affect the removal of the particles that come into a rainwater tank. Hence, the slight differences in the physicochemical characteristics identi‐ fied at the tank inlet and outlet in this study appear to be due to these design factors. In addition, the physicochemical conditions appear to influence the microbes in the water, as can be seen in the different microbial numbers.

#### *3.2.2. Comparing cell dynamics on coupon: cell number*

To compare the biofilm development at the tank inlet and outlet, we also investigated the number of viable cells on the coupons immersed at each site (**Figure 9**). The two sites exhibit‐ ed a similar number of cells until the fourth week. At week 9, however, the number at the inlet was 2.5 times higher than that at the outlet (2.7 × 105 and 1.1 × 105 CFU/cm2 , respectively),

suggesting there was greater biofilm growth at the former. After 15 weeks, the correspond‐ ing figures were 3.6 CFU/cm2 at the inlet and 3.0 CFU/cm2 at the outlet, and the difference in cell numbers between the two sites had been reduced to 1.2 times.

**Figure 9.** Number of viable cells on the coupons immersed at the tank inlet and outlet for 15 weeks.

Mature biofilm development may take anywhere from several hours to several weeks, depending on the system [27]. Biofilm formation is one possible survival strategy for bacte‐ ria, and one of the advantages of bacterial adherence is the greater availability of nutrients attached to the surface [28]. Geesey et al. [29] reported high rates of biofilm development in oligotrophic environments. In the current study, biofilm formation was observed on the coupons after 1 week of immersion. Considering the oligotrophic nature of rainwater tanks [2], it appears that biofilms develop within 1 week in this environment.

In this study, cell number in outlet site would be smaller than that in inlet part because of attachment to the existing biofilms on the wall in inlet part and sedimentation with small particles. The difference in biofilm formation between inlet and outlet part would be results of nutrient concentration and planktonic cell number in rain water flowing from inlet part. The influence of flow velocity and the substratum effect was most likely excluded in this study because, during the experiment period, a coupon of identical material was placed in the middle of the tank and to minimize the effect of water flow, the water flow was controlled without supply by overflowing.

#### *3.2.3. Biofilm thickness*

the particle and nutrient parameters of the rainwater, namely turbidity, SS, TOC, and TP, at

**Figure 8** shows the number of viable cells at the inlet and outlet of the tank during the experimental period. The difference between the two sites was significant (P < 0.05): the

**Figure 8.** Comparison of the viable cell quantities at the tank inlet and outlet. The difference between them is statisti‐

The rainwater tank used in this study was designed with an internal wall in the inlet section and a baffle in the middle to improve sediment efficiency. Ryu [6] reported that such design factors as inlet barrier and baffles can affect the removal of the particles that come into a rainwater tank. Hence, the slight differences in the physicochemical characteristics identi‐ fied at the tank inlet and outlet in this study appear to be due to these design factors. In addition, the physicochemical conditions appear to influence the microbes in the water, as can be seen

To compare the biofilm development at the tank inlet and outlet, we also investigated the number of viable cells on the coupons immersed at each site (**Figure 9**). The two sites exhibit‐ ed a similar number of cells until the fourth week. At week 9, however, the number at the inlet

and 1.1 × 105

CFU/cm2

, respectively),

versus 1 × 105

CFU/mL,

the outlet of the tank were slightly lower than those at the inlet.

respectively).

156 Microbial Biofilms - Importance and Applications

cally significant (Student's t-test; P < 0.05, n = 12).

in the different microbial numbers.

*3.2.2. Comparing cell dynamics on coupon: cell number*

was 2.5 times higher than that at the outlet (2.7 × 105

number of viable cells at the inlet was triple that at the outlet (3 × 105

The CLSM images of the biofilm thickness on the coupons exhibited similar viable cell patterns (**Figure 10**). At the tank inlet, the thickness was 4.5 ± 0.1 μm at the end of the first week, increasing to 48.4 ± 1.3 μm at week 9 and then decreasing to 25.0 ± 2.8 μm at week 15.

Apilanez et al. [30] demonstrated that once biofilm has attained a certain weight, which can be related to a certain thickness, detachment occurs. The development of greater biofilm thickness can thus lead to earlier, and a great extent of detachment. Several processes can lead to detachment: erosion or shearing, and sloughing and abrasion. Donlan [31] wrote that when biofilm increases in thickness, its rate of erosion also increases. The detached biofilm possi‐ bly settles at the bottom of the tank, but it also provides a way for cells to migrate and colonize a less populated area.

At the tank outlet, in contrast, a biofilm thickness of 5.7 ± 0.7 μm was seen at the end of the first week and that thickness continued to increase until it reached 29.5 ± 2.0 μm at week 15. No detachment phase was observed at the outlet in this study. It seems that because the nutrient concentration is lower at the outlet, biofilm development is slower at that site.

**Figure 11.** DGGE profiles at each sampling time point and the closest phylogenetic relatives found in the DGGE bands.

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 159

Following a high number of bands at the initial stage, reductions occurred later, possibly arising from the competitive dominance of a few populations. The biofilms at the inlet and outlet demonstrated a reduction in the number of bands after the first sample date. The populations that were initially detected may still have been present in later biofilms, but the rapid growth of other populations made them more difficult to detect. As biofilm matures, the number of available microhabitats may increase (for example, from the formation of an anaerobic pocket within the biofilm), thereby supporting a greater number of bacterial

Despite the overall differences in banding patterns, a number of bands appeared at the same position in the DGGE gels in almost all of the samples, including *Methylophilus methylotro‐ phus*(Band No. 1), *Methylocella palustris*(Band No. 9) and *Nitrospira* sp. (Band No. 10), although their intensity differed. *Methylophilus* sp. (Band No. 2) was found only at the outlet site at all stages, whereas *Methylotenera mobilis* (Band No. 5), *Microbacterium pumilum* (Band No. 6) and *Bacillus* sp. (Band No. 8) were identified in the earlier samples (week 1 and/or week 4). Some

In the initial stage of biofilm formation, free-swimming bacteria attach to the surface through hydrophobic and electrostatic interactions and through the use of flagella [32]. In this study, for example, both *Methylotenera mobilis* (Band No. 5) and *Microbacterium pumilum* (Band No. 6) were detected in the earlier samples. The former is mobile by means of a single flagellum [33], and the latteris non-motile [34]. Initial colonization on the surface may not be entirely random,

bands, such as *Nitrospira* sp. (Band No. 10), faded over time.

populations.

**Figure 10.** Comparison of biofilm thickness obtained by CSLM (average ± standard deviation, n = 4).

#### *3.2.4. Dynamics in bacterial community*

Two sets of biofilms displayed changes in their DGGE banding patterns and number of bands as they developed (**Figures 11** and **12**). Differences were apparent between the inlet and outlet samples, both in the individual samples of a specific age and in the overall pattern of bacteri‐ al community development. The biofilm at the inlet exhibited a greater number of bands in the earliest sample (1 week), displaying a decrease by the fourth week and then increasing again. At the outlet, there were also a greater number of bands after 1 week, a decrease by the ninth week, and then a slight increase. The band patterns appeared similar between the initial two stages (weeks 1 and 4) and later two stages (weeks 9 and 15), and seemed to simplify over time as the biofilm developed.

Apilanez et al. [30] demonstrated that once biofilm has attained a certain weight, which can be related to a certain thickness, detachment occurs. The development of greater biofilm thickness can thus lead to earlier, and a great extent of detachment. Several processes can lead to detachment: erosion or shearing, and sloughing and abrasion. Donlan [31] wrote that when biofilm increases in thickness, its rate of erosion also increases. The detached biofilm possi‐ bly settles at the bottom of the tank, but it also provides a way for cells to migrate and colonize

At the tank outlet, in contrast, a biofilm thickness of 5.7 ± 0.7 μm was seen at the end of the first week and that thickness continued to increase until it reached 29.5 ± 2.0 μm at week 15. No detachment phase was observed at the outlet in this study. It seems that because the nutrient concentration is lower at the outlet, biofilm development is slower at that site.

**Figure 10.** Comparison of biofilm thickness obtained by CSLM (average ± standard deviation, n = 4).

Two sets of biofilms displayed changes in their DGGE banding patterns and number of bands as they developed (**Figures 11** and **12**). Differences were apparent between the inlet and outlet samples, both in the individual samples of a specific age and in the overall pattern of bacteri‐ al community development. The biofilm at the inlet exhibited a greater number of bands in the earliest sample (1 week), displaying a decrease by the fourth week and then increasing again. At the outlet, there were also a greater number of bands after 1 week, a decrease by the ninth week, and then a slight increase. The band patterns appeared similar between the initial two stages (weeks 1 and 4) and later two stages (weeks 9 and 15), and seemed to simplify over

*3.2.4. Dynamics in bacterial community*

time as the biofilm developed.

a less populated area.

158 Microbial Biofilms - Importance and Applications

**Figure 11.** DGGE profiles at each sampling time point and the closest phylogenetic relatives found in the DGGE bands.

Following a high number of bands at the initial stage, reductions occurred later, possibly arising from the competitive dominance of a few populations. The biofilms at the inlet and outlet demonstrated a reduction in the number of bands after the first sample date. The populations that were initially detected may still have been present in later biofilms, but the rapid growth of other populations made them more difficult to detect. As biofilm matures, the number of available microhabitats may increase (for example, from the formation of an anaerobic pocket within the biofilm), thereby supporting a greater number of bacterial populations. Following a high number of bands at the initial reductions occurred arising from the competitive dominance of a few The biofilms at the inlet outlet demonstrated a reduction in the number of bands after the first sample rapid growth of other populations made them more difficult to As biofilm number of available microhabitats may (for from the formation of anaerobic pocket within the thereby supporting a greater number of position in the DGGE gels in almost all of the including *Methylophilus* 

Despite the overall differences in banding patterns, a number of bands appeared at the same position in the DGGE gels in almost all of the samples, including *Methylophilus methylotro‐ phus*(Band No. 1), *Methylocella palustris*(Band No. 9) and *Nitrospira* sp. (Band No. 10), although their intensity differed. *Methylophilus* sp. (Band No. 2) was found only at the outlet site at all stages, whereas *Methylotenera mobilis* (Band No. 5), *Microbacterium pumilum* (Band No. 6) and *Bacillus* sp. (Band No. 8) were identified in the earlier samples (week 1 and/or week 4). Some bands, such as *Nitrospira* sp. (Band No. 10), faded over time.

In the initial stage of biofilm formation, free-swimming bacteria attach to the surface through hydrophobic and electrostatic interactions and through the use of flagella [32]. In this study, for example, both *Methylotenera mobilis* (Band No. 5) and *Microbacterium pumilum* (Band No. 6) were detected in the earlier samples. The former is mobile by means of a single flagellum [33], and the latteris non-motile [34]. Initial colonization on the surface may not be entirely random, in that certain bacterial species may have greater colonization aptitude than others, such as greater mobility.

outlet site, most likely due to the difference in nutrient concentrations. As the biofilm development process was faster at the tank inlet, more sludge from the detachment was also

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 161

**Figure 13.** Rank-abundance distributions of bacteria in different periods of biofilm development.

1 week −0.113 0.90 −0.106 0.96 4 weeks −0.194 0.89 −0.105 0.97 9 weeks −0.111 0.84 −0.150 0.98 15 weeks −0.141 0.81 −0.123 0.88

**Table 3.** Regression statistics for rank-abundance distributions of bacteria in the biofilm at different sites and stages of

The persistence of the *P. aeruginosa* cells inoculated into the pilot tanks resulted from the interaction between the cell growth and death rates and from that among the attachment, detachment, and sedimentation processes. The total number of inoculated cells in the water samples and on the bottoms and walls of the tanks decreased in both tanks (**Figure 14**). Cell death contributed more to the observed cell decline than did growth in the tanks due to low-

**Figure 15** shows the removal rate of *P. aeruginosa* from the water of the two pilot tanks. Ninetynine percent of the inoculated *P. aeruginosa* was removed after 4 days in Pilot Tank 2 and after 5 days in Pilot Tank 1. The faster removal rate in Pilot Tank 2 was due to its higher S/V ratio.

**Slope r2 Slope r2**

**Sample Inlet Outlet**

**3.3. The effect of biofilms on microbial quality in rainwater tanks**

development (P < 0.05; n varies by sample).

*3.3.1. P. aeruginosa removal in water*

nutrient conditions.

seen at this site.

**Figure 12.** Changes in the number of DGGE bands in the biofilm during the experimental period.

Rank-abundance distributions provide insights into both richness and evenness. In this study, the rank-abundance plots displayed a trend towards a geometric distribution (**Figure 13**), and linear regression was performed to examine changes in the pattern of evenness during biofilm development (**Table 3**). Lower slope values indicate greater evenness, and higher values indicate greater dominance by certain populations. At the tank inlet, the slope value was −0.113 at the end of the first week. It then increased steeply after 4 weeks, decreased after the ninth week, and then increased again. At the outlet, in contrast, the slope value increased sharply at the 9-week sample and then exhibited a decrease at the last sample.

The pattern of biofilm development seems to follow three major stages. Jackson et al. [35] suggested an initial stage characterized by the colonization of different populations, an intermediate stage characterized by a limited number of dominant populations utilizing similar resources, and a late or mature stage characterized by mature biofilm of a complex spatial structure that facilitates greater diversity through increased variation in habitat and available resources. However, in the current study, this characterization appeared to apply only up until the mature stage, after which detachment occurred. Following detachment, biofilm development appeared to return to the intermediate and/or mature stage, and the process was then repeated. In this study, this pattern/cycle was confirmed at the tank inlet site, where biofilm development was more rapid throughout the experimental period than at the outlet site, most likely due to the difference in nutrient concentrations. As the biofilm development process was faster at the tank inlet, more sludge from the detachment was also seen at this site.

**Figure 13.** Rank-abundance distributions of bacteria in different periods of biofilm development.


**Table 3.** Regression statistics for rank-abundance distributions of bacteria in the biofilm at different sites and stages of development (P < 0.05; n varies by sample).

#### **3.3. The effect of biofilms on microbial quality in rainwater tanks**

#### *3.3.1. P. aeruginosa removal in water*

in that certain bacterial species may have greater colonization aptitude than others, such as

**Figure 12.** Changes in the number of DGGE bands in the biofilm during the experimental period.

the 9-week sample and then exhibited a decrease at the last sample.

Rank-abundance distributions provide insights into both richness and evenness. In this study, the rank-abundance plots displayed a trend towards a geometric distribution (**Figure 13**), and linear regression was performed to examine changes in the pattern of evenness during biofilm development (**Table 3**). Lower slope values indicate greater evenness, and higher values indicate greater dominance by certain populations. At the tank inlet, the slope value was −0.113 at the end of the first week. It then increased steeply after 4 weeks, decreased after the ninth week, and then increased again. At the outlet, in contrast, the slope value increased sharply at

The pattern of biofilm development seems to follow three major stages. Jackson et al. [35] suggested an initial stage characterized by the colonization of different populations, an intermediate stage characterized by a limited number of dominant populations utilizing similar resources, and a late or mature stage characterized by mature biofilm of a complex spatial structure that facilitates greater diversity through increased variation in habitat and available resources. However, in the current study, this characterization appeared to apply only up until the mature stage, after which detachment occurred. Following detachment, biofilm development appeared to return to the intermediate and/or mature stage, and the process was then repeated. In this study, this pattern/cycle was confirmed at the tank inlet site, where biofilm development was more rapid throughout the experimental period than at the

greater mobility.

160 Microbial Biofilms - Importance and Applications

The persistence of the *P. aeruginosa* cells inoculated into the pilot tanks resulted from the interaction between the cell growth and death rates and from that among the attachment, detachment, and sedimentation processes. The total number of inoculated cells in the water samples and on the bottoms and walls of the tanks decreased in both tanks (**Figure 14**). Cell death contributed more to the observed cell decline than did growth in the tanks due to lownutrient conditions.

**Figure 15** shows the removal rate of *P. aeruginosa* from the water of the two pilot tanks. Ninetynine percent of the inoculated *P. aeruginosa* was removed after 4 days in Pilot Tank 2 and after 5 days in Pilot Tank 1. The faster removal rate in Pilot Tank 2 was due to its higher S/V ratio.

*3.3.2. Microbial behaviour of P. aeruginosa put into the rainwater tanks*

shown in the tanks with higher S/V ratios.

0.14); (C) P < 0.01 except Day 7 (P = 0.10)].

The number of *P. aeruginosa* in the water decreased by 3–4 log units, indicating that the death, attachment and sedimentation processes dominated the overall dynamics (**Figure 16A**). The removal rate of *P. aeruginosa* in the water phase was −0.57 log10 cells ml−1 day−1 (r2 = 0.93) in Pilot Tank 1 and −0.74 log10 cells ml−1 day−1 (r2 = 0.98) in Pilot Tank 2. A faster removal rate was

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 163

**Figure 16.** Behavior of Pseudomonas aeruginosa inoculated in (A) the water and on the (B) wall and (C) bottom of the8 pilot-scale tanks. [Student's t-test; (A) P < 0.05 except Day 0 (P = 0.51); (B) P < 0.1 except Day 0 (P = 0.17) and 1 (P =

**Figure 14.** Total amount of *Pseudomonas aeruginosa* inoculated in pilot-scale tanks. . [Student's t-test; P < 0.05 except Day 0 (P = 0.55) and 3 (P = 0.15)].

**Figure 15.** Removal rate of *Pseudomonas aeruginosa* inoculated in the water of the two pilot tanks.

#### *3.3.2. Microbial behaviour of P. aeruginosa put into the rainwater tanks*

**Figure 14.** Total amount of *Pseudomonas aeruginosa* inoculated in pilot-scale tanks. . [Student's t-test; P < 0.05 except Day

**Figure 15.** Removal rate of *Pseudomonas aeruginosa* inoculated in the water of the two pilot tanks.

0 (P = 0.55) and 3 (P = 0.15)].

162 Microbial Biofilms - Importance and Applications

The number of *P. aeruginosa* in the water decreased by 3–4 log units, indicating that the death, attachment and sedimentation processes dominated the overall dynamics (**Figure 16A**). The removal rate of *P. aeruginosa* in the water phase was −0.57 log10 cells ml−1 day−1 (r2 = 0.93) in Pilot Tank 1 and −0.74 log10 cells ml−1 day−1 (r2 = 0.98) in Pilot Tank 2. A faster removal rate was shown in the tanks with higher S/V ratios.

**Figure 16.** Behavior of Pseudomonas aeruginosa inoculated in (A) the water and on the (B) wall and (C) bottom of the8 pilot-scale tanks. [Student's t-test; (A) P < 0.05 except Day 0 (P = 0.51); (B) P < 0.1 except Day 0 (P = 0.17) and 1 (P = 0.14); (C) P < 0.01 except Day 7 (P = 0.10)].

The number of attached *P. aeruginosa* cells increased over 4 days in Pilot Tank 1 and over 3 days in Pilot Tank 2 (**Figure 16B**). Their attachment to the biofilm on the wall was initially dominant, and more bacteria were attached in Pilot Tank 2 because of the higher S/V ratio.

After 4 days, the number of attached *P. aeruginosa* cells declined by 2–3 log units, indicating that the death or detachment processes were the dominant bacterial dynamics on the wall (**Figure 16B**). Established biofilms developed from indigenous river water bacteria have been shown to reduce the persistence of introduced *E. coli* and other enteric pathogens [36]. Banning et al. [37] showed that, under certain conditions, the presence of mixed-populated biofilms may limit the survival potential of enteric bacteria pathogens introduced into groundwater. In addition, biofilm dynamics changes and pathogen persistence are affected by increasing in nutrient levels. It was reported that a significant decrease in the survival rate of the *Campylo‐ bacter jejuni* strain in heterogeneous tap-water biofilms following the addition of serine, a carbon source favoured by *C. jejuni*, and a concurrent increase in the number of indigenous biofilm microflora [38]. These studies demonstrate that, under certain conditions, biofilms represent sites of intensified competition for limited nutrients. Therefore, for the biofilms in oligotrophic rainwater tanks, a decrease in *P. aeruginosa* cells may result from the nutrients competition with indigenous microbial communities.

Inoculated *P. aeruginosa* were found on the bottom in tanks and decayed over time (**Figure 16C**). More bacteria observed at the bottom of Tank 1, which had a lower S/V ratio, and more bacteria observed on the wall in Tank 2. The number of *P. aeruginosa* increased slightly on days 3 and 6 in Tank 2, probably due to detachment from the wall rather than bacterial regrowth, as this effect was not observed in Tank 1.

**Figure 17.** Number of *Pseudomonas aeruginosa* inoculated in the rainwater of the full-scale tanks. [Student's t-test; P < 0.1

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 165

It has been suggested that rainwater tanks are unique ecosystems that support functional ecosystems comprising complex communities of environmental bacteria [2]. This study showed that a wider surface area for biofilm formation led to a higher removal rate of *P. aeruginosa* in rainwater. When opportunistic pathogens such as *P. aeruginosa* introduced to rainwater tanks with limited nutrient conditions, it seem to be removed due to their attach‐ ment to biofilms and die both naturally because of competition with indigenous microbial

**4. Suggestion of a design and maintenance guideline for rainwater system**

From this study, it was suggested the expected role of biofilms for improving water quality in rainwater tank. Contaminants including microorganisms in rainwater are possibly attached on biofilm, and the biofilms are grown by nutrient degradation and additional attachment. Then, followed sloughing and sedimentation processes, the rainwater quality seemed be

In addition, four recommendations were suggested for design and maintenance of RWH

except Day 0 (P = 0.67)].

*3.3.4. Biofilm's role in rainwater tank*

communities for nutrients.

sustained by certain level.

system as followed description.

#### *3.3.3. Microbial behaviour of P. aeruginosa put into the full-scale tanks*

The number of *P. aeruginosa* in the water decreased by 1.5 log units in Tank 1 and by 2 log units in Tank 2 (**Figure 17**). The removal rate was −0.604 log10 cells ml−1 day−1 (r2 = 0.99) in Tank 1 and −0.854 log10 cells ml−1 day−1 (r2 = 0.98) in Tank 2. In line with the results of the pilot test, a faster removal rate was shown in Tank 2 due to its higher S/V ratio. Thus, it can be conclud‐ ed that increasing the S/V ratio in rainwater tanks to a certain level is possibly effective to remove bacteria from rainwater.

In this study, the removal rates of *P. aeruginosa* were determined by calculating the slope and correlation coefficient (r2 ) of the linear regression of the log-transformed cell concentration data according to the first-order decay equation. Crane and Moore [39] reviewed a variety of modified models of first-order decay kinetics and concluded that the simplest model is the most advantageous. As noted, the findings of the current study suggest that increasing the S/ V ratio in rainwater tanks is an effective way of improving their microbial quality. Accord‐ ingly, additional research aimed at identifying which range of S/V ratios is most effective in improving such quality may benefit from modifying the first-order kinetics. The resulting information would help in the development of appropriate guidelines for the design of rainwater tanks.

**Figure 17.** Number of *Pseudomonas aeruginosa* inoculated in the rainwater of the full-scale tanks. [Student's t-test; P < 0.1 except Day 0 (P = 0.67)].

#### *3.3.4. Biofilm's role in rainwater tank*

The number of attached *P. aeruginosa* cells increased over 4 days in Pilot Tank 1 and over 3 days in Pilot Tank 2 (**Figure 16B**). Their attachment to the biofilm on the wall was initially dominant, and more bacteria were attached in Pilot Tank 2 because of the higher S/V ratio.

After 4 days, the number of attached *P. aeruginosa* cells declined by 2–3 log units, indicating that the death or detachment processes were the dominant bacterial dynamics on the wall (**Figure 16B**). Established biofilms developed from indigenous river water bacteria have been shown to reduce the persistence of introduced *E. coli* and other enteric pathogens [36]. Banning et al. [37] showed that, under certain conditions, the presence of mixed-populated biofilms may limit the survival potential of enteric bacteria pathogens introduced into groundwater. In addition, biofilm dynamics changes and pathogen persistence are affected by increasing in nutrient levels. It was reported that a significant decrease in the survival rate of the *Campylo‐ bacter jejuni* strain in heterogeneous tap-water biofilms following the addition of serine, a carbon source favoured by *C. jejuni*, and a concurrent increase in the number of indigenous biofilm microflora [38]. These studies demonstrate that, under certain conditions, biofilms represent sites of intensified competition for limited nutrients. Therefore, for the biofilms in oligotrophic rainwater tanks, a decrease in *P. aeruginosa* cells may result from the nutrients

Inoculated *P. aeruginosa* were found on the bottom in tanks and decayed over time (**Figure 16C**). More bacteria observed at the bottom of Tank 1, which had a lower S/V ratio, and more bacteria observed on the wall in Tank 2. The number of *P. aeruginosa* increased slightly on days 3 and 6 in Tank 2, probably due to detachment from the wall rather than bacterial

The number of *P. aeruginosa* in the water decreased by 1.5 log units in Tank 1 and by 2 log units in Tank 2 (**Figure 17**). The removal rate was −0.604 log10 cells ml−1 day−1 (r2 = 0.99) in Tank 1

faster removal rate was shown in Tank 2 due to its higher S/V ratio. Thus, it can be conclud‐ ed that increasing the S/V ratio in rainwater tanks to a certain level is possibly effective to

In this study, the removal rates of *P. aeruginosa* were determined by calculating the slope and

according to the first-order decay equation. Crane and Moore [39] reviewed a variety of modified models of first-order decay kinetics and concluded that the simplest model is the most advantageous. As noted, the findings of the current study suggest that increasing the S/ V ratio in rainwater tanks is an effective way of improving their microbial quality. Accord‐ ingly, additional research aimed at identifying which range of S/V ratios is most effective in improving such quality may benefit from modifying the first-order kinetics. The resulting information would help in the development of appropriate guidelines for the design of

= 0.98) in Tank 2. In line with the results of the pilot test, a

) of the linear regression of the log-transformed cell concentration data

competition with indigenous microbial communities.

regrowth, as this effect was not observed in Tank 1.

and −0.854 log10 cells ml−1 day−1 (r2

164 Microbial Biofilms - Importance and Applications

remove bacteria from rainwater.

correlation coefficient (r2

rainwater tanks.

*3.3.3. Microbial behaviour of P. aeruginosa put into the full-scale tanks*

It has been suggested that rainwater tanks are unique ecosystems that support functional ecosystems comprising complex communities of environmental bacteria [2]. This study showed that a wider surface area for biofilm formation led to a higher removal rate of *P. aeruginosa* in rainwater. When opportunistic pathogens such as *P. aeruginosa* introduced to rainwater tanks with limited nutrient conditions, it seem to be removed due to their attach‐ ment to biofilms and die both naturally because of competition with indigenous microbial communities for nutrients.

### **4. Suggestion of a design and maintenance guideline for rainwater system**

From this study, it was suggested the expected role of biofilms for improving water quality in rainwater tank. Contaminants including microorganisms in rainwater are possibly attached on biofilm, and the biofilms are grown by nutrient degradation and additional attachment. Then, followed sloughing and sedimentation processes, the rainwater quality seemed be sustained by certain level.

In addition, four recommendations were suggested for design and maintenance of RWH system as followed description.

**1.** There seems to be a unique microbial ecosystem which is able to control the microbial quality to the certain level by themselves. Thus, it is recommended that avoid mixing with chlorinated tap water which might disturb the microbial ecosystem in rainwater tank. When rainwater and tap water connected system designs, it is advisable to arrange separate supply tank without direct connection of tap water to the main storage tank.

[4] Meera, V. and M.M. Ahammed. Water quality of rooftop rainwater harvesting systems: a review. Journal of Water Supply: Research and Technology – AQUA. 2006;55(4):257–

Role of Biofilm in Rainwater Tank http://dx.doi.org/10.5772/63373 167

[5] Han, M.Y. and J.S. Mun. Particle behavior consideration to maximize the settling capacity of rainwater storage tanks. Water Science and Technology. 2008;56(11):73–79.

[6] Ryu, H. The effects on design factors for water quality and management in a rainwa‐

[7] Geesey, G.G. and J.D. Bryers. Biofouling of engineered materials and systems. In: Bryers, J.D., editor. Biofilms II. Process analysis and applications. Hoboken, NJ: Wiley-

[8] Lazarova V. and J. Manem. Innovative biofilm treatment technologies for water and wastewater treatment. In: Bryers, J.D., editor. Biofilms II. Process analysis and

[9] Sutherland, I.W. Novel and established applications of microbial polysaccharide.

[10] Han, M.Y., S. Park, and S.R. Kim. Analysis of rainwater quality in rainwater harvest‐ ing systems at dormitories in Seoul National University, Seoul, Korea. In Proceed‐

[11] Muyzer, G., E.C. de Waal, and A.G. Uitterlinden. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental

[12] Lewandowski, Z. and H. Beyenal. Fundamentals of biofilm research. Boca Raton, FL:

[13] Apilanez, I., A. Gutiérrez, and M. Diaz. Effect of surface materials on initial biofilm

[14] Wahl, M. Marine epibiosis. I. Fouling and antifouling: some basic aspects. Marine

[15] Williams, M.M., J.W.S. Domingo, M.C. Meckes, C.A. Kelty, and H.S. Rochon. Phylogenetic diversity of drinking water bacteria in a distribution system simulator.

[16] Coenye, T., J. Goris, P. De Vos, P. Vandamme, and J.J. Lipuma. Classification of *Ralstonia pickettii*-like isolates from the environment and clinical samples as *Ralstonia insidiosa* sp. nov. International Journal of Systematic and Evolutionary Microbiology. 2003;53(4):

[17] White, D.C., S.D. Suttont, and D.B. Ringelberg. The genus Sphingomonas: physiolo‐

gy and ecology. Current Opinion in Biotechnology. 1996;7:301–306.

ter storage tank, Master thesis, Seoul National University; 2009.

applications. Hoboken, NJ: Wiley-Liss; 2000. pp. 281–325.

development. Bioresource Technology. 1998;66:225–230.

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1075–1080.


### **Author details**

Mikyeong Kim1 and Mooyoung Han2\*

\*Address all correspondence to: myhan@snu.ac.kr

1 Institute of Construction and Environmental Engineering (ICEE), Seoul National University, Seoul, Republic of Korea

2 Department of Civil and Environmental Engineering, Seoul National University, Seoul, Republic of Korea

### **References**


[4] Meera, V. and M.M. Ahammed. Water quality of rooftop rainwater harvesting systems: a review. Journal of Water Supply: Research and Technology – AQUA. 2006;55(4):257– 268.

**1.** There seems to be a unique microbial ecosystem which is able to control the microbial quality to the certain level by themselves. Thus, it is recommended that avoid mixing with chlorinated tap water which might disturb the microbial ecosystem in rainwater tank. When rainwater and tap water connected system designs, it is advisable to arrange separate supply tank without direct connection of tap water to the main storage tank.

**2.** Design of baffle and inlet barrier is recommended because they not only induce the sedimentation of inflow particle but also control bacterial quality in rainwater by

**3.** Increasing the S/V ratio bacterial quality is possibly controlled by inducing more bio‐ film development. Therefore, it is recommended to consider the parameter of S/V ratio

**4.** Frequent cleaning and/or disinfection of rainwater tank inside seems to be counterpro‐ ductive because biofilm developed in rainwater tank improve the bacterial quality in

1 Institute of Construction and Environmental Engineering (ICEE), Seoul National University,

2 Department of Civil and Environmental Engineering, Seoul National University, Seoul,

[1] Krampitz, E. and R. Hollander. Longevity of pathogenic bacteria especially Salmonel‐

[2] Evans, C.A., P.J. Coombes, R.H. Dunstan, and T. Harrison. Extensive bacterial diversity indicates the potential operation of a dynamic micro-ecology within domestic rainwa‐

[3] Cunliffe, D.A. Guidance on the use of rainwater tanks. National Environmental Health

ter storage systems. Science of the Total Environment. 2009;407:5206–5215.

la in cistern water. Zentralbl Hyg Umweltmed. 1999;202(5):389–397.

increasing the surface for biofilm development.

rainwater tank by adhesion of bacteria in rainwater.

and Mooyoung Han2\*

\*Address all correspondence to: myhan@snu.ac.kr

when rainwater tank is designed.

166 Microbial Biofilms - Importance and Applications

**Author details**

Mikyeong Kim1

Seoul, Republic of Korea

Forum. 1998. p. 8.

Republic of Korea

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168 Microbial Biofilms - Importance and Applications


**Chapter 9**

**Biofilms in Beverage Industry**

Additional information is available at the end of the chapter

**Keywords:** biofilms, water, soft drinks, *Aeromonas* , *Asaia*

trial and aquatic environments in very high abundance.

Over the years, numerous studies have been conducted into the possible links between biofilms in beverage industry and health safety. Consumers trust that the soft drinks they buy are safe and their quality is guaranteed. This chapter provides an overview of available scientific knowledge and cites numerous studies on various aspects of biofilms in drinking water technology and soft drinks industry and their implications for health safety. Particular attention is given to *Proteobacteria*, including two different genera: *Aeromo‐ nas*, which represents *Gammaproteobacteria*, and *Asaia*, a member of *Alphaproteobacteria*.

In water systems, both natural and industrial dominate *Proteobacteria*. This is the main group (phylum) of Gram-negative bacteria, taxonomically very diverse, consisting of more than 200 genera. Its membership includes both pathogenic bacteria of the genera *Escherichia*, *Salmonel‐ la*, *Vibrio*, *Helicobacter*, and many other types of free-living or symbiotic, motile or nonmotile, chemoautotrophic or heterotrophic bacteria from outstanding aerobes to obligatory anaerobes. Although bacteria are physiologically and morphologically diverse, they constitute a coher‐ ent set of six main classes: *Alphaproteobacteria*, *Betaproteobacteria*, *Gammaproteobacteria*, *Deltaproteobacteria*, *Epsilonproteobacteria*, and *Zetaproteobacteria*. Taxonomy of the group is determined primarily on the basis of ribosomal RNA sequences [1]. Species belonging to the classes *Alphaproteobacteria*, *Betaproteobacteria*, and *Gammaproteobacteria* are very heterogene‐ ous in their physiological characteristics. Each of the three classes includes aerobes and anaerobes, photosynthetic and nonphotosynthetic cells. They are distributed in both terres‐

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.

Dorota Kregiel and Hubert Antolak

http://dx.doi.org/10.5772/62940

**1. Drinking water systems**

**Abstract**

### **Chapter 9**

## **Biofilms in Beverage Industry**

Dorota Kregiel and Hubert Antolak

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62940

#### **Abstract**

Over the years, numerous studies have been conducted into the possible links between biofilms in beverage industry and health safety. Consumers trust that the soft drinks they buy are safe and their quality is guaranteed. This chapter provides an overview of available scientific knowledge and cites numerous studies on various aspects of biofilms in drinking water technology and soft drinks industry and their implications for health safety. Particular attention is given to *Proteobacteria*, including two different genera: *Aeromo‐ nas*, which represents *Gammaproteobacteria*, and *Asaia*, a member of *Alphaproteobacteria*.

**Keywords:** biofilms, water, soft drinks, *Aeromonas* , *Asaia*

### **1. Drinking water systems**

In water systems, both natural and industrial dominate *Proteobacteria*. This is the main group (phylum) of Gram-negative bacteria, taxonomically very diverse, consisting of more than 200 genera. Its membership includes both pathogenic bacteria of the genera *Escherichia*, *Salmonel‐ la*, *Vibrio*, *Helicobacter*, and many other types of free-living or symbiotic, motile or nonmotile, chemoautotrophic or heterotrophic bacteria from outstanding aerobes to obligatory anaerobes.

Although bacteria are physiologically and morphologically diverse, they constitute a coher‐ ent set of six main classes: *Alphaproteobacteria*, *Betaproteobacteria*, *Gammaproteobacteria*, *Deltaproteobacteria*, *Epsilonproteobacteria*, and *Zetaproteobacteria*. Taxonomy of the group is determined primarily on the basis of ribosomal RNA sequences [1]. Species belonging to the classes *Alphaproteobacteria*, *Betaproteobacteria*, and *Gammaproteobacteria* are very heterogene‐ ous in their physiological characteristics. Each of the three classes includes aerobes and anaerobes, photosynthetic and nonphotosynthetic cells. They are distributed in both terres‐ trial and aquatic environments in very high abundance.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In natural systems, freshwater or potable water distribution networks, *Betaproteobacteria* dominate (87–99%), while *Alphaproteobacteria* are in marine waters [2]. *Proteobacteria* predomi‐ nated in biofilms present in drinking water distribution systems, but the compositions of the dominant proteobacterial classes and genera and their proportions varied among biofilm samples [3]. The majority of strains isolated from biofilms in water distribution networks is *Alpha-* or *Gammaproteobacteria* [4]. Except *Proteobacteria*, *Firmicutes*, *Bacteroidetes*, *Actinobacte‐ ria*, *Nitrospirae*, and *Cyanobacteria* are usually the major components of biofilm bacterial community.

Physicochemical nature of such consortia implies differentiation of the physiological condi‐ tion of individuals forming them [8]. Creating consortium is an effective adaptation strategy, including cell protection against adverse environmental factors; increased nutrient availabil‐ ity; increased binding of water molecules, thereby reducing the risk of dehydration; and

Biofilms in Beverage Industry http://dx.doi.org/10.5772/62940 173

Microbial consortia exhibit altered phenotypic characteristics compared to planktonic cells, particularly with respect to growth and gene expression. All these factors increase the survival of cells forming biofilms. As a result, the inactivation of bacterial cells by conventional methods such as the use of antibiotics and disinfectants is often ineffective [9]. Especially this exopoly‐ mer matrix confers resistant properties to the whole system via the limitation of the effective‐ ness of disinfection by consuming the oxidants used, such as chlorine and chloramines [10].

At high cell concentration, a series of cell signaling mechanisms are employed by the biofilm, and this is collectively termed *quorum sensing*. *Quorum sensing* describes a process where a number of autoinducers (chemical and peptide signals in high concentrations, e.g., homoser‐ ine lactones) are used to stimulate genetic expression of both mechanical and enzymatic processes. In mature biofilms, enzymes are produced by the community itself which break‐ down polysaccharides holding the biofilm together, actively releasing surface bacteria for colonization of fresh substrates. For example, alginate lyase produced by *Pseudomonas fluorescens* and *Pseudomonas aeruginosa*, N-acetyl-heparosan lyase by *Escherichia coli*, and hyaluronidase by *Streptococcus equi* are used in the breakdown of the biofilm matrix [6].

Biofilms are polymicrobial communities, therefore the potential for cell coaggregation plays an integral role in spatiotemporal biofilm development and the moderation of biofilm composition. Coaggregation is mediated by the interaction between specific macromolecules on the cell surface of one species and cognate macromolecules expressed on the cell surface of the partner species. Microbial cells may also come into contact through hydrophobic interac‐ tions or electrostatic forces, but these last associations are relatively weak. Coaggregationmediating proteins are referred as adhesins. Coaggregation may occur between lectin-like protein adhesins and their polysaccharide receptors or by protein–protein (adhesin–adhesin) interactions. These interactions may be unimodal, but in some cases are bimodal, involving

Cell aggregation, as well as biofilm formation may have both intrageneric and intergeneric character [11]. Consortia are very changeable and their components depend on the environ‐ mental conditions. The study conducted by Rickard et al. [12] revealed that intergeneric and intraspecies coaggregation between water bacteria are common phenomena, and expression of coaggregation is dependent on cells being in the optimum physiological state for coaggre‐ gation, which usually occurs in stationary phase. Therefore, it is possible that since cells grow very slowly in nutrient-limited biofilms, these biofilms would provide suitable conditions for

Different materials such as cast iron galvanized steel, stainless steel, copper, and polyethy‐ lene are used to manufacture water distribution pipes. It is worth noting that these materials favor biofilm formation in the water distribution systems. The presence of biofilms in drinking

two different interacting pairs of macromolecules [7].

expression of coaggregation.

increased ability to transfer DNA.

One of the common features of *Proteobacteria* is the ability of biofilm formation and/or aggregation and formation of the so-called "flocs". An important component of such struc‐ tures, in addition to microbial cells, is water – it represents about 97%. Besides water, the biofilm or flocs matrix are extracellular polymeric substances (EPSs). The bacterial cells in biofilms are embedded in a heteropolymeric matrix containing humic substances, glycopro‐ teins, polysaccharides, and nucleic acids [5].

A first step in the successional development of biofilms is the coating of uncolonized surfa‐ ces with many particles, organic or inorganic (conditioning film), which enhances attach‐ ment of initial colonizing bacteria. Anything that may be present within the bulk fluid can through gravitational force or movement of flow settle onto a surface and become part of a conditioning layer. Surface charge, potential, and tensions can be altered favorably by the interactions between the conditioning layer and the surface. Factors such as available energy, surface functionality, bacterial orientation, temperature and pressure conditions are local environmental variables which contribute to bacterial adhesion. Physical forces associated to bacterial adhesion include the van der Waals' forces, steric interactions, and electrostatic (double layer) interactions, collectively known as the DVLO (Derjaguin, Verwey, Landau, and Overbeek) forces [6]. An extended DVLO theory takes into consideration hydrophobic/ hydrophilic and osmotic interactions.

In real time, a number of the reversibly adsorbed cells remain immobilized and become irreversibly adsorbed. The physical appendages of bacteria (flagella, fimbriae, and pili) overcome the physical repulsive forces of the electrical double layer. Some evidence has shown that microbial adhesion strongly depends on the hydrophobic–hydrophilic properties of interacting surfaces. The first colonizers grow in surface-attached microcolonies and pro‐ duce EPS. After an initial lag phase, a rapid increase in population is observed, which is described as the exponential growth phase. As the microcolonies develop, additional species, the so-called secondary colonizers, are recruited through coaggregation and nonspecific aggregation interactions, increasing the biofilm biomass and species complexity [7].

Simultaneously, expression of a number of genes for the production of cell surface proteins and excretion products increases. Surface proteins (porins) such as Opr C and Opr E allow the transport of extracellular products into the cell and excretion materials out of the cell, e.g., polysaccharides. EPS molecules impart mechanical stability and are pivotal to biofilm adhesion and cohesion, and evasion from harsh dynamic environmental conditions. The differences in gene expression of planktonic and sessile cells were identified, and as many as 57 biofilm-associated proteins were not found in the planktonic profile [6].

Physicochemical nature of such consortia implies differentiation of the physiological condi‐ tion of individuals forming them [8]. Creating consortium is an effective adaptation strategy, including cell protection against adverse environmental factors; increased nutrient availabil‐ ity; increased binding of water molecules, thereby reducing the risk of dehydration; and increased ability to transfer DNA.

In natural systems, freshwater or potable water distribution networks, *Betaproteobacteria* dominate (87–99%), while *Alphaproteobacteria* are in marine waters [2]. *Proteobacteria* predomi‐ nated in biofilms present in drinking water distribution systems, but the compositions of the dominant proteobacterial classes and genera and their proportions varied among biofilm samples [3]. The majority of strains isolated from biofilms in water distribution networks is *Alpha-* or *Gammaproteobacteria* [4]. Except *Proteobacteria*, *Firmicutes*, *Bacteroidetes*, *Actinobacte‐ ria*, *Nitrospirae*, and *Cyanobacteria* are usually the major components of biofilm bacterial

One of the common features of *Proteobacteria* is the ability of biofilm formation and/or aggregation and formation of the so-called "flocs". An important component of such struc‐ tures, in addition to microbial cells, is water – it represents about 97%. Besides water, the biofilm or flocs matrix are extracellular polymeric substances (EPSs). The bacterial cells in biofilms are embedded in a heteropolymeric matrix containing humic substances, glycopro‐

A first step in the successional development of biofilms is the coating of uncolonized surfa‐ ces with many particles, organic or inorganic (conditioning film), which enhances attach‐ ment of initial colonizing bacteria. Anything that may be present within the bulk fluid can through gravitational force or movement of flow settle onto a surface and become part of a conditioning layer. Surface charge, potential, and tensions can be altered favorably by the interactions between the conditioning layer and the surface. Factors such as available energy, surface functionality, bacterial orientation, temperature and pressure conditions are local environmental variables which contribute to bacterial adhesion. Physical forces associated to bacterial adhesion include the van der Waals' forces, steric interactions, and electrostatic (double layer) interactions, collectively known as the DVLO (Derjaguin, Verwey, Landau, and Overbeek) forces [6]. An extended DVLO theory takes into consideration hydrophobic/

In real time, a number of the reversibly adsorbed cells remain immobilized and become irreversibly adsorbed. The physical appendages of bacteria (flagella, fimbriae, and pili) overcome the physical repulsive forces of the electrical double layer. Some evidence has shown that microbial adhesion strongly depends on the hydrophobic–hydrophilic properties of interacting surfaces. The first colonizers grow in surface-attached microcolonies and pro‐ duce EPS. After an initial lag phase, a rapid increase in population is observed, which is described as the exponential growth phase. As the microcolonies develop, additional species, the so-called secondary colonizers, are recruited through coaggregation and nonspecific

aggregation interactions, increasing the biofilm biomass and species complexity [7].

57 biofilm-associated proteins were not found in the planktonic profile [6].

Simultaneously, expression of a number of genes for the production of cell surface proteins and excretion products increases. Surface proteins (porins) such as Opr C and Opr E allow the transport of extracellular products into the cell and excretion materials out of the cell, e.g., polysaccharides. EPS molecules impart mechanical stability and are pivotal to biofilm adhesion and cohesion, and evasion from harsh dynamic environmental conditions. The differences in gene expression of planktonic and sessile cells were identified, and as many as

community.

teins, polysaccharides, and nucleic acids [5].

172 Microbial Biofilms - Importance and Applications

hydrophilic and osmotic interactions.

Microbial consortia exhibit altered phenotypic characteristics compared to planktonic cells, particularly with respect to growth and gene expression. All these factors increase the survival of cells forming biofilms. As a result, the inactivation of bacterial cells by conventional methods such as the use of antibiotics and disinfectants is often ineffective [9]. Especially this exopoly‐ mer matrix confers resistant properties to the whole system via the limitation of the effective‐ ness of disinfection by consuming the oxidants used, such as chlorine and chloramines [10]. to

At high cell concentration, a series of cell signaling mechanisms are employed by the biofilm, and this is collectively termed *quorum sensing*. *Quorum sensing* describes a process where a number of autoinducers (chemical and peptide signals in high concentrations, e.g., homoser‐ ine lactones) are used to stimulate genetic expression of both mechanical and enzymatic processes. In mature biofilms, enzymes are produced by the community itself which break‐ down polysaccharides holding the biofilm together, actively releasing surface bacteria for colonization of fresh substrates. For example, alginate lyase produced by *Pseudomonas fluorescens* and *Pseudomonas aeruginosa*, N-acetyl-heparosan lyase by *Escherichia coli*, and hyaluronidase by *Streptococcus equi* are used in the breakdown of the biofilm matrix [6]. e.g.,

Biofilms are polymicrobial communities, therefore the potential for cell coaggregation plays an integral role in spatiotemporal biofilm development and the moderation of biofilm composition. Coaggregation is mediated by the interaction between specific macromolecules on the cell surface of one species and cognate macromolecules expressed on the cell surface of the partner species. Microbial cells may also come into contact through hydrophobic interac‐ tions or electrostatic forces, but these last associations are relatively weak. Coaggregationmediating proteins are referred as adhesins. Coaggregation may occur between lectin-like protein adhesins and their polysaccharide receptors or by protein–protein (adhesin–adhesin) interactions. These interactions may be unimodal, but in some cases are bimodal, involving two different interacting pairs of macromolecules [7]. potential through

Cell aggregation, as well as biofilm formation may have both intrageneric and intergeneric character [11]. Consortia are very changeable and their components depend on the environ‐ mental conditions. The study conducted by Rickard et al. [12] revealed that intergeneric and intraspecies coaggregation between water bacteria are common phenomena, and expression of coaggregation is dependent on cells being in the optimum physiological state for coaggre‐ gation, which usually occurs in stationary phase. Therefore, it is possible that since cells grow very slowly in nutrient-limited biofilms, these biofilms would provide suitable conditions for expression of coaggregation.

Different materials such as cast iron galvanized steel, stainless steel, copper, and polyethy‐ lene are used to manufacture water distribution pipes. It is worth noting that these materials favor biofilm formation in the water distribution systems. The presence of biofilms in drinking

water distribution pipes usually leads to a number of undesirable effects on the quality of water that is supplied to consumers. For example, the development of biofilms in copper pipes facilitates cuprosolvency which increases the release of copper into the distribution system. What's more, increased carbon influences the growth of heterotrophic plate count bacteria which are also involved in the corrosion of copper [13]. Silhan et al. [14] showed that among drinking water pipe materials such as galvanized steel, cross-linked polyethylene, copper pipes, and medium-density polyethylene, the most dense biofilm of *E. coli* was formed on the steel surface.

Molecular analysis of microbial communities by Yu et al. [15] indicated the presence of *Alpha*and *Betaproteobacteria*, *Actinobacteria*, and *Bacteroidetes* in biofilms on the pipe materials. Moreover, the DGGE profile of bacterial 16S rDNA fragments showed significant differen‐ ces among different surfaces, suggesting that the pipe materials affect not only biofilm formation potential but also microbial diversity.

The development of biofilms inside water distribution pipes facilitates the propagation of mixed microbial populations and is considered the main source of planktonic bacteria in water supply systems. Among the heterotrophic bacteria in drinking water systems, the pathogen‐ ic bacteria or at least opportunistic pathogens often appear. Enteropathogenic *E. coli* or other members of *Enterobacteriaceae* may appear in water supply systems due to contamination as a result of flooding, water supply failure, or insufficient disinfection. Other opportunistic bacteria such as *P. aeruginosa*, *Burkholderia* spp., *Stenotrophomonas maltophilia*, and *Legionella* spp. were quite often detected [16]. They increase the health risks associated with the con‐ sumption of water [13].

**Figure 1.** Gram-negative rods of *Aeromonas hydrophila*.

gastroenteritis caused by *Aeromonas*.

they were confirmed by luminometric measurements.

[22].

The vast majority of bacteria isolated from biofilms belonged to *Aeromonas hydrophila*. They showed the major virulence factors such as surface polysaccharides (capsule, lipopolysac‐ charide, and glucan), S-layers, iron-binding systems, exotoxins and extracellular enzymes, secretion systems, fimbriae, and other nonfilamentous adhesins, motility, and flagella [21, 22]. Despite the demonstration of the enterotoxic potential of some *Aeromonas* spp. strains, there is still a debate on its consideration as an etiological agent, as there were no big epidemical outbreaks described and no adequate animal model is available to reproduce the

Biofilms in Beverage Industry http://dx.doi.org/10.5772/62940 175

In clinical and environmental isolates of *Aeromonas* species, two distinct types of fimbriae have been found based on their morphology: short, rigid fimbriae (0,6–2 μm) that can be found in high numbers on the bacterial cell and long, wavy fimbriae (4–7 nm) found in smaller numbers. The short fimbriae are able to cause autoaggregation, and large ones considered hemaggluti‐ nins. Amino-acid sequence analysis indicates that they correspond to type IV pili, known as important structures for adhesion to epithelial cells and involved in biofilm formation. Some of them exhibit highest homology with the type IV pili of *Pseudomonas* and *Neisseria* species

In studies conducted by Kregiel et al., *A. hydrophila* isolated from water distribution system, adhered to different abiotic surfaces such as glass, polystyrene, polyvinyl chloride, and gumosil, commonly used as packaging and installation materials [23–25]. After 3 weeks in an aqueous environment with a small amount of organic matter, bacteria formed numerous microcolonies surrounded extracellular mucilaginous substance (**Figure 2**). The results of microscopic examination demonstrated the strong adhesion properties of *A. hydrophila* and

In the last decade, a group of new, potentially dangerous pathogens forming biofilms were classified as *Aeromonas* spp. rods from class *Gammaproteobacteria* [17, 18] (**Figure 1**). The experimental data and clinical and epidemiological evidence show that *Aeromonas* spp. may be an etiological factor of bacterial gastroenteritis in children and people with reduced immunity.

Bacteria *Aeromonas* spp. are capable not only of survival, but also propagation in water at temperatures up to 10°C and show a greater ability to utilize different carbon compounds than other Gram-negative bacteria.

According to Sautour et al. [19], the genus *Aeromonas* shows the ability to use not only carbohydrates, amino acids, and carboxylic acids, but also fatty acids and saturated hydro‐ carbons. Growth of these bacteria in an aqueous medium follows in the presence of even a small amount of biodegradable dissolved organic carbon compounds.

It was noted that there was an intense increase in the number of heterotrophic bacteria in the summer months. The results obtained by Craveiro et al. [20] demonstrated that *Aeromonas* spp. strains were able to form biofilm at both room and refrigeration temperatures. The chlorinebased disinfectant demonstrated to be efficient in removing preformed biofilm, but both were unsuccessful in preventing biofilm formation, highlighting the importance of adequate cleaning and disinfection procedures, with emphasis on food processing surfaces.

**Figure 1.** Gram-negative rods of *Aeromonas hydrophila*.

water distribution pipes usually leads to a number of undesirable effects on the quality of water that is supplied to consumers. For example, the development of biofilms in copper pipes facilitates cuprosolvency which increases the release of copper into the distribution system. What's more, increased carbon influences the growth of heterotrophic plate count bacteria which are also involved in the corrosion of copper [13]. Silhan et al. [14] showed that among drinking water pipe materials such as galvanized steel, cross-linked polyethylene, copper pipes, and medium-density polyethylene, the most dense biofilm of *E. coli* was formed on the

Molecular analysis of microbial communities by Yu et al. [15] indicated the presence of *Alpha*and *Betaproteobacteria*, *Actinobacteria*, and *Bacteroidetes* in biofilms on the pipe materials. Moreover, the DGGE profile of bacterial 16S rDNA fragments showed significant differen‐ ces among different surfaces, suggesting that the pipe materials affect not only biofilm

The development of biofilms inside water distribution pipes facilitates the propagation of mixed microbial populations and is considered the main source of planktonic bacteria in water supply systems. Among the heterotrophic bacteria in drinking water systems, the pathogen‐ ic bacteria or at least opportunistic pathogens often appear. Enteropathogenic *E. coli* or other members of *Enterobacteriaceae* may appear in water supply systems due to contamination as a result of flooding, water supply failure, or insufficient disinfection. Other opportunistic bacteria such as *P. aeruginosa*, *Burkholderia* spp., *Stenotrophomonas maltophilia*, and *Legionella* spp. were quite often detected [16]. They increase the health risks associated with the con‐

In the last decade, a group of new, potentially dangerous pathogens forming biofilms were classified as *Aeromonas* spp. rods from class *Gammaproteobacteria* [17, 18] (**Figure 1**). The experimental data and clinical and epidemiological evidence show that *Aeromonas* spp. may be an etiological factor of bacterial gastroenteritis in children and people with reduced

Bacteria *Aeromonas* spp. are capable not only of survival, but also propagation in water at temperatures up to 10°C and show a greater ability to utilize different carbon compounds than

According to Sautour et al. [19], the genus *Aeromonas* shows the ability to use not only carbohydrates, amino acids, and carboxylic acids, but also fatty acids and saturated hydro‐ carbons. Growth of these bacteria in an aqueous medium follows in the presence of even a

It was noted that there was an intense increase in the number of heterotrophic bacteria in the summer months. The results obtained by Craveiro et al. [20] demonstrated that *Aeromonas* spp. strains were able to form biofilm at both room and refrigeration temperatures. The chlorinebased disinfectant demonstrated to be efficient in removing preformed biofilm, but both were unsuccessful in preventing biofilm formation, highlighting the importance of adequate

cleaning and disinfection procedures, with emphasis on food processing surfaces.

small amount of biodegradable dissolved organic carbon compounds.

steel surface.

174 Microbial Biofilms - Importance and Applications

sumption of water [13].

other Gram-negative bacteria.

immunity.

formation potential but also microbial diversity.

The vast majority of bacteria isolated from biofilms belonged to *Aeromonas hydrophila*. They showed the major virulence factors such as surface polysaccharides (capsule, lipopolysac‐ charide, and glucan), S-layers, iron-binding systems, exotoxins and extracellular enzymes, secretion systems, fimbriae, and other nonfilamentous adhesins, motility, and flagella [21, 22]. Despite the demonstration of the enterotoxic potential of some *Aeromonas* spp. strains, there is still a debate on its consideration as an etiological agent, as there were no big epidemical outbreaks described and no adequate animal model is available to reproduce the gastroenteritis caused by *Aeromonas*. The vast majority of bacteria isolated from biofilms belonged to *Aeromonas* showed the major virulence factors such as surface and iron-binding exotoxins and extracellular secretion and other nonfilamentous and Despite the demonstration of the enterotoxic potential of some is still a debate on its consideration as an etiological as there were no big outbreaks described and no adequate animal model is available to reproduce

In clinical and environmental isolates of *Aeromonas* species, two distinct types of fimbriae have been found based on their morphology: short, rigid fimbriae (0,6–2 μm) that can be found in high numbers on the bacterial cell and long, wavy fimbriae (4–7 nm) found in smaller numbers. The short fimbriae are able to cause autoaggregation, and large ones considered hemaggluti‐ nins. Amino-acid sequence analysis indicates that they correspond to type IV pili, known as important structures for adhesion to epithelial cells and involved in biofilm formation. Some of them exhibit highest homology with the type IV pili of *Pseudomonas* and *Neisseria* species [22]. In clinical and environmental isolates of two distinct types of fimbriae high numbers on the bacterial cell and wavy found in smaller of them exhibit highest homology with the type IV pili of and

In studies conducted by Kregiel et al., *A. hydrophila* isolated from water distribution system, adhered to different abiotic surfaces such as glass, polystyrene, polyvinyl chloride, and gumosil, commonly used as packaging and installation materials [23–25]. After 3 weeks in an aqueous environment with a small amount of organic matter, bacteria formed numerous microcolonies surrounded extracellular mucilaginous substance (**Figure 2**). The results of microscopic examination demonstrated the strong adhesion properties of *A. hydrophila* and they were confirmed by luminometric measurements. In studies conducted by Kregiel et isolated from water distribution adhered to different abiotic surfaces such as polyvinyl aqueous environment with a small amount of organic bacteria formed microcolonies surrounded extracellular mucilaginous The results

For example, the flavored drinking water samples with sucrose and natural fruit flavors showing signs of turbidity and the characteristic "flocs" formed by heterotrophic bacteria [28]. The developed specific methods allowed for the isolation of bacteria belonging to the *Asaia* spp. – a new, previously unknown in Poland, microbial contamination of mineral water and flavored beverages. Isolated bacteria were Gram-negative, aerobic rods with dimensions of 0.4–1.0 × 08–2.5 μm. These bacteria formed characteristic small (1–3 mm in diameter), pale pink or pink colonies in agar plates. The isolates were identified based on 16S rRNA gene sequen‐ ces. It is worth noting that the same morphotypes and genotypes were isolated from fruit

Biofilms in Beverage Industry http://dx.doi.org/10.5772/62940 177

*Asaia* sp. was established in 2000 as the fifth genera of acetic acid bacteria of the class *Alphaproteobacteria*. Bacteria *Asaia* sp. were first isolated from the orchid tree flower (*Bauhinia purpurea*) and flowers of *Pueraria* (*Plumbago*), growing in tropical climates. Currently, Genera *Asaia* contains eight species named as: *As. bogorensis, As. siamensis, As. krungthhepensis, As. lannensis, As. spathodeae, As. astilbis, As. platycodi* and *As. prunellae.*. It is distinguished from other types of acetic acid bacteria not only by genetic features, but also by biochemical properties. The optimum pH and temperature of these bacteria are 5.5 and 30°C, respective‐ ly. Nevertheless, the strains belonging to *Asaia* sp. isolated from environments in tropical

*Asaia* spp. belongs to the risk 1 group, which means that it is a group of saprophytic microor‐ ganisms without causing diseases in humans. However, according to the literature, these bacteria can cause opportunistic infections when they get into the bloodstream of a man with weakened immune systems. Several cases of bacteremia caused by *Asaia* spp. were document‐ ed, especially in chronically ill adults and pediatric patients with cardiomyopathy or cancer. The first documented case of bacteremia caused by *As. bogorensis* was reported in a young patient with a history of intravenous-drug abuse. *As. bogorensis* was identified by sequenc‐ ing the 16S rRNA gene. The isolate was resistant to almost all antibiotics routinely tested for Gram-negative rods, but was susceptible to gentamicin and doxycycline [30]. One of the last reports describes transient bacteremia due to *As. lannensis* in a patient with a psychiatric disorder and compulsive self-injection of different substances. Only restriction fragment length polymorphism of PCR-amplified 16S rRNA gene allowed for proper identification of

*Asaia* spp. show strong ability to aggregate and form characteristic "flocs" and to create biofilms on selected surfaces commonly used in the food industry: glass, polyethylene terephthalate,

It was found that the hydrophobicity of the cells decreased with increasing the age of the population. The higher hydrophobicity of young cells stimulates the process of aggregation and formation of flocs. The studies proved that the adhesive abilities of *As. lannensis* depend on the carbon source, nutrient availability, and physicochemical properties of abiotic surface. The strongest adhesion properties were characterized by cells in the minimal medium with

concentrates, which were previously used for production of flavored waters.

Indonesia, Thailand, and Japan have optimum growth even at 37°C [29].

isolate. The strain was also highly resistant to most antibiotics [31].

and polypropylene [32] (**Figure 3**).

sucrose.

**Figure 2.** Biofilm of *Aeromonas hydrophila* on a glass surface.

The studies have found that both due to the strong adhesive properties of *A. hydrophila*, and the possibility of the virulence factors determining its pathogenicity, it should be considered the inclusion of *Aeromonas* rods for routine microbiological water analysis, especially for monitoring water or beverage distribution systems.

### **2. Soft drinks**

When a change in the chemical nature of a fluid occurred, there is usually a qualitative shift created microbial consortia [8]. While the succession is a well-known process in classical ecology, in the case of biofilms or cell aggregates it is not fully understood. Despite many researches, the full knowledge on the formation of microbial consortia is still lacking. However, succession processes seem to be rather stochastic (reproduction and death) [26]. During growth of consortia, competition for resources makes that weak individuals are eliminated, and stronger competitors become dominant. Finally, in the mature consortia, cells are becoming more diverse by individual differences and "internal recycling."

Environmental factors may also shape the succession in microbial consortia. Changes in pH, the presence of carbon sources in the form of saccharides, and other additional substances cause significant qualitative changes in biofilms [27].

For example, the flavored drinking water samples with sucrose and natural fruit flavors showing signs of turbidity and the characteristic "flocs" formed by heterotrophic bacteria [28]. The developed specific methods allowed for the isolation of bacteria belonging to the *Asaia* spp. – a new, previously unknown in Poland, microbial contamination of mineral water and flavored beverages. Isolated bacteria were Gram-negative, aerobic rods with dimensions of 0.4–1.0 × 08–2.5 μm. These bacteria formed characteristic small (1–3 mm in diameter), pale pink or pink colonies in agar plates. The isolates were identified based on 16S rRNA gene sequen‐ ces. It is worth noting that the same morphotypes and genotypes were isolated from fruit concentrates, which were previously used for production of flavored waters.

*Asaia* sp. was established in 2000 as the fifth genera of acetic acid bacteria of the class *Alphaproteobacteria*. Bacteria *Asaia* sp. were first isolated from the orchid tree flower (*Bauhinia purpurea*) and flowers of *Pueraria* (*Plumbago*), growing in tropical climates. Currently, Genera *Asaia* contains eight species named as: *As. bogorensis, As. siamensis, As. krungthhepensis, As. lannensis, As. spathodeae, As. astilbis, As. platycodi* and *As. prunellae.*. It is distinguished from other types of acetic acid bacteria not only by genetic features, but also by biochemical properties. The optimum pH and temperature of these bacteria are 5.5 and 30°C, respective‐ ly. Nevertheless, the strains belonging to *Asaia* sp. isolated from environments in tropical Indonesia, Thailand, and Japan have optimum growth even at 37°C [29].

*Asaia* spp. belongs to the risk 1 group, which means that it is a group of saprophytic microor‐ ganisms without causing diseases in humans. However, according to the literature, these bacteria can cause opportunistic infections when they get into the bloodstream of a man with weakened immune systems. Several cases of bacteremia caused by *Asaia* spp. were document‐ ed, especially in chronically ill adults and pediatric patients with cardiomyopathy or cancer. The first documented case of bacteremia caused by *As. bogorensis* was reported in a young patient with a history of intravenous-drug abuse. *As. bogorensis* was identified by sequenc‐ ing the 16S rRNA gene. The isolate was resistant to almost all antibiotics routinely tested for Gram-negative rods, but was susceptible to gentamicin and doxycycline [30]. One of the last reports describes transient bacteremia due to *As. lannensis* in a patient with a psychiatric disorder and compulsive self-injection of different substances. Only restriction fragment length polymorphism of PCR-amplified 16S rRNA gene allowed for proper identification of isolate. The strain was also highly resistant to most antibiotics [31].

**Figure 2.** Biofilm of *Aeromonas hydrophila* on a glass surface.

176 Microbial Biofilms - Importance and Applications

monitoring water or beverage distribution systems.

more diverse by individual differences and "internal recycling."

cause significant qualitative changes in biofilms [27].

**2. Soft drinks**

The studies have found that both due to the strong adhesive properties of *A. hydrophila*, and the possibility of the virulence factors determining its pathogenicity, it should be considered the inclusion of *Aeromonas* rods for routine microbiological water analysis, especially for

When a change in the chemical nature of a fluid occurred, there is usually a qualitative shift created microbial consortia [8]. While the succession is a well-known process in classical ecology, in the case of biofilms or cell aggregates it is not fully understood. Despite many researches, the full knowledge on the formation of microbial consortia is still lacking. However, succession processes seem to be rather stochastic (reproduction and death) [26]. During growth of consortia, competition for resources makes that weak individuals are eliminated, and stronger competitors become dominant. Finally, in the mature consortia, cells are becoming

Environmental factors may also shape the succession in microbial consortia. Changes in pH, the presence of carbon sources in the form of saccharides, and other additional substances *Asaia* spp. show strong ability to aggregate and form characteristic "flocs" and to create biofilms on selected surfaces commonly used in the food industry: glass, polyethylene terephthalate, and polypropylene [32] (**Figure 3**).

It was found that the hydrophobicity of the cells decreased with increasing the age of the population. The higher hydrophobicity of young cells stimulates the process of aggregation and formation of flocs. The studies proved that the adhesive abilities of *As. lannensis* depend on the carbon source, nutrient availability, and physicochemical properties of abiotic surface. The strongest adhesion properties were characterized by cells in the minimal medium with sucrose.

**3. New antiadhesion strategy: organosilanes**

and inorganic materials (**Figure 4**).

**Figure 4.** Model structure of organosilanes.

of abiotic surfaces or bioactive properties of consumption waters.

It is known that it is best to prevent than to fight against biofilm formed on the internal surface of a distribution system. For drinking waters and soft drinks, reduction or elimination of the formation of cell consortia can be obtained only by changing the physicochemical properties

Biofilms in Beverage Industry http://dx.doi.org/10.5772/62940 179

Compounds of the biocidal and/or antiadhesive properties applied in potable water systems have to inhibit effectively the growth of microorganisms without releasing toxic compounds with low molecular weight into aquatic environment. Such compounds may be organosi‐ lanes containing at least one bond between the carbon and silicon atom Si–CH3. A carbon– silicon bond is very durable, and the presence of an alkyl group causes a change in surface tension. Additionally, organosilanes can contain other functional groups with antimicrobial

Organofunctional silanes are hybrid compounds that combine the functionality of a reactive organic group and the inorganic functionality of an alkyl silicate in a single molecule. This special property means they can be used as 'molecular bridges' between organic substrates

These compounds are relatively environmentally friendly, improve adhesion, and provide better protection against corrosion. Surfaces on which they can be used include metal, plastic, glass, rubber, ceramic, porcelain, marble, cement, granite, tile, silica, sand, appliances that have been enameled, polyester, polyurethane, polyacrylic, resins that are melamine or phenolic,

The growth of many microorganisms can be reduced on surfaces treated with alkylsilanes. In general, the reactivity of hydroxylated surfaces with organofunctional silanes decreases in the following order: Si–NR2 > Si–Cl > Si–NH–Si > Si–O2CCH3 > Si–OCH3 > Si–OCH2CH3. The methoxy and ethoxysilanes are the most widely used organofunctional silanes for surface modification. The methoxysilanes are capable of reacting with substrates under dry, protic

siliceous, polycarbonate and wood, as well as painted surfaces.

properties, for example, methoxy, ethoxy, amino, methacrylic, and sulfide [35].

Definitely, the level of cell adhesion decreased in media that is rich in nutrients. Biofilm creation in a specific medium which was the commercial mineral flavored water had a dynamic character [32].

It is difficult to determine the origin of the contamination of soft drinks with the *Asaia* spp. However, the fruits and fruit concentrates are regarded as the source of the contamination [28, 33]. Most strains were isolated from the reclaimed fruit beverages and flavored mineral waters. This spoilage often occurs in the acid products preserved by the benzoate, sorbate, and dimethyldicarbonate. Horsakova et al. [34] found that these bacteria occur in the processing equipment in the form of biofilm, which is persistent and hardly removable by the common sanitation. The isolated bacteria *Asaia* spp. exhibit the polysaccharide encapsulation. The presence of preservatives is almost no effect on the *Asaia* spp. growth. The minimum inhibi‐ tion concentration for sorbic and benzoic acid under the conditions of the model fruit drink (pH 3.45; Rf 10 Brix) were between 250 and 500 mg/l, while the concentration 250 mg/l is used for the stabilization of similar fruit beverage production.

The resistance of *Asaia* spp. to common preservatives limits the available possibilities to prevent spoilage of similar drinks. Additionally, the contamination of the technological equipment always brings the serious problem. The common sanitation procedures used in the beverage production may be insufficient to eliminate the very rigid biofilm, which is formed by *Asaia* spp. in the equipment. According to Horsakova et al. [34], the reliable elimination of such biofilm may require more forcing condition (e.g., hot sodium hydroxide and detergent and enzyme solutions) and in any hardly accessible points (pipe bends, branches, connec‐ tions, and valves) mechanical treatment is the only possibility.

### **3. New antiadhesion strategy: organosilanes**

It is known that it is best to prevent than to fight against biofilm formed on the internal surface of a distribution system. For drinking waters and soft drinks, reduction or elimination of the formation of cell consortia can be obtained only by changing the physicochemical properties of abiotic surfaces or bioactive properties of consumption waters. It is known that it is best to prevent than to fight against biofilm formed on the internal

Compounds of the biocidal and/or antiadhesive properties applied in potable water systems have to inhibit effectively the growth of microorganisms without releasing toxic compounds with low molecular weight into aquatic environment. Such compounds may be organosi‐ lanes containing at least one bond between the carbon and silicon atom Si–CH3. A carbon– silicon bond is very durable, and the presence of an alkyl group causes a change in surface tension. Additionally, organosilanes can contain other functional groups with antimicrobial properties, for example, methoxy, ethoxy, amino, methacrylic, and sulfide [35]. with low molecular weight into aquatic Such compounds may be lanes containing at least one bond between the carbon and silicon atom A silicon bond is very and the presence of an alkyl group causes a change in organosilanes can contain other functional groups with

Organofunctional silanes are hybrid compounds that combine the functionality of a reactive organic group and the inorganic functionality of an alkyl silicate in a single molecule. This special property means they can be used as 'molecular bridges' between organic substrates and inorganic materials (**Figure 4**). organic group and the inorganic functionality of an alkyl silicate in a single special property means they can be used 'molecular between organic

**Figure 4.** Model structure of organosilanes.

**Figure 3.** "Flocs" formed by cells of *Asaia* spp.

178 Microbial Biofilms - Importance and Applications

for the stabilization of similar fruit beverage production.

tions, and valves) mechanical treatment is the only possibility.

character [32].

Definitely, the level of cell adhesion decreased in media that is rich in nutrients. Biofilm creation in a specific medium which was the commercial mineral flavored water had a dynamic

It is difficult to determine the origin of the contamination of soft drinks with the *Asaia* spp. However, the fruits and fruit concentrates are regarded as the source of the contamination [28, 33]. Most strains were isolated from the reclaimed fruit beverages and flavored mineral waters. This spoilage often occurs in the acid products preserved by the benzoate, sorbate, and dimethyldicarbonate. Horsakova et al. [34] found that these bacteria occur in the processing equipment in the form of biofilm, which is persistent and hardly removable by the common sanitation. The isolated bacteria *Asaia* spp. exhibit the polysaccharide encapsulation. The presence of preservatives is almost no effect on the *Asaia* spp. growth. The minimum inhibi‐ tion concentration for sorbic and benzoic acid under the conditions of the model fruit drink (pH 3.45; Rf 10 Brix) were between 250 and 500 mg/l, while the concentration 250 mg/l is used

The resistance of *Asaia* spp. to common preservatives limits the available possibilities to prevent spoilage of similar drinks. Additionally, the contamination of the technological equipment always brings the serious problem. The common sanitation procedures used in the beverage production may be insufficient to eliminate the very rigid biofilm, which is formed by *Asaia* spp. in the equipment. According to Horsakova et al. [34], the reliable elimination of such biofilm may require more forcing condition (e.g., hot sodium hydroxide and detergent and enzyme solutions) and in any hardly accessible points (pipe bends, branches, connec‐ These compounds are relatively environmentally friendly, improve adhesion, and provide better protection against corrosion. Surfaces on which they can be used include metal, plastic, glass, rubber, ceramic, porcelain, marble, cement, granite, tile, silica, sand, appliances that have been enameled, polyester, polyurethane, polyacrylic, resins that are melamine or phenolic, siliceous, polycarbonate and wood, as well as painted surfaces. These compounds are relatively environmentally improve and appliances that been resins that are melamine or

The growth of many microorganisms can be reduced on surfaces treated with alkylsilanes. In general, the reactivity of hydroxylated surfaces with organofunctional silanes decreases in the following order: Si–NR2 > Si–Cl > Si–NH–Si > Si–O2CCH3 > Si–OCH3 > Si–OCH2CH3. The methoxy and ethoxysilanes are the most widely used organofunctional silanes for surface modification. The methoxysilanes are capable of reacting with substrates under dry, protic the reactivity of hydroxylated surfaces with organofunctional silanes decreases in following methoxy and ethoxysilanes are the most widely used organofunctional silanes for

conditions, while the less reactive ethoxysilanes require catalysis. The low toxicity of ethanol, a byproduct of the reaction, favors the use of ethoxysilanes in many commercial applications [35].

ries: fructose, which inhibits the adherence of type 1 fimbriae, and proanthocyanidins, which inhibits the adherence of p-fimbriae. The binding of the proteinaceous bacterial fimbrial tips to mucosal surfaces on the uroepithelium occurs as a specific receptor‐ligand association favored by hydrophobic interactions. This possible mechanism is that the cranberry com‐ pounds, acting as receptor analogs, competitively inhibit the adhesion of *E. coli* to host cells by binding to the fimbrial tips. Another mechanism of cranberry activity is the in vitro reduction in the expression of p-fimbriae in *E. coli* by changing the conformation of surface molecules [38]. Zafriri et al. were the first to postulate that compounds in cranberry could affect p and type 1 fimbriae of *E. coli* [39]. In 1998, Howell et al. [40] identified specific proanthocya‐

Biofilms in Beverage Industry http://dx.doi.org/10.5772/62940 181

Proanthocyanidins are one of many plant phenols, which are aromatic secondary metabo‐ lites found in the plant kingdom. They are mainly found in *Vaccinium* berries such as cran‐ berries and blueberries. They are dimers or oligomers of catechin and epicatechin and their gallic acid esters. Proanthocyanidins are in the first place very strong antioxidants. Studies have shown that proanthocyanidins act as anticancer and antiallergic agents, and that they improve heart health. These flavonoids have several potential clinical effects, including antiatherosclerotic, anti-inflammatory, antitumor, antithrombogenic, antiosteoporotic, and antiviral. Some of these effects, such as antitumor, are still up for discussion, and the role of

They are also known as olgoflavanoids, and consist of monomerflavan-3-ol units. When linked through either C4 to C8 or C4 to C6 bonds, the linkages are called B-linked. When the linkages were through a C2 and C7 compound, they are called A type [41]. While B-linked proantho‐ cyanidins can be found in different fruit products including apple juice, purple grape juice, green tea, and dark chocolate, A-linked ones are found in cranberries and it is a linkage with

The antiadhesive properties of cranberry were demonstrated against different microorgan‐ isms: *E. coli*, *Proteus mirabilis*, or *Helicobacter pylori*, responsible for urinary tract infections and

nidin compounds in cranberry responsible to antiadhesive properties.

unique antiadhesion properties associated with them [42] (**Figure 5**).

flavonoids in different effects is not fully known.

**Figure 5.** Proanthocyanidins: type A (left) and type B (right).

One of the most established and successful uses of the application of organosilanes is preven‐ tion against biofilm formation. The use of the proper quaternary amine-based organosilane can provide durable antimicrobial protection against a wide variety of microorganisms [36].

Adhesion abilities of *A. hydrophila* to the glass surface modified by coating with four differ‐ ent organosilanes with active functional groups were described by Kregiel [23]. The pres‐ ence of active functional groups had an impact on a significantreduction in the surface tension of the test surfaces due to reduced participation polarforces – one of the components of surface forces. Among the modifiers, organosilanes containing methoxy groups and quaternary ammonium salts showed the best antiadhesive and antibacterial properties. Organosilanes were stable in an aqueous environment. Interesting results from the modification of the surface of the glass gave impulsion to extend the study on modification of plastic materials common‐ ly used as pipe materials in water systems [24, 25]. The modified PVC surfaces were made by silane coupling on the native material. Modifications of silicone elastomer were carried by cocrosslinking organosilane with silicone. Almost all of the modified surfaces were character‐ ized by antimicrobial and antiadhesive features. Among the modifications, especially polydimethylsiloxane with a quaternary ammonium salt and a methoxy group in the silicone elastomer showed the greatest antiadhesive and antibacterial properties against *A. hydrophila*.

### **4. New antiadhesion strategy: proanthocyanidins**

Scientific studies showed that natural compounds from different fruits have potential health benefits against cancer, aging and neurological diseases, inflammation, diabetes, and bacteri‐ al infections. For example, cranberry juice was recognized for benefits of maintenance of a healthy urinary tract. Cranberry is a term derived from the contraction of "crane berry." This name is derived from the nickname of the bilberry flower, and the sand crane, a bird that often feeds on the berries of this plant. The cranberry is part of the *Ericaceae* family and naturally grows in acidic swamps full of peat moss in humid forests [37].

Bacterial adhesion is accomplished by the binding of lectins exposed on the cell surfaces of pili and fimbriae to complementary carbohydrates on the host tissues. Pili are small filaments that can be either mannose-resistant or mannose-sensitive. The mannose-sensitive pili, called type 1 pili, permit bacterial adhesion to the urothelium. The fimbriae (p-fimbriae) are inhibited by fructose, present in cranberries. The more virulent strains of *E. coli*, isolated from patients with urinary tract infections, have other types of these structures that bind to glycosphingolipids of the lipid double membrane of renal cells, which precedes renal parenchymal invasion.

The current hypothesis is that cranberries work principally by preventing the adhesion of type 1 and p-fimbriae *E. coli* strains to the urothelium. Without adhesion, the bacteria cannot infect the mucosal surface. In vitro, this adhesion is mediated by two components of cranber‐ ries: fructose, which inhibits the adherence of type 1 fimbriae, and proanthocyanidins, which inhibits the adherence of p-fimbriae. The binding of the proteinaceous bacterial fimbrial tips to mucosal surfaces on the uroepithelium occurs as a specific receptor‐ligand association favored by hydrophobic interactions. This possible mechanism is that the cranberry com‐ pounds, acting as receptor analogs, competitively inhibit the adhesion of *E. coli* to host cells by binding to the fimbrial tips. Another mechanism of cranberry activity is the in vitro reduction in the expression of p-fimbriae in *E. coli* by changing the conformation of surface molecules [38]. Zafriri et al. were the first to postulate that compounds in cranberry could affect p and type 1 fimbriae of *E. coli* [39]. In 1998, Howell et al. [40] identified specific proanthocya‐ nidin compounds in cranberry responsible to antiadhesive properties.

Proanthocyanidins are one of many plant phenols, which are aromatic secondary metabo‐ lites found in the plant kingdom. They are mainly found in *Vaccinium* berries such as cran‐ berries and blueberries. They are dimers or oligomers of catechin and epicatechin and their gallic acid esters. Proanthocyanidins are in the first place very strong antioxidants. Studies have shown that proanthocyanidins act as anticancer and antiallergic agents, and that they improve heart health. These flavonoids have several potential clinical effects, including antiatherosclerotic, anti-inflammatory, antitumor, antithrombogenic, antiosteoporotic, and antiviral. Some of these effects, such as antitumor, are still up for discussion, and the role of flavonoids in different effects is not fully known. to mucosal surfaces on the uroepithelium occurs as a specific favored by hydrophobic This possible mechanism is that the cranberry acting as receptor competitively inhibit the adhesion of to host by binding to the fimbrial Another mechanism of cranberry activity is the in reduction in the expression of p-fimbriae in by changing the conformation of Zafriri et were the first to postulate that compounds in cranberry could Proanthocyanidins are one of many plant which are aromatic secondary lites found in the plant They are mainly found in berries such as berries and They are dimers or oligomers of catechin and epicatechin and gallic acid Proanthocyanidins are in the first place very strong have shown that proanthocyanidins act as anticancer and antiallergic and that improve heart These flavonoids have several potential clinical antithrombogenic, Some of these such as are still up for and the role 

They are also known as olgoflavanoids, and consist of monomerflavan-3-ol units. When linked through either C4 to C8 or C4 to C6 bonds, the linkages are called B-linked. When the linkages were through a C2 and C7 compound, they are called A type [41]. While B-linked proantho‐ cyanidins can be found in different fruit products including apple juice, purple grape juice, green tea, and dark chocolate, A-linked ones are found in cranberries and it is a linkage with unique antiadhesion properties associated with them [42] (**Figure 5**). They are also known as and consist of monomer flavan-3-ol When through either to or to the linkages are called When the

**Figure 5.** Proanthocyanidins: type A (left) and type B (right).

conditions, while the less reactive ethoxysilanes require catalysis. The low toxicity of ethanol, a byproduct of the reaction, favors the use of ethoxysilanes in many commercial applications

One of the most established and successful uses of the application of organosilanes is preven‐ tion against biofilm formation. The use of the proper quaternary amine-based organosilane can provide durable antimicrobial protection against a wide variety of microorganisms [36].

Adhesion abilities of *A. hydrophila* to the glass surface modified by coating with four differ‐ ent organosilanes with active functional groups were described by Kregiel [23]. The pres‐ ence of active functional groups had an impact on a significantreduction in the surface tension of the test surfaces due to reduced participation polarforces – one of the components of surface forces. Among the modifiers, organosilanes containing methoxy groups and quaternary ammonium salts showed the best antiadhesive and antibacterial properties. Organosilanes were stable in an aqueous environment. Interesting results from the modification of the surface of the glass gave impulsion to extend the study on modification of plastic materials common‐ ly used as pipe materials in water systems [24, 25]. The modified PVC surfaces were made by silane coupling on the native material. Modifications of silicone elastomer were carried by cocrosslinking organosilane with silicone. Almost all of the modified surfaces were character‐ ized by antimicrobial and antiadhesive features. Among the modifications, especially polydimethylsiloxane with a quaternary ammonium salt and a methoxy group in the silicone elastomer showed the greatest antiadhesive and antibacterial properties against *A. hydrophila*.

Scientific studies showed that natural compounds from different fruits have potential health benefits against cancer, aging and neurological diseases, inflammation, diabetes, and bacteri‐ al infections. For example, cranberry juice was recognized for benefits of maintenance of a healthy urinary tract. Cranberry is a term derived from the contraction of "crane berry." This name is derived from the nickname of the bilberry flower, and the sand crane, a bird that often feeds on the berries of this plant. The cranberry is part of the *Ericaceae* family and naturally

Bacterial adhesion is accomplished by the binding of lectins exposed on the cell surfaces of pili and fimbriae to complementary carbohydrates on the host tissues. Pili are small filaments that can be either mannose-resistant or mannose-sensitive. The mannose-sensitive pili, called type 1 pili, permit bacterial adhesion to the urothelium. The fimbriae (p-fimbriae) are inhibited by fructose, present in cranberries. The more virulent strains of *E. coli*, isolated from patients with urinary tract infections, have other types of these structures that bind to glycosphingolipids of the lipid double membrane of renal cells, which precedes renal parenchymal invasion.

The current hypothesis is that cranberries work principally by preventing the adhesion of type 1 and p-fimbriae *E. coli* strains to the urothelium. Without adhesion, the bacteria cannot infect the mucosal surface. In vitro, this adhesion is mediated by two components of cranber‐

**4. New antiadhesion strategy: proanthocyanidins**

grows in acidic swamps full of peat moss in humid forests [37].

[35].

180 Microbial Biofilms - Importance and Applications

The antiadhesive properties of cranberry were demonstrated against different microorgan‐ isms: *E. coli*, *Proteus mirabilis*, or *Helicobacter pylori*, responsible for urinary tract infections and gastritis, as well as other pathogenic Gram-negative and Gram-positive bacteria: *P*. *aerugino‐ sa*, *Staphylococcus aureus*, or *Listeria monocytogenes* [43–45].

[2] Emtiazi F, Schwartz T, Marten SM, Krolla-Sidenstein P, Obst U. Investigation of natural biofilms formed during the production of drinking water from surface water embank‐ ment filtration. Water Research 2004;38:1197–1206. DOI: 10.1016/j.watres.2003.10.056

Biofilms in Beverage Industry http://dx.doi.org/10.5772/62940 183

[3] Wu HT, Mi ZL, Zhang JX, Chen C, Xie SG. Bacterial communities associated with an occurrence of colored water in an urban drinking water distribution system. Biomed‐

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[12] Rickard AH, Leach SA, Hall LS, Buswell CM, High NJ, Handley PS. Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria. Applied and Environmental Microbiology 2002;68:73644–73650. DOI: 10.1128/AEM.

[13] Mulamattathil SG, Bezuidenhout C, Mbewe M. Biofilm formation in surface and drinking water distribution systems in Mafikeng, South Africa. South African Journal

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It was also noted that the adhesion of *Asaia* spp. cells in the presence of cranberry juice was much lower, especially for the packaging material – polystyrene [37]. In the presence of 10% cranberry juice, attachment of bacterial cells was three times lower. The obtained results suggested that compounds of cranberry inhibit both biofilm formation and coaggregation of microbial cells. This fact would help to utilize antioxidant-rich cranberry juice as a natural antiadhesive protectant and microbiological stability enhancing agent for functional soft drinks.

### **5. Conclusion**

Problems related to microbial contamination in the beverage industry have been studied for more than a century. However, most of the knowledge acquired over the years relates to singlecells, but today it is generally accepted that microorganisms grow and survive in organized communities where their physiology is very different. This paper has given an overview of the most widely used research on the controlled attachment of specific bacteria present in drinking water or soft drinks. Both surfaces modified by organosilanes and cranberry juice supplementation are the latest developments in this area. Particularly, cranberry juice and cranberry extracts may be investigated as a natural solution for food industry by creating an additional barrier to inhibit the growth of spoilage bacteria and providing additional health benefits.

### **Author details**

Dorota Kregiel\* and Hubert Antolak

\*Address all correspondence to: dorota.kregiel@p.lodz.pl

Institute of Fermentation Technology and Biotechnology, Lodz University of Technology, Lodz, Poland

### **References**

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[2] Emtiazi F, Schwartz T, Marten SM, Krolla-Sidenstein P, Obst U. Investigation of natural biofilms formed during the production of drinking water from surface water embank‐ ment filtration. Water Research 2004;38:1197–1206. DOI: 10.1016/j.watres.2003.10.056

gastritis, as well as other pathogenic Gram-negative and Gram-positive bacteria: *P*. *aerugino‐*

It was also noted that the adhesion of *Asaia* spp. cells in the presence of cranberry juice was much lower, especially for the packaging material – polystyrene [37]. In the presence of 10% cranberry juice, attachment of bacterial cells was three times lower. The obtained results suggested that compounds of cranberry inhibit both biofilm formation and coaggregation of microbial cells. This fact would help to utilize antioxidant-rich cranberry juice as a natural antiadhesive protectant and microbiological stability enhancing agent for functional soft

Problems related to microbial contamination in the beverage industry have been studied for more than a century. However, most of the knowledge acquired over the years relates to singlecells, but today it is generally accepted that microorganisms grow and survive in organized communities where their physiology is very different. This paper has given an overview of the most widely used research on the controlled attachment of specific bacteria present in drinking water or soft drinks. Both surfaces modified by organosilanes and cranberry juice supplementation are the latest developments in this area. Particularly, cranberry juice and cranberry extracts may be investigated as a natural solution for food industry by creating an additional barrier to inhibit the growth of spoilage bacteria and providing additional health

Institute of Fermentation Technology and Biotechnology, Lodz University of Technology,

[1] Lee K-B, Liu C-T, Anzai Y, Kim H, Aono T, Oyaizu H. The hierarchical system of the '*Alphaproteobacteria*': description of *Hyphomonadaceae* fam. nov., *Xanthobacteraceae* fam. nov. and *Erythrobacteraceae* fam. nov. International Journal of Systematic and

Evolutionary Microbiology. 2005;55:1907–1919. DOI: 10.1099/ijs.0.63663-0

*sa*, *Staphylococcus aureus*, or *Listeria monocytogenes* [43–45].

182 Microbial Biofilms - Importance and Applications

drinks.

benefits.

**Author details**

Dorota Kregiel\*

Lodz, Poland

**References**

and Hubert Antolak

\*Address all correspondence to: dorota.kregiel@p.lodz.pl

**5. Conclusion**

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	- [12] Rickard AH, Leach SA, Hall LS, Buswell CM, High NJ, Handley PS. Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria. Applied and Environmental Microbiology 2002;68:73644–73650. DOI: 10.1128/AEM. 68.7.3644-3650.2002
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[14] Silhan J, Corfitzen CB, Albrechtsen HJ. Effect of temperature and pipe material on biofilm formation and survival of *Escherichia coli* in used drinking water pipes: a laboratory-based study. Water Science and Technology 2006;54:48–56. DOI: 10.2166/ wst.2006.447

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Biofilms in Beverage Industry http://dx.doi.org/10.5772/62940 185

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**Section 3**

**Biofilm in Health and Diseases**


**Biofilm in Health and Diseases**

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**Chapter 10**

**Staphylococcal Biofilms: Pathogenicity, Mechanism and**

**Regulation of Biofilm Formation by Quorum-Sensing**

Staphylococcal infections are reported to cause very important problems in hospital‐ ized and immunocompressed patients worldwide due to their tough and irresponsive treatment by antibiotics. Biofilm-embedded bacteria that gain resistance to immune defense and antibiotics by antibiotic degrading enzymes, efflux pumps, and certain gene products of which expression are changed by the quorum sensing cause chronic and recurrent infections such as indwelling device–associated infections. Biofilm-embed‐ ded sessile community has heterogeneous cells that have wide range of different responds to each antimicrobials. *Staphylococcus epidermidis* (*S. epidermidis*) and *Staphylococcus aureus* (*S. aureus*) that are mostly known pathogenic strains can induce gene expression of biofilm that has an important role in the pathogenesis of staphylo‐ coccal infections and causes bacterial attachment and colonization on biotic such as tissues or abiotic surfaces such as prosthetic surfaces that may act as a substrate for microbial adhesion when microorganisms exposed to stress conditions. This ex‐ pressed and matured biofilm causes bacterial spread to whole body, consequently, spread of infection in to whole body. It is hard to treat biofilm infections, and new agents are being researched to prevent formation and dissemination of biofilm. Defining the virulence and the role of biofilm of *S. epidermidis* and *S. aureus* in chronic and recur‐ rent infections such as indwelling device–associated infections, the mechanism and the global regulation of biofilm production by quorum-sensing system, inactivation of biofilm formation, and the resistance patterns of biofilm-embedded microorganism

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.

**System and Antibiotic Resistance Mechanisms of**

**Biofilm-Embedded Microorganisms**

Additional information is available at the end of the chapter

against antimicrobials are important.

Sahra Kırmusaoğlu

**Abstract**

http://dx.doi.org/10.5772/62943

**Chapter 10**
