**Removal of Heavy Metals from Aqueous Solutions by Aerobic and Anaerobic Biomass**

Onofre Monge-Amaya, María Teresa Certucha-Barragán, Francisco Javier Almendariz-Tapia and Gonzalo Mauricio Figueroa-Torres

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61330

## **1. Introduction**

Industrialization and urbanization have promoted the generation of great quantities of aqueous effluents that may contain high levels of toxic compounds [1]. Every day, 2,000,000 tons of wastes (from sewers or agricultural and industrial residues) are released into rivers and seas, spreading disease and damage to ecosystems. Achim Steiner, executive chief of the United Nations Program for the Environment stated: "If the world is to thrive, let alone to survive on a planet of 6 billion people heading to over 9 billion by 2050, we need to be collectively smarter about how we manage waste, including wastewaters" [2].

Heavy metals, or potentially toxic elements, constitute a specific group of pollutants that are released into the environment as a result of industrial activities, such as the mining industry. These elements can cause health problems. In México, the mining industry is one of the most important economic activities, with gold, silver, and copper being the precious metals with higher production rates [3].

The metallurgical process of the mining industry involves a series of extraction and purification techniques that result in the disposal of metals into water bodies through acid mine drainage (DAM). Heavy metals can then accumulate at toxic concentrations for a functional ecosystem, which constitutes an economic problem of public health [4].

Controlling and reducing water pollution is a significant concern for our society. Wastewater spills create eutrophication and toxic problems. The wastewater penetrates the soil, contami‐ nates groundwater, and reduces the quality necessary for human consumption [5].

Discharge limits have been established for heavy metals, among many other water pollutants. Most heavy metals are soluble and form aqueous solutions; hence, they cannot be separated by ordinary physical treatments [6].

© 2015 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.

Contamination of soil and water is the result of numerous industrial activities such as mining, melting, fabrication of jewelry, batteries, and automobiles, and volatile ashes from incineration processes. This type of contamination poses a serious threat for human and animal health since heavy metals remain in the environment for an indefinite time [7].

México has several sites contaminated by heavy metals and other residues from the mining industry. A particular example of pollution is found in the San Pedro River, located in the state of Sonora, México, where silver and copper production has been exploited for decades. The San Pedro River stream originates near Cananea – a mining town known for having the biggest mining districts of the state – and culminates in the state of Arizona, in the United States. Surface water pollution in the San Pedro river was reported in 1997 and 1999 [8]. In 2008, the presence of heavy metals in the river sediments was also evaluated [9]. The river has been contaminated by heavy metals due to its proximity with the metallurgical activity of the state. Metals found in the river are: cadmium, cobalt, chromium, iron, manganese, copper, zinc, nickel, and lead. However, two of the metals with higher concentrations were copper and iron, which exceeded the maximum permissible values established in the Mexican laws for water quality. These laws consider lead, zinc, mercury, silver, nickel, cadmium, aluminum, copper, and arsenic, as water pollutants due to the toxicity they pose for aquatic and terrestrial organisms (NOM-001-ECOL-1996; NOM-002-ECOL-1996; NOM-003-ECOL-1996).

More recently, on August 7, 2014, the Buenavista Copper Mine in Cananea was under the spotlight when approximately 40,000 cubic meters of sulfuric acid were spilled into the Bacanuchi River (also situated in Sonora). This toxic leakage affected an estimate of about 800,000 people [10]. Heavy metals pollution has been reported, but the remediation projects aiming to recover the quality of these sites have been extremely scarce. Thus, it is of great importance for research institutions and industries to evaluate technological alternatives for the removal and stabilization of inorganic contaminants, keeping into consideration the specific environmental conditions of each polluted site [11].

## **2. Heavy metal removal processes**

The removal of heavy metals can be carried out by a number of conventional treatments, such as reverse osmosis, electrodialysis, ultrafiltration, chemical precipitation, and ionic exchange. These methods, however, have the disadvantage of requiring high operation costs. The ionic exchange resins, for example, have been commercially known for their effectiveness as pollutant adsorbents in wastewater treatments, but their high cost hinders their application at industrial levels [1]. Chemical processes, although simple to perform, end up being even more expensive because of the active agent that cannot be recovered for future uses. Besides, the final product is a high concentrated sludge difficult to handle [4].

Heavy metals sources are not renewable, and the natural reserves are being consumed. Therefore, it is imperative that those elements considered dangerous to the environment or those of technological importance and economic value are withdrawn and recovered at their point of origin through appropriate treatments.

A brief description of the before mentioned processes is presented next:

Contamination of soil and water is the result of numerous industrial activities such as mining, melting, fabrication of jewelry, batteries, and automobiles, and volatile ashes from incineration processes. This type of contamination poses a serious threat for human and animal health since

México has several sites contaminated by heavy metals and other residues from the mining industry. A particular example of pollution is found in the San Pedro River, located in the state of Sonora, México, where silver and copper production has been exploited for decades. The San Pedro River stream originates near Cananea – a mining town known for having the biggest mining districts of the state – and culminates in the state of Arizona, in the United States. Surface water pollution in the San Pedro river was reported in 1997 and 1999 [8]. In 2008, the presence of heavy metals in the river sediments was also evaluated [9]. The river has been contaminated by heavy metals due to its proximity with the metallurgical activity of the state. Metals found in the river are: cadmium, cobalt, chromium, iron, manganese, copper, zinc, nickel, and lead. However, two of the metals with higher concentrations were copper and iron, which exceeded the maximum permissible values established in the Mexican laws for water quality. These laws consider lead, zinc, mercury, silver, nickel, cadmium, aluminum, copper, and arsenic, as water pollutants due to the toxicity they pose for aquatic and terrestrial

organisms (NOM-001-ECOL-1996; NOM-002-ECOL-1996; NOM-003-ECOL-1996).

specific environmental conditions of each polluted site [11].

final product is a high concentrated sludge difficult to handle [4].

**2. Heavy metal removal processes**

point of origin through appropriate treatments.

More recently, on August 7, 2014, the Buenavista Copper Mine in Cananea was under the spotlight when approximately 40,000 cubic meters of sulfuric acid were spilled into the Bacanuchi River (also situated in Sonora). This toxic leakage affected an estimate of about 800,000 people [10]. Heavy metals pollution has been reported, but the remediation projects aiming to recover the quality of these sites have been extremely scarce. Thus, it is of great importance for research institutions and industries to evaluate technological alternatives for the removal and stabilization of inorganic contaminants, keeping into consideration the

The removal of heavy metals can be carried out by a number of conventional treatments, such as reverse osmosis, electrodialysis, ultrafiltration, chemical precipitation, and ionic exchange. These methods, however, have the disadvantage of requiring high operation costs. The ionic exchange resins, for example, have been commercially known for their effectiveness as pollutant adsorbents in wastewater treatments, but their high cost hinders their application at industrial levels [1]. Chemical processes, although simple to perform, end up being even more expensive because of the active agent that cannot be recovered for future uses. Besides, the

Heavy metals sources are not renewable, and the natural reserves are being consumed. Therefore, it is imperative that those elements considered dangerous to the environment or those of technological importance and economic value are withdrawn and recovered at their

heavy metals remain in the environment for an indefinite time [7].

32 Biomass Production and Uses

*Reverse osmosis*: a process where heavy metals are separated through a semipermeable membrane by using a pressure higher than the osmotic pressure, which is caused by the dissolved solids in wastewaters. The high pressures required for this process are the main reason for the high operating costs of reverse osmosis.

*Electrodialysis*: in this process, metallic ions are separated by selective semipermeable mem‐ branes. An electric current is applied between two electrodes located at each side of the membranes, which produces a migration of cations and anions toward their respective electrodes. The migration of ions results in the formation of metal salts that precipitate out of solution. However, a major disadvantage of electrodialysis is membrane clogging, caused mainly by the formation of metal hydroxides.

*Ultrafiltration*: this process involves the use of porous membranes and high pressures for the separation of metal ions. Sludge generation is the main disadvantage in this treatment.

*Ionic exchange*: metallic ions in diluted solutions are exchanged with the ions located in the active sites of synthetic resins by electrostatic forces. Sludge generation and the high costs of exchange resins are the main disadvantages.

*Chemical precipitation*: precipitation of metallic ions is achieved by the addition of coagulants such as calcium salts, iron, and other organic polymers. The inconvenience of this method is the excessive amounts of sludge (it might include toxic compounds) produced during the precipitation.

*Phytoremediation:* it involves the use of certain plants as removing or stabilizing agents in contaminated soils, sediments, and water. The time required for effective stabilization of heavy metals is large and can be a constraint in this process; furthermore, plant regeneration is even more complex.

All of the disadvantages previously mentioned, such as incomplete removal, high energy consumption, excessive residual sludge, and formation of other toxic residues requiring careful disposal protocols justify the need for a cost-effective treatment for the removal of heavy metals from wastewater [12].

New technologies are currently being developed, taking into consideration the processing costs and direct scaling up and implementation [13]. The search for effective removal technol‐ ogies has directed attention toward biosorption, an ecological alternative that uses different biological materials for binding and concentrating metal ions.

*Biosorption:* This process is based on the capacity of biological materials to concentrate heavy metals by either metabolic or physical–chemical pathways.

Developments in the field of environmental biotechnology have allowed the identification of several species of algae, bacteria, fungi, and yeast as effective metal biosorbents [14]. The main advantages of biosorption over conventional treatments include: lower costs, high removal yields, minimum residual sludge formation, and potential biosorbent regeneration and metal recovery [15].

The biosorption process involves a solid phase – the biosorbent, or biomass – and a liquid phase – the solvent (commonly water). The liquid phase contains the sorbate, i.e., the species to be sorbed (metallic ions). During biosorption, the sorbate is attracted and bound to the biosorbent through a variety of mechanisms. This "binding" process continues until a state of equilibrium is achieved between the amount of sorbate present and the available active sites of the biosorbent [16].

The two mechanisms by which biosorption can take place are [13]:


**Figure 1.** General experimental setup for biosorption of heavy metals.

The biosorption process can be carried out in a bioreactor, where the wastewater flows through a bed of microorganisms which bind the heavy metals. Bioreactors are useful tools where high volumes of wastewaters may be treated continuously, transferring the contaminated "portion" to a considerable smaller volume. However, certain problems can arise during the operation of bioreactors, such as biomass washout, liquid–solid separation difficulties, and pressure drops. These problems originate due to the fact that microbial biomass generally consists of small particles with low density and poor mechanical strength [17]. Immobilization of biomass in a suitable matrix (or material supports) can overcome washout problems by inducing cellular growth in the form of a stable biofilm constituted by microbial cells and extracellular polymeric substances.

## **3. Heavy metals removal by aerobic biomass**

Nowadays, the use of microorganisms for environmental remediation and recovery purposes has grown as a research field. It is believed that the most fitted microorganisms for removal treatments are the ones isolated from the same environment where they were naturally selected; however, genetic manipulation techniques can be used to enhance the capacity of different microorganisms [18].

Bioremediation utilizes the catalytic abilities of living organisms to degrade and transform pollutants from aquatic and terrestrial ecosystems. This alternative can be potentially applied to mitigate environmental contamination. Bioremediation has focused on the exploitation of genetic diversity and metabolic versatility, characteristic traits that make bacteria suitable for the transformation of pollutants into harmless products, or less toxic compounds, that can be reintegrated in the natural biochemical cycles. On the other hand, there are other microorgan‐ isms such as fungi or plants that have been isolated and used in removal processes like phytoremediation [19].

Microorganisms are naturally exposed to heavy metals in essential or toxic quantities, and the amount of heavy metals in certain sites can be so high that microorganism growth is not possible. Metal toxicity forces microorganisms to develop various strategies to defend themselves against high concentrations of heavy metals [20].

There are several experimental protocols important to effectively examine metal biosorption by aerobic biomass. These protocols are described below.

## **3.1. Isolation / Inoculation**

The biosorption process involves a solid phase – the biosorbent, or biomass – and a liquid phase – the solvent (commonly water). The liquid phase contains the sorbate, i.e., the species to be sorbed (metallic ions). During biosorption, the sorbate is attracted and bound to the biosorbent through a variety of mechanisms. This "binding" process continues until a state of equilibrium is achieved between the amount of sorbate present and the available active sites

**•** Bioaccumulation: based in the intracellular transport of metallic ions by living biomass. **•** Bioadsorption: based on the adsorption of metallic ions on the cell surface. This process can occur by ionic exchange, precipitation, complexation, or electrostatic attraction. Figure 1 shows a basic experimental approach that can be used to determine the biosorption capacity,

The biosorption process can be carried out in a bioreactor, where the wastewater flows through a bed of microorganisms which bind the heavy metals. Bioreactors are useful tools where high volumes of wastewaters may be treated continuously, transferring the contaminated "portion" to a considerable smaller volume. However, certain problems can arise during the operation of bioreactors, such as biomass washout, liquid–solid separation difficulties, and pressure drops. These problems originate due to the fact that microbial biomass generally consists of small particles with low density and poor mechanical strength [17]. Immobilization of biomass in a suitable matrix (or material supports) can overcome washout problems by inducing cellular growth in the form of a stable biofilm constituted by microbial cells and extracellular

Nowadays, the use of microorganisms for environmental remediation and recovery purposes has grown as a research field. It is believed that the most fitted microorganisms for removal

The two mechanisms by which biosorption can take place are [13]:

*q*, a measure of the metal uptake by biomass.

**Figure 1.** General experimental setup for biosorption of heavy metals.

**3. Heavy metals removal by aerobic biomass**

of the biosorbent [16].

34 Biomass Production and Uses

polymeric substances.

Isolation is used to identify microorganisms able to grow in polluted environments. Waste‐ water samples are generally collected from damaged sites, and yeast or bacteria (biomass) cells are grown by inoculating them into a nutrient-rich environment. Inoculation is usually done in cell-culture dishes by the streaking method using selective enriched nutritive media for each microorganism. Commonly, 10 mL of wastewater sample is inoculated in a specific culture medium at 37°C for 24 h.

## **3.2. Batch biosorption and kinetics of heavy metals**

The biosorption batch tests with aerobic biomass are carried out in experimental vessels, such as Erlenmeyer flasks. Wastewater samples are mixed with a known amount of biomass. Flasks are placed in an incubator at specific conditions and tests are carried out in duplicate, using two flasks for every sampling time. For aerobic microorganisms such as yeast, the conditions are usually set as follows: pH 3–4, 37°C, and 100 rpm. Samples are taken at regular intervals until equilibrium is achieved. Every sample is then centrifuged to separate biomass from the solution. Concentration of metals is usually determined by atomic absorption spectrometry.

Biosorption efficiency (E) is calculated as follows:

$$E = \left(\frac{Co-\mathcal{C}f}{Co}\right) \times 100\tag{1}$$

where:

*Co, Cf* are the initial and final metal concentration (mg/L).

The biosorption capacity of the biomass at any given time is calculated as follows:

$$q\_{eq} = \frac{m\_o - m\_{eq}}{V\_{ads}}\tag{2}$$

where

*mo* is the initial mass (mg), equal to the initial concentration (mg/L) times initial volume;

*meq* is the mass at equilibrium (mg), equal to the concentration (mg/L) times volume at equilibrium;

*vads* is the volume of biomass used (L).

#### **3.3. Continuous biosorption studies**

Continuous studies are carried out in bioreactors. Bioreactors consist commonly of acrylic or glass columns with lateral sampling points. Perhaps, the simplest configuration is the Upflow Aerobic Reactor packed with material supports and biomass recirculation. An example of material support is clinoptilolite, a zeolite with a particle size of 4.76 mm, a pore diameter of 3.22E–03μ m and a Si/Al ratio of 4.53. Aerobic conditions are met by supplying air from the bottom of the column through peristaltic pumps.

#### **3.4. Biosorption tests in aerobic reactors**

Once reactors are inoculated with the selected aerobic biomass, mineral medium is used for biomass acclimation at pH levels optimum for growth. Mineral medium is only used during startup as a source of nutrients for biomass growth and immobilization. In the case of yeasts, pH is generally 3–4, and the medium consists of the following compounds: (g/L): ammonium phosphate 1, glucose 5, sodium chloride 5, magnesium sulfate 0.2, and phosphate potassium 1 [21].

Figure 2 shows a schematic diagram of two Upflow Aerobic Reactors connected in series that were used to remove heavy metals by Hernández-Mata et al., 2014 [22]. In this scheme, the first reactor (R1) was inoculated with biomass and the effluent was recirculated until the biomass reached a concentration of 1 g/L. When the desired biomass concentration was achieved, the biosorption stage was initiated with mining effluents. After the biosorption stage, a desorption (purification) step was carried out to remove the metallic ions adsorbed by the biomass. Biomass concentration was measured once again until the concentration reached 1 g/L. The effluent of R1 was then fed to R2 (containing the same biomass produced in R1) and biosorption was examined in both reactors. Samples were taken at regular intervals at the inlet and outlet points until column saturation was evident [22].

Removal of Heavy Metals from Aqueous Solutions by Aerobic and Anaerobic Biomass http://dx.doi.org/10.5772/61330 37

**Figure 2.** Schematic diagram of two upflow aerobic reactors packed with zeolite.

*Co, Cf*

36 Biomass Production and Uses

where

equilibrium;

*vads* is the volume of biomass used (L).

**3.3. Continuous biosorption studies**

bottom of the column through peristaltic pumps.

and outlet points until column saturation was evident [22].

**3.4. Biosorption tests in aerobic reactors**

phosphate potassium 1 [21].

are the initial and final metal concentration (mg/L).

The biosorption capacity of the biomass at any given time is calculated as follows:

*<sup>q</sup> <sup>V</sup>*

*eq*

*o eq*


*m m*

*mo* is the initial mass (mg), equal to the initial concentration (mg/L) times initial volume;

*meq* is the mass at equilibrium (mg), equal to the concentration (mg/L) times volume at

Continuous studies are carried out in bioreactors. Bioreactors consist commonly of acrylic or glass columns with lateral sampling points. Perhaps, the simplest configuration is the Upflow Aerobic Reactor packed with material supports and biomass recirculation. An example of material support is clinoptilolite, a zeolite with a particle size of 4.76 mm, a pore diameter of 3.22E–03μ m and a Si/Al ratio of 4.53. Aerobic conditions are met by supplying air from the

Once reactors are inoculated with the selected aerobic biomass, mineral medium is used for biomass acclimation at pH levels optimum for growth. Mineral medium is only used during startup as a source of nutrients for biomass growth and immobilization. In the case of yeasts, pH is generally 3–4, and the medium consists of the following compounds: (g/L): ammonium phosphate 1, glucose 5, sodium chloride 5, magnesium sulfate 0.2, and

Figure 2 shows a schematic diagram of two Upflow Aerobic Reactors connected in series that were used to remove heavy metals by Hernández-Mata et al., 2014 [22]. In this scheme, the first reactor (R1) was inoculated with biomass and the effluent was recirculated until the biomass reached a concentration of 1 g/L. When the desired biomass concentration was achieved, the biosorption stage was initiated with mining effluents. After the biosorption stage, a desorption (purification) step was carried out to remove the metallic ions adsorbed by the biomass. Biomass concentration was measured once again until the concentration reached 1 g/L. The effluent of R1 was then fed to R2 (containing the same biomass produced in R1) and biosorption was examined in both reactors. Samples were taken at regular intervals at the inlet

*ads*

## **4. Heavy metals removal by living anaerobic biomass**

Anaerobic microorganisms perform as part of their metabolism a process known as anaerobic digestion, which has been widely implemented in the treatment and stabilization of effluents with high organic loads. Two of the main bacterial groups that participate in anaerobic digestion are acidogenic microorganisms (responsible for the conversion of organic matter into volatile fatty acids, VFAs) and methanogenic microorganisms (methane producers).

Generally, it is considered that methanogenic bacteria are less resistant to external changes in their growing conditions such as pH, temperature, and/or presence of toxic metals [23]. It was also reported in a previous study that inhibition by heavy metals was less noticeable for acidogenic bacteria [24].

#### **4.1. Biomass treatment (Acidogenic phase)**

To achieve acidogenic conditions, biomass can be inoculated in Erlenmeyer flasks for a large period of time (up to 8 weeks), mixing anaerobic sludge and material supports (if desirable). The flasks are kept at 30°C. The feed medium is changed continuously and prepared according to the requirements of the microorganism [25]. The medium pH is kept at acidic levels (3–4) to inhibit the growth of methanogenic organisms, which is favored at neutral pH.

Dextrose is generally used as substrate. This substrate is the source of organic matter that enhances volatile fatty acids (VFAs) formation, mainly: acetic acid, propionic acid, and butyric acid. In order to verify that the anaerobic sludge is carrying out the acidogenic phase of digestion, VFAs formation and concentration can be measured by HPLC (high performance liquid chromatography) taking samples from the flasks at regular intervals. pH can be measured daily and the growth of biomass can be indirectly calculated by determining the volatile suspended solids (VSS), which are obtained according to the gravimetric method [26].

#### **4.2. Toxicity studies**

Toxicity tests are carried out prior to any biosorption test with living biomass to obtain inhibitory concentrations. For acidogenic biomass, VFAs formation or substrate consumption are a direct measurement of microbial activity. During a toxicity experiment, a known amount of biomass (or immobilized biomass, if desirable) is put into a series of flasks and mixed with fixed volumes of metallic solutions and a selected substrate. The concentration of heavy metals in the metallic solutions varies according to each experimental setup and metallic ion, but one flask must be selected as a blank. The concentration of the organic substrate is kept constant in all flasks. Solution pH has to be adjusted to acidic levels (3–5) to avoid metal precipitation. Once the biomass and solutions are mixed, the flasks are closed and placed in an incubator at a specific temperature and rpm (for instance, 35°C and 50 rpm). Small liquid samples are taken from each flask at regular intervals to determine substrate or VFAs concentration. Sampling can stop when concentrations in all flasks remain constant for at least two consecutive points.

Once all measurements are done, toxicity is determined in terms of the half-inhibitory concen‐ tration,IC50,whichistheconcentrationatwhichmicrobialactivityisdecreasedby50%.Microbial activity is determined by calculating the difference between the initial concentrations and final concentrationsineachflaskanddividingitbytheconcentrationdifferenceoftheblank(Equation 3). IC50 is then determined graphically from a plot of "% activity" versus "metallic concentra‐ tion". The blank is considered to have a 100% microbial activity since no metallic inhibition takes place, but the activity decreases with increasing heavy metal concentration.

$$A\left(\%\right) = \frac{\mathcal{C}\_{0-}\mathcal{C}}{\mathcal{C}\_{B0-}\mathcal{C}\_{Bt}} \times 100\% \tag{3}$$

where

A(%): microbial activity.

D0, D48: concentration of substrate or VFAs at times 0, and t, respectively. Dc0, Dc48: concentration of substrate or VFAs in the blank flask at times 0, and 7, respectively.

#### **4.3. Biosorption isotherms**

Biosorption isotherms are plots of biosorption capacity versus metallic concentration at equilibrium. Isotherms can be adjusted to adsorption models to determine other parameters useful in the scaling up of biosorption processes, such as maximum biosorption capacity and affinity coefficients. To determine biosorption capacity, batch tests are carried out in a similar fashion to toxicity tests, but the variable of importance is the heavy metals concentration. A known amount of biomass (or immobilized biomass, if desirable) is put into a series of flasks and mixed with fixed volumes of metallic solutions. Metallic ions concentrations are deter‐ mined by atomic absorption spectrometry. Biosorption equilibrium takes place when concen‐ trations in all flasks remain constant for at least two consecutive points, and sampling can stop. Biosorption capacity can then be calculated according to Equation 4 [27].

$$q = \frac{V(\mathbb{C}\_0 - \mathbb{C}\_f)}{S} \tag{4}$$

where

Dextrose is generally used as substrate. This substrate is the source of organic matter that enhances volatile fatty acids (VFAs) formation, mainly: acetic acid, propionic acid, and butyric acid. In order to verify that the anaerobic sludge is carrying out the acidogenic phase of digestion, VFAs formation and concentration can be measured by HPLC (high performance liquid chromatography) taking samples from the flasks at regular intervals. pH can be measured daily and the growth of biomass can be indirectly calculated by determining the volatile suspended solids (VSS), which are obtained according to the gravimetric method [26].

Toxicity tests are carried out prior to any biosorption test with living biomass to obtain inhibitory concentrations. For acidogenic biomass, VFAs formation or substrate consumption are a direct measurement of microbial activity. During a toxicity experiment, a known amount of biomass (or immobilized biomass, if desirable) is put into a series of flasks and mixed with fixed volumes of metallic solutions and a selected substrate. The concentration of heavy metals in the metallic solutions varies according to each experimental setup and metallic ion, but one flask must be selected as a blank. The concentration of the organic substrate is kept constant in all flasks. Solution pH has to be adjusted to acidic levels (3–5) to avoid metal precipitation. Once the biomass and solutions are mixed, the flasks are closed and placed in an incubator at a specific temperature and rpm (for instance, 35°C and 50 rpm). Small liquid samples are taken from each flask at regular intervals to determine substrate or VFAs concentration. Sampling can stop when concentrations in all flasks remain constant for at least two consecutive points.

Once all measurements are done, toxicity is determined in terms of the half-inhibitory concen‐ tration,IC50,whichistheconcentrationatwhichmicrobialactivityisdecreasedby50%.Microbial activity is determined by calculating the difference between the initial concentrations and final concentrationsineachflaskanddividingitbytheconcentrationdifferenceoftheblank(Equation 3). IC50 is then determined graphically from a plot of "% activity" versus "metallic concentra‐ tion". The blank is considered to have a 100% microbial activity since no metallic inhibition

takes place, but the activity decreases with increasing heavy metal concentration.

0 t <sup>C</sup> % 100% *B B*

D0, D48: concentration of substrate or VFAs at times 0, and t, respectively. Dc0, Dc48: concentration

Biosorption isotherms are plots of biosorption capacity versus metallic concentration at equilibrium. Isotherms can be adjusted to adsorption models to determine other parameters

= ´ (3)

*C C* - -

( ) <sup>0</sup>

*<sup>C</sup> <sup>A</sup>*

of substrate or VFAs in the blank flask at times 0, and 7, respectively.

**4.2. Toxicity studies**

38 Biomass Production and Uses

where

A(%): microbial activity.

**4.3. Biosorption isotherms**

q = biosorption capacity, (mg metal/g VSS);

C0 = initial metal concentration (mg metal/L);

Cf = final metal concentration (mg metal/L);

S = biosorbent (biomass) used (g);

V = volume of metallic solution (L).

The data at equilibrium (concentration and biosorption capacity) can be adjusted to established adsorption models. A correlation factor can be calculated by lineal regression to determine which model fits best to the experimental values. The most commonly used models in the literature are the Langmuir and Freundlich models.

#### **4.4. Continuous studies**

Continuous studies can be carried out in bioreactors of all shapes and sizes, but the most commonly used configuration is the anaerobic packed bed reactor (APBR). Generally, wastewater flows upward through the reactor bed, and the use of a material support prevents from biomass losses and enhances bed stability. Environmental conditions depend upon the type of biomass used. Figure 3 shows the schematic diagram of an APBR used for the bio‐ sorption of heavy metals [28]. Bioreactors startup times are varied, and the parameters commonly measured during operation are pH, chemical oxygen demand (COD), substrate consumption, methane formation, VFAs formation, volatile suspended solids (VSS). Recircu‐ lation of the effluent can be added to the reactors configuration to enhance biomass growth before biosorption takes place.

The COD values are a measure of the organic load of wastewaters. When both the influent and effluent points are sampled, the COD analysis provides a quantifiable measurement of the removal efficiency of organic matter in the bioreactor. The most common COD method involves digestion of the sample at 120°C followed by a colorimetric analysis. The procedure is thoroughly described in the Standard Methods for the Examination of Water and Waste‐ water [26].

VFAs concentrations are indicative of the acidogenic activity of anaerobic biomass. Total VFAs can be analyzed by a simple titration method (using hydrochloric acid and sodium hydroxide) proposed by [29] Powell and Archer (1989). Specific VFAs, such as acetic acid, propionic acid, or butyric acid can be analyzed by HPLC. For the determination of substrate consumption, most methods are relatively simple and involve colorimetric techniques. A method utilized for glucose concentration is the DNS (3,5-dinitro-salicyclic acid) method, where the free sugar reduces the DNS reagent at high temperature, resulting in the formation of a colored product that absorbs light at 540 nm [30].

**Figure 3.** Example of an APBR used for heavy metals biosorption.

Once the startup stage is complete, heavy metals can be fed to the bioreactor to initiate the biosorption stage. A plot of C/C0 versus time is known as a rupture curve, where Co is the inlet concentration and C is the outlet concentration. Rupture curves provide information about the quality of a biosorbent in terms of the breakthrough time, saturation time, and retention capacities. The breakthrough time, *t*b, is defined as the time in which the outlet concentration is equal to a maximum permissible value (usually 10% of the inlet concentration or lower). Saturation time, *t*s, is the time in which the column is completely saturated by the metallic ions. Metallic retention capacity, *Q*ads, can be calculated according to the following equation:

$$Q\_{\rm ads} = \frac{\mathbf{C}\_0 F}{m\_s} \int\_{t=t\_0}^{t=t\_s} \left(1 - \frac{\mathbf{C}}{\mathbf{C}\_o}\right) \mathbf{d}t \qquad \Rightarrow \qquad Q\_{\rm ads} = \frac{F}{m\_s} \int\_{t=t\_0}^{t=t\_s} \mathbf{C}\_{\rm ads} \mathbf{d}t \tag{5}$$

where

Qads: Retention capacity [mg/gVSS];

Cads: Co-C [mg/L];

is thoroughly described in the Standard Methods for the Examination of Water and Waste‐

VFAs concentrations are indicative of the acidogenic activity of anaerobic biomass. Total VFAs can be analyzed by a simple titration method (using hydrochloric acid and sodium hydroxide) proposed by [29] Powell and Archer (1989). Specific VFAs, such as acetic acid, propionic acid, or butyric acid can be analyzed by HPLC. For the determination of substrate consumption, most methods are relatively simple and involve colorimetric techniques. A method utilized for glucose concentration is the DNS (3,5-dinitro-salicyclic acid) method, where the free sugar reduces the DNS reagent at high temperature, resulting in the formation of a colored product

Once the startup stage is complete, heavy metals can be fed to the bioreactor to initiate the biosorption stage. A plot of C/C0 versus time is known as a rupture curve, where Co is the inlet concentration and C is the outlet concentration. Rupture curves provide information about the quality of a biosorbent in terms of the breakthrough time, saturation time, and retention capacities. The breakthrough time, *t*b, is defined as the time in which the outlet concentration is equal to a maximum permissible value (usually 10% of the inlet concentration or lower). Saturation time, *t*s, is the time in which the column is completely saturated by the metallic ions. Metallic retention capacity, *Q*ads, can be calculated according to the following equation:

> 0 0 <sup>0</sup> 1 d d *s s t t t t ads ads ads t t t t s o <sup>s</sup> C F C F <sup>Q</sup> t Q Ct mC m* = = = =

è ø ò ò (5)

æ ö = - Þ= ç ÷

water [26].

40 Biomass Production and Uses

where

that absorbs light at 540 nm [30].

**Figure 3.** Example of an APBR used for heavy metals biosorption.

t0: Initial time [d];

ts: Saturation time [d];

F: Volumetric flow [L/d].

Removal efficiency can also be determined simply by calculating the total metallic load and final metallic retention.

## **4.5. Bed characterization**

Bed characterization in anaerobic reactors is usually achieved by the following techniques: fraction of solids, Gram staining, microscopic observation via optical microscopy or scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive spectroscopy (EDS). These analyses supply plenty of information about the morphology and structure of the microorganisms and extracellular polymeric substances of the biofilm. XRD and EDS are especially helpful when a material support is utilized since these analyses provide the elemental composition of the different solid phases of the bioreactor bed.

## **5. Sulfate-reducing process and metal bioprecipitation**

The microbial sulfate-reducing process (SRP) has been utilized as a potential tool for heavy metals removal during the final steps of wastewater treatments and effluent recovery of several industries. Under anaerobic conditions, sulfate-reducing bacteria (SRB) reduce sulfate to sulfur, which reacts with the metallic ions, resulting in the formation of metallic sulfurs. Metallic sulfurs are universally identified because of their low solubility in aqueous systems, making the sulfate-reducing process an effective alternative for wastewater treatment [31]. Furthermore, selective recovery of economically important metals is also possible [32]. The sulfate-reducing process is successfully applied in the removal of metallic ions and sulfates in acid mine drainage (AMD), and can be useful in the removal of the remaining metals in industrial wastewaters [31].

During the SRP, sulfate ions (SO4 2− ) are enzymatically reduced to sulfur (H2S, HS-, and S2–) to obtain the energy required for the growth and maintenance of SRB. In order for this process to take place, cells carry out an enzymatic oxidation of organic matter (electron donor) to carbon dioxide and water [33, 34]. The SRP is strictly an anaerobic process, since it can only occur in the absence of electron acceptors with high redox potential such as oxygen or nitrate [35]. The SRP for heavy metals removal is based on the formation of metallic sulfurs with low solubility and the neutralization of water as a result of the alkalinity produced during the microbial oxidation of the electron donors [36]. This phenomenon has been defined as bioprecipitation [37], and can be described by the following equations [38].

Formation of sulfur and alkalinity (sulfidogenic oxidation) is defined by Equation 6, where CH2O represents the electron donor:

$$2\text{ }\text{CH}\_2\text{O} + \text{SO}\_4^{2-} \rightarrow \text{H}\_2\text{S} + 2\text{ }\text{HCO}\_3^- \tag{6}$$

When H2 is used as electron donor, the reaction generates hydroxide ions:

$$2\text{ }8H\_2 + 2\text{SO}\_4^{2-} \rightarrow \text{H}\_2\text{S} + \text{HS}^- + 5\text{H}\_2\text{O} + 3\text{OH}^-\tag{7}$$

The formation of biogenic sulfur (H2S, HS-, S2-) enhances precipitation of dissolved metals, where M2+ represents metallic ions such as: Zn2+, Cu2+, Ni2+, Co2+, Fe2+, Hg2+, Pb2+, Cd2+, o Ag+:

$$\text{CH}\_2\text{S} + \text{M}^{2+} \rightarrow \text{MS}\_{(s)} + 2\text{ H}^\* \tag{8}$$

The precipitation of metallic ions releases protons which acidify the water. Consequently, it is necessary to reduce the excess of sulfate to compensate acidity. The alkalinity of the hydroxide ions or bicarbonate produced during the sulfidogenic oxidation neutralizes the acidity of water.

$$\rm{HCO\_3^- + H^+ \to CO\_{2(g)} + H\_2O} \tag{9}$$

$$\text{OH}^- + \text{H}^+ \rightarrow \text{H}\_2\text{O} \tag{10}$$

#### **5.1. Advantages of the sulfate reducing process in wastewater treatment**

The SRP is a valuable biotechnological tool for heavy metals removal in mining lixiviates and industrial effluents. It is considered potentially superior to other biological processes due to its capacity to produce alkalinity, neutralize the pH of acidic water, and simultaneously remove organic matter, sulfates and heavy metals [39, 32, 40, and 38]. Furthermore, recent studies of the SRP have revealed potential immobilization for metalloids (arsenic), radioactive isotopes (uranium), and cyanides [41, 42, and 43]. The SRP has also shown applications in organic matter removal and degradation of xenobiotic and toxic compounds [44].

The most commonly known advantages of the SRP are the low formation of metallic sulfur sludge (small volume and low solubility) compared to hydroxide precipitation and the recovery of economically important metals and precipitated metallic ions [45]. Recently, some methods have been implemented to selectively recover metals through pH and sulfur control [33].

## **5.2. Toxicity of metals**

Formation of sulfur and alkalinity (sulfidogenic oxidation) is defined by Equation 6, where

The formation of biogenic sulfur (H2S, HS-, S2-) enhances precipitation of dissolved metals, where M2+ represents metallic ions such as: Zn2+, Cu2+, Ni2+, Co2+, Fe2+, Hg2+, Pb2+, Cd2+,

The precipitation of metallic ions releases protons which acidify the water. Consequently, it is necessary to reduce the excess of sulfate to compensate acidity. The alkalinity of the hydroxide ions or bicarbonate produced during the sulfidogenic oxidation neutralizes the acidity of

( ) <sup>2</sup>

OH H H O2

organic matter removal and degradation of xenobiotic and toxic compounds [44].

The SRP is a valuable biotechnological tool for heavy metals removal in mining lixiviates and industrial effluents. It is considered potentially superior to other biological processes due to its capacity to produce alkalinity, neutralize the pH of acidic water, and simultaneously remove organic matter, sulfates and heavy metals [39, 32, 40, and 38]. Furthermore, recent studies of the SRP have revealed potential immobilization for metalloids (arsenic), radioactive isotopes (uranium), and cyanides [41, 42, and 43]. The SRP has also shown applications in

The most commonly known advantages of the SRP are the low formation of metallic sulfur sludge (small volume and low solubility) compared to hydroxide precipitation and the recovery of economically important metals and precipitated metallic ions [45]. Recently, some methods have been implemented to selectively recover metals through pH and sulfur

**5.1. Advantages of the sulfate reducing process in wastewater treatment**

2 42 3 2 CH O SO H S 2 HCO - - + ®+ (6)

2 42 2 8 2 SO H S HS 5 O 3 *H H OH* -- - + ®++ + (7)

<sup>2</sup> s H S M MS 2 H + + +® + (8)

3 2 2 g( ) HCO H CO H O - + +® + (9)


2

When H2 is used as electron donor, the reaction generates hydroxide ions:

2

CH2O represents the electron donor:

42 Biomass Production and Uses

o Ag+:

water.

control [33].

It has been reported that metals are inhibitory agents for anaerobic microorganisms, including SRB [46, 47]. The inhibition is mostly due to the capacity of metals to deactivate enzymes by reacting with other sulfhydryl groups (-SH) and replacing the metals that constitute the active sites, such as Cu(II), Zn(II), Co(II), Ni(II). The deactivation of enzymes implies a negative impact on bacterial growth and activity [48]. There are some discrepancies in the literature with regard to the inhibitory levels of heavy metals over SRB because the majority of experi‐ ments are carried out at different environmental conditions [49].

Biogenic sulfur (produced during the SRP) forms complexes insoluble with heavy metals, resulting in the precipitation of metallic sulfur and, in turn, a toxicity reduction [46]. Sulfur inhibition may be decreased by precipitating sulfur with iron [50]. Several studies have focused on the use of SRP for the precipitation of metallic sulfurs within the same reactors where the sulfate-reducing activity takes place. However, this method might increase the inhibition of SRB [51].

To reduce inhibitory effects and increase pH in anaerobic reactors, a portion of the wastewater can be recycled and mixed with the influent. The remaining sulfur in the recirculating effluent reacts with heavy metals and causes precipitation of metallic ions before they get in contact with the anaerobic sludge [52]. The search of new strains tolerant to sulfurs or the special designs of bioreactors can help to prevent the toxic effect of heavy metals on SRB [53].

Another problem associated with heavy metal precipitation within the reactor is that metallic sulfurs are deposited on the biomass, and the contaminated sediments generate an increase in volume [54]. Moreover, contrary to general belief that only soluble metallic ions cause inhibition, it has been proven that metallic sulfurs affect the metabolic activity of SRB. Metallic sulfurs are not toxic, but they block substrate and nutrients access into the cells by forming a barrier on the cellular walls of SRB [47]. A proper alternative to separate the biological process from the precipitation is to use a two-step process, where metallic precipitation is isolated from the biological process [54].

#### **5.3. Selective precipitation of heavy metals**

Metallic sulfurs are generally highly insoluble at neutral pH, whereas some compounds, such as CuS, are insoluble at pH values as low as 2. The great advantage of precipitation is the possibility for selective recovery of metallic sulfurs. It has been shown that each metal precipitates at a unique sulfur concentration S2–, or potential (pS), directly related to the solubility of the metallic sulfur formed. Controlling these concentrations within a precipitator can be carried out using pH electrodes and sulfide ion selective electrodes (pS electrode). The unique quality of the potential level (pS) of each metal has been successfully applied as a controlling parameter for the selective precipitation of metals and formation of pure metallic sulfurs suitable for reutilization. The success of the precipitation process depends not only on the heavy metal removal from the soluble phase but also on its separation from the liquid phase. Thus, solid–liquid separation processes (for instance, sedimentation and filtration) are of great importance for a successful removal [55].

## **5.4. Types of reactors used for the sulfate-reducing process**

Biomass is retained within bioreactors according to the adherence properties of cells. Thus, bioreactors can be classified into two groups [56]: fluidized bed reactors and fixed bed reactors. In a fixed bed reactor, biomass is retained either by the formation of biofilms on static or suspended inert materials or by the obstruction of biological particles on packing materials (Figure 4). A biofilm is defined as a complex structure constituted by cells and extracellular products in elongated or granular forms [57]. In fluidized bed reactors (or free bed reactors), biomass is retained by forming biological particles of high density and sedimentability: granules. Methanogenic granular sludge and sulfate-reducing sludge are composed of microbial aggregates that grow by mutual bonding of bacterial cells in the absence of a support material [58].

**Figure 4.** Anaerobic reactors used in sulfate-reducing applications.

Numerous literature studies have applied multiple reactors designs of the sulfate-reducing process for the treatment of water with high concentrations of sulfates and heavy metals. Some of these designs include batch reactors (BR), sequencing batch reactors (SBR), continuously stirred tank reactors (CSTR), anaerobic contact processes (ACP), anaerobic baffled reactors (ABR), anaerobic filter reactors (AFR), fluidized bed reactors (FBR), gas lift reactors (GLR), anaerobic hybrid reactors (AHR), membrane bioreactors (MBR), and upflow anaerobic sludge blanket reactors (UASB) [38].

## **6. Biosorption models**

The kinetic model of a microbial process is defined as: the verbal or mathematical correlation between velocities and concentrations of reagents products, inserted into mass balances for the prediction of substrate conversion level and individual yields at specific operating conditions [59].

The complexity of the kinetic models used to describe the changes within the cell during a microbial transformation can be very broad. Several kinetic models proposed in the literature are summarized in Table 1 [60].


**Table 1.** Classification of kinetic models.

The simplest models, unstructured-non segregated models, have been used for numerous engineering troubleshooting applications. However, in order to have a better system descrip‐ tion it is necessary to use models that take into account complex reaction schemes, i.e., models that take into account the metabolic pathways of each microorganism.

A mathematical model is the abstract representation of a specific aspect of reality. Its structure is composed of two parts. The first part corresponds to all those characteristic aspects of an idealized reality, and the second part refers simply to the existing relationships between the aforementioned elements [61].

The order of reaction is an experimental magnitude dependent of the way in which velocity relates to concentration [62]. Any typical reaction in nature will occur at a rate dependent of certain factors, the reaction rate is indicated by a constant value (k). It is found that reaction rates are related to the reaction order according to the following mathematical expression [63]:

$$-\frac{dA}{dt} = k \left[\mathbf{A}\right]^n\tag{11}$$

where

**5.4. Types of reactors used for the sulfate-reducing process**

**Figure 4.** Anaerobic reactors used in sulfate-reducing applications.

blanket reactors (UASB) [38].

**6. Biosorption models**

conditions [59].

material [58].

44 Biomass Production and Uses

Biomass is retained within bioreactors according to the adherence properties of cells. Thus, bioreactors can be classified into two groups [56]: fluidized bed reactors and fixed bed reactors. In a fixed bed reactor, biomass is retained either by the formation of biofilms on static or suspended inert materials or by the obstruction of biological particles on packing materials (Figure 4). A biofilm is defined as a complex structure constituted by cells and extracellular products in elongated or granular forms [57]. In fluidized bed reactors (or free bed reactors), biomass is retained by forming biological particles of high density and sedimentability: granules. Methanogenic granular sludge and sulfate-reducing sludge are composed of microbial aggregates that grow by mutual bonding of bacterial cells in the absence of a support

Numerous literature studies have applied multiple reactors designs of the sulfate-reducing process for the treatment of water with high concentrations of sulfates and heavy metals. Some of these designs include batch reactors (BR), sequencing batch reactors (SBR), continuously stirred tank reactors (CSTR), anaerobic contact processes (ACP), anaerobic baffled reactors (ABR), anaerobic filter reactors (AFR), fluidized bed reactors (FBR), gas lift reactors (GLR), anaerobic hybrid reactors (AHR), membrane bioreactors (MBR), and upflow anaerobic sludge

The kinetic model of a microbial process is defined as: the verbal or mathematical correlation between velocities and concentrations of reagents products, inserted into mass balances for the prediction of substrate conversion level and individual yields at specific operating *n* = reaction order;

*k* = rate constant;

*A* = concentration of component A;

*t* = time.

This equation is integrated for every order of reaction (zero order, first order, second order, pseudo first order, and pseudo second order) as follows:

**•** *Zero order reaction:*

Differential equation:

$$-\frac{\text{dA}}{\text{dt}} = \text{kA}^{\circ}\tag{12}$$

Separating variables:

$$\int \mathbf{dA} = \mathbf{k} \Big[ \mathbf{dt} \tag{13}$$

Solving the integral:

$$\mathbf{A} = \mathbf{k}\mathbf{t} + \mathbf{C} \tag{14}$$

**•** *First-order reaction:*

Differential equation:

$$-\frac{\text{dA}}{\text{dt}} = \text{kA}^1\tag{15}$$

Separating variables:

$$\int \frac{\mathbf{dA}}{\mathbf{A}} = \mathbf{k} \int \mathbf{dt} \tag{16}$$

Solving the integral:

$$\text{LnA} = \text{kt} + \text{C} \tag{17}$$

**•** *Second-order reaction:*

Differential equation:

$$-\frac{dA}{dt} = \mathbf{k} \mathbf{A}^2\tag{18}$$

Separating variables:

$$\int \frac{\mathbf{dA}}{\mathbf{A}^2} = \mathbf{k} \left[ \mathbf{dt} \right] \tag{19}$$

Solving the integral:

Removal of Heavy Metals from Aqueous Solutions by Aerobic and Anaerobic Biomass http://dx.doi.org/10.5772/61330 47

$$\frac{1}{A} = \text{kt} + \text{C} \tag{20}$$

where C: Integration constant

**•** *Pseudo first-order reaction:*

Differential equation:

**•** *Zero order reaction:*

46 Biomass Production and Uses

Differential equation:

Separating variables:

Solving the integral:

**•** *First-order reaction:*

Differential equation:

Separating variables:

Solving the integral:

**•** *Second-order reaction:*

Differential equation:

Separating variables:

Solving the integral:

dA kA dt

dA <sup>1</sup> kA

dA k dt

<sup>2</sup> kA

dt

2 dA k dt


dA k dt <sup>=</sup> ò ò (13)

A kt C = + (14)

dt - = (15)

<sup>A</sup> <sup>=</sup> ò ò (16)

LnA kt C = + (17)

*dA* - = (18)

<sup>A</sup> <sup>=</sup> ò ò (19)

$$\frac{d\mathbf{x}}{dt} = \mathbf{k}\_2(\mathbf{C}\_{A0} - \mathbf{x})\mathbf{b} \tag{21}$$

Solving the equation:

$$\mathbf{k} = \mathbf{b} \mathbf{k}\_2 = \frac{1}{\mathbf{t}} \left| \frac{1}{\mathbf{C}\_{\text{A}}} - \frac{1}{\mathbf{C}\_{\text{A0}}} \right| \tag{22}$$

**•** *Pseudo second-order reaction:*

$$\frac{d\mathbf{x}}{dt} = \mathbf{k}\_2 (\mathbf{C}\_{A0} - \mathbf{x})^2 \mathbf{b} \tag{23}$$

Solving the equation:

$$\mathbf{k} = \mathbf{b}\mathbf{k}\_2 = \frac{1}{\mathbf{t}} \left[ \frac{1}{\mathbf{C}\_A} - \frac{1}{\mathbf{C}\_{A0}} \right]^2 \tag{24}$$

where

*CA* = amount of metal adsorbed (mg/L)

*CAo* = initial concentration (mg/L)

*t* = time (min)

*k* = equation constant (mg/L-min)

*b* = initial concentration of component b, constant throughout the reaction time.

If the lineal model properly fits the experimental values (i.e., a correlation factor, R2 , close to 1) the adsorption process can be described as chemisorption [64].

The development of biosorption systems is dependent of many factors including: temperature, pH, biosorption capacities and selectivities, recovery efficiency, and resistance to other components or operating conditions. Nevertheless, most biosorption studies focus on the measurement of the biosorption capacities of biomass [65]. The quantification of the sorbate– biosorbent interactions is fundamental for the evaluation of the biosorption capacity. Due to the similarity between the biosorption process and the adsorption process, biosorption capacity can be analyzed by sorption isotherms. Sorption isotherms are model equations that represent the behavior of experimental data.

The Langmuir and Freundlich equations are two of the most utilized adsorption models. These models are described by the following equations [1]:

#### **6.1. Langmuir model**

$$q\_{\epsilon} = \frac{q\_{\max} b \mathbf{C}\_{\epsilon}}{1 + b \mathbf{C}\_{\epsilon}} \tag{25}$$

$$\frac{1}{q\_c} = \left(\frac{1}{bq\_{\text{max}}}\right)\left(\frac{1}{\mathcal{C}\_c}\right) + \left(\frac{1}{q\_{\text{max}}}\right) \tag{26}$$

where

*qe*: biosorption capacity at equlibrium (mg/g VSS).

*qmax*: máximum biosorption capacity (mg/g VSS).

*Ce*: metallic concentration at equilibrium (mg/L).

*b*: affinity coefficient between the sorbate and the biosorbent (L/mg).

*qe* and *Ce* are obtained at the equilibrium point, whereas *qmax* and *b* can be determined graphi‐ cally by a plot of (1/*qe*) versus (1/*Ce*).

#### **6.2. Freundlich model**

$$\mathbf{q}\_e = \mathbf{k} \mathbf{C}\_e^{\mathbf{V}\_n} \tag{27}$$

$$\operatorname{Lnn}(q\_{\epsilon}) = \frac{1}{n}\operatorname{Lnn}(\mathbb{C}\_{\epsilon}) + \operatorname{Lnn}(k) \tag{28}$$

where

*qe*: biosorption capacity at equilibrium;

*Ce*: metallic concentration at equilibrium;

*k,n*: Freundlich constants.

The parameters *k* and *n* can be graphically determined from a plot of Ln(*qe*) versus Ln(*Ce*).

## **7. Conclusion**

Environmental pollution is one of the main problems of our society. Heavy metals constitute a major group of contaminants characterized by having a density five times greater than that of water. One of the main sources of heavy metals pollution is the acid mine drainage (AMD) generated by mining industries. The AMD is an acid lixiviate that may contain high concen‐ trations of sulfates, iron, calcium, zinc, manganese, aluminum, copper, and other types of toxic elements such as arsenic and lead. In México, several regions have been affected due to the presence of heavy metals in wastewaters, which generates the necessity of implementing economic and efficient remediation techniques. The review focuses on biological methods and the advantages they offer over conventional treatments. One particular alternative studied in recent years is biosorption – based on the ability of biomass to bind and concentrate heavy metals – because of its economic nature and high removal efficiencies in dilute wastewaters.

capacity can be analyzed by sorption isotherms. Sorption isotherms are model equations that

The Langmuir and Freundlich equations are two of the most utilized adsorption models. These

max 1

1 11 1 *e e q bq C q*

*e*

*b*: affinity coefficient between the sorbate and the biosorbent (L/mg).

*e*

*q bC <sup>q</sup> bC* <sup>=</sup> <sup>+</sup> (25)

*e e q kC* = (27)

= + (28)

(26)

*e*

max max

*qe* and *Ce* are obtained at the equilibrium point, whereas *qmax* and *b* can be determined graphi‐

1 *n*

<sup>1</sup> Ln( ) Ln( ) Ln( ) *e e q Ck n*

The parameters *k* and *n* can be graphically determined from a plot of Ln(*qe*) versus Ln(*Ce*).

Environmental pollution is one of the main problems of our society. Heavy metals constitute a major group of contaminants characterized by having a density five times greater than that

æ öæ ö æ ö = + ç ÷ç ÷ ç ÷ è øè ø è ø

represent the behavior of experimental data.

**6.1. Langmuir model**

48 Biomass Production and Uses

where

where

models are described by the following equations [1]:

*qe*: biosorption capacity at equlibrium (mg/g VSS). *qmax*: máximum biosorption capacity (mg/g VSS). *Ce*: metallic concentration at equilibrium (mg/L).

cally by a plot of (1/*qe*) versus (1/*Ce*).

*qe*: biosorption capacity at equilibrium; *Ce*: metallic concentration at equilibrium;

*k,n*: Freundlich constants.

**7. Conclusion**

**6.2. Freundlich model**

Biological technologies provide plenty of advantages and can be just as effective and economic as other technologies (Table 2). However, it is of upmost importance to continue with scientific research to acquire an improved understanding of the bioremediation processes and optimize industrial applications.


**Table 2.** A comparison between the existing methods for heavy metals removal.

## **Acknowledgements**

The authors would like to thank the Department of Chemical Engineering and Metallurgy of the University of Sonora for the space and equipment facilitated during the realization of this work.

## **Author details**

Onofre Monge-Amaya\* , María Teresa Certucha-Barragán, Francisco Javier Almendariz-Tapia and Gonzalo Mauricio Figueroa-Torres

\*Address all correspondence to: onofrem@iq.uson.mx

Department of Chemical Engineering and Metallurgy, University of Sonora, Hermosillo, Sonora, México

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plexation. Soil Sediment Contamin J 2006; 11(6): 841–859.

methanogenic bioreactors. FEMS Microbiol Rev 1994; 15:119–36.

anaerobic treatment. Biotechnol Bioeng 1980; 22:699–734.

sulfate reduction. Waste Manage 2001; 21: 197–203.

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Leeuwenhoek 1995; 67:3–28.

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drid). 1979; 43–7.

Canada. 2005; 148.

9.

54 Biomass Production and Uses

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61507

## **1. Introduction**

The amount of heavy metal ions released into the environment has been increased due to industrial activities and technological development. Furthermore, indiscriminate disposal has caused worldwide concern for many years because of the toxicity, accumulation in the food chain, persistence in nature, and concentration by organisms [1–3]. Heavy metals are not biodegradable and tend to accumulate in living organisms, causing various diseases and disorders [4, 5]. It is, therefore, important to reduce the levels of toxic metals or to completely remove them from wastewaters before being discharged into the environment [6]. Then, the minimization and assessment of harmful pollutants such as lead (Pb) and uranium (U) in the environment are very significant from the viewpoint of environmental protection.

There are many processes for the treatment of metal-contaminated wastewaters, including chemical precipitation, membrane filtration, reverse osmosis, ion exchange, and adsorption. However, their use is limited due to various disadvantages [7]. Adsorption has been proved as one of the most efficient methods for the removal of heavy metals from aqueous media [8].

Recently, adsorption based on carbonaceous materials including activated carbon (AC) [9], biochar [10], and carbon nanotubes [11] has been gradually applied to this area. Activated carbon has shown great potential for the removal of various inorganic and organic pollutants and radionuclides due to properties such as large surface area, microporous structure, and high adsorption capacity. As a promising material among nanostructured carbon materials, powdered activated charcoal continues to attract tremendous attention due to its unique physical and chemical properties [3, 12]. In particular, the chemical functionalization of activated carbon can modify its physical and chemical properties, leading to an improved performance in various applications [13–15]. The activation of AC is known to play a key role

© 2015 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 enhancing adsorption efficiency via surface morphology modifications with certain functional groups [15–18].

Zeolites are three-dimensional aluminosilicate minerals with a porous structure that have valuable merits, such as cation exchange, molecular sieving, catalysis, and adsorption [19–21]. However, in many cases, these materials do not exhibit high adsorption efficiencies for target metals, and therefore their modification has been reported to enhance their adsorption potential [6, 21–23]. Chitosan, as an abundant natural polysaccharide, has attracted much attention in the biomaterial area because of its biocompatibility, biodegradability, and nonantigenicity [20, 21, 24]. Then, it is expected that the zeolite/chitosan hybrid material, which can incorporate the merits of both materials, may exhibit promising capability for the removal of heavy metal from aqueous solution.

Considering the above, adsorption experiments for heavy metals from aqueous solutions by modified carbon and zeolite/chitosan hybrid material were carried out using Inductively Coupled Plasma Atom Emission Spectrometer (ICP-AES) and ICP-MS in this work to obtain the optimum conditions for heavy metal adsorption process. In this paper, we first present the results of adsorption experiments for U(VI) using "Yukitsubaki" carbon (special product for Aga Town in Niigata Prefecture; abbreviated to YKC herein after) modified with nitric acid as part of the adsorption study of metals using relatively low-cost biomass. Second, the results of adsorption experiments for Pb by activated carbon modified with nitric acid are shown. Then, we finally present the results of adsorption experiments for Pb using zeolite/chitosan hybrid composite (abbreviated to ZCHC herein after).

Our aim was to investigate the efficiency of biomass (or the material prepared by biomass) as an adsorbent for heavy metals for more practical use in the future. Adsorption isotherms of heavy metals were studied and analyzed using Langmuir and Freundlich equations. Further‐ more, to evaluate the characteristics of the adsorbents used in this work, the surface morphol‐ ogy, specific surface area, and functional groups of these materials were determined by SEM (Scanning Electron Microscopy), N2-BET (Brunaeur, Emmet, and Teller) method, and FT-IR (Fourier Transform Infrared Spectroscopy).

## **2. Experimental Work**

## **2.1. Samples**

## *2.1.1. The charcoal (Yukitsubaki carbon, YKC) modified with nitric acid (HNO3)*

YKC was made from the trunk of *Camellia japonica L.*, which grows in the northeast of Japan, a particularly special product of Aga Town in Niigata Prefecture. YKC was pestled and sieved with 60 mesh. For the removal of ash in YKC, deashing was carried out with boiled ultrapure water. Then, it was dried at 110 °C in an oven. For enhancing the adsorption capacity, YKC carbon was oxidized in 10% HNO3 and 30% HNO3 at 90 °C for 4 h and washed with ultrapure water in order to completely remove the residue. Subsequently the sample was heated at 300 °C for 2 h to remove the residual nitrate ions on YKC. In this paper, the pristine YKC, and the modified YKCs oxidized by 10% and 30% HNO3 were named YKC0, YKC10, and YKC30, respectively.

## *2.1.2. The Activated Carbon (AC) modified with potassium permanganate (KMnO4)*

Activated carbon was washed with deionized water (at 80 °C) to remove fine powders and contaminants, and was then dried at 110 °C for 2 h before use. Five grams of activated carbon was placed in a 200-mL conical flask, which contained 50 mL KMnO4 solution (0.01 or 0.03 mol/L). After adjusting the temperature to 25 °C and stirring for 12 h, the resulting solution was filtrated through a 0.45-μm membrane filter. Then, the filtrate was washed with deionized water until the pH of the filtrate was constant. The activated carbon was dried at 70 °C for 6 h. The untreated and treated (i.e., modified with 0.01 mol/L and 0.03 mol/L KMnO4 solution) activated carbon were described as AC0, ACK1, and ACK3, respectively.

## *2.1.3. The Zeolite/Chitosan Hybrid material (ZCHC)*

in enhancing adsorption efficiency via surface morphology modifications with certain

Zeolites are three-dimensional aluminosilicate minerals with a porous structure that have valuable merits, such as cation exchange, molecular sieving, catalysis, and adsorption [19–21]. However, in many cases, these materials do not exhibit high adsorption efficiencies for target metals, and therefore their modification has been reported to enhance their adsorption potential [6, 21–23]. Chitosan, as an abundant natural polysaccharide, has attracted much attention in the biomaterial area because of its biocompatibility, biodegradability, and nonantigenicity [20, 21, 24]. Then, it is expected that the zeolite/chitosan hybrid material, which can incorporate the merits of both materials, may exhibit promising capability for the removal

Considering the above, adsorption experiments for heavy metals from aqueous solutions by modified carbon and zeolite/chitosan hybrid material were carried out using Inductively Coupled Plasma Atom Emission Spectrometer (ICP-AES) and ICP-MS in this work to obtain the optimum conditions for heavy metal adsorption process. In this paper, we first present the results of adsorption experiments for U(VI) using "Yukitsubaki" carbon (special product for Aga Town in Niigata Prefecture; abbreviated to YKC herein after) modified with nitric acid as part of the adsorption study of metals using relatively low-cost biomass. Second, the results of adsorption experiments for Pb by activated carbon modified with nitric acid are shown. Then, we finally present the results of adsorption experiments for Pb using zeolite/chitosan

Our aim was to investigate the efficiency of biomass (or the material prepared by biomass) as an adsorbent for heavy metals for more practical use in the future. Adsorption isotherms of heavy metals were studied and analyzed using Langmuir and Freundlich equations. Further‐ more, to evaluate the characteristics of the adsorbents used in this work, the surface morphol‐ ogy, specific surface area, and functional groups of these materials were determined by SEM (Scanning Electron Microscopy), N2-BET (Brunaeur, Emmet, and Teller) method, and FT-IR

YKC was made from the trunk of *Camellia japonica L.*, which grows in the northeast of Japan, a particularly special product of Aga Town in Niigata Prefecture. YKC was pestled and sieved with 60 mesh. For the removal of ash in YKC, deashing was carried out with boiled ultrapure water. Then, it was dried at 110 °C in an oven. For enhancing the adsorption capacity, YKC carbon was oxidized in 10% HNO3 and 30% HNO3 at 90 °C for 4 h and washed with ultrapure water in order to completely remove the residue. Subsequently the sample was heated at 300

*2.1.1. The charcoal (Yukitsubaki carbon, YKC) modified with nitric acid (HNO3)*

functional groups [15–18].

56 Biomass Production and Uses

of heavy metal from aqueous solution.

hybrid composite (abbreviated to ZCHC herein after).

(Fourier Transform Infrared Spectroscopy).

**2. Experimental Work**

**2.1. Samples**

Zeolite was heated at 700 °C for 3 h to activate the surface in a muffle furnace and then washed with hydrochloric acid (5%, volume) and deionized water (at 80 °C) to remove fine powders and contaminants and was then dried at 110 °C for 2 h before use.

ZCHC was prepared by mixing solutions of chitosan and dispersions of zeolite in water. The general procedure of the synthesis is based on sol–gel method [25, 26]. First, 1 g chitosan was dissolved in 20 mL of 0.2 M acetic acid with constant stirring at temperature of 50 °C. Then, 10 mL of deionized water was added into 10 mL chitosan sol solution and was heated and stirred for 1 h. These solutions were mixed, while zeolite was dispersed in the chitosan solution with constant stirring for 5 h at a temperature of 50 °C. Then, the solution was transferred into five 10-mL centrifuge tubes, which were centrifuged at 9000 rpm for 5 min, and then washed with deionized water to remove contaminants. The mixed solutions were put on Petri dishes and were left to dry at room temperature for 24 h. The obtained films with a thickness of 0.1 mm were used for the following adsorption experiments.

#### **2.2. Adsorption experiment for heavy metals using natural materials prepared by biomass**

The following adsorption experiments were performed in a batch system using the above‐ mentioned samples. Experimental conditions (i.e., pH, contact time, sorbent amount, and temperature) in this work were optimized and determined based on our preliminary experi‐ ments and other studies [15, 20, 22]. The pH of each solution was adjusted by using 0.1 molL −1 NH3 aq/0.1 molL−1 HNO3. Adsorption isotherms of heavy metals onto these materials were measured at varying initial concentrations under optimized condition.

## *2.2.1. The charcoal (Yukitsubaki carbon, YKC) modified with nitric acid (HNO3)*

Each YKC sample of 30 mg was contacted with 50 mL of a known amount of U(VI) in a 100 mL conical flask, and the suspensions were shaken in a water bath. Adsorption experiments were conducted in the pH range of 2–10, with an adsorbent dosage of 0.1–0.6 gL−1, at a temperature of 5–45 °C, contact time from 1 to 24 h, and an initial concentration from 0 to 200 μgL−1.

## *2.2.2. The Activated Carbon (AC) modified with potassium permanganate (KMnO4)*

Activated carbon was thoroughly mixed with 50 mL of a known amount of Pb2+ in a 200-mL conical flask, and the suspensions were shaken in a water bath at room temperature (25 ± 2 °C). Adsorption experiments were conducted in the pH range of 3–7, with an adsorbent dosage of 0.1–1.5 gL−1, contact time from 1 to 24 h, and an initial concentration from 20 to 200 mgL−1.

## *2.2.3. The zeolite/chitosan hybrid material (ZCHC)*

Each ZCHC was thoroughly mixed with 50 mL of a known amount of Pb2+ in a 200-mL conical flask, and the suspensions were shaken in a water bath at room temperature (25 ± 2 °C). Adsorption experiments were conducted in the pH range of 3–7, a contact time from 1 to 48 h, and an initial concentration from 20 to 200 mgL−1, with an adsorbent dosage of 1.0 gL−1.

Following each sorption experiment, the suspension containing modified carbon (or ZCHC) and heavy metal solution was filtered through a 0.45-μm membrane filter (Advantec Mixed Cellulose Ester, 47 mm) to remove heavy metals that have been adsorbed into the adsorbent, and the concentration of these metals in the filtrate was determined with ICP-MS (X2, Ther‐ moFisher) or AAS (Z-5000, HITACHI).

The metal uptake by each adsorbent was calculated using the following equation:

$$q = \frac{(\mathbb{C}\_l - \mathbb{C}\_s)}{m} \cdot V \tag{1}$$

where *q* is the adsorption capacity of heavy metal with the adsorbent at equilibrium (mg g−1), *C*i and *C*e are the initial and equilibrium concentrations of heavy metal in a batch system, respectively (mg L−1), *V* is the volume of the solution (*L*), and *m* is the weight of the adsorbent (*g*).

#### **2.3. Langmuir and Freundlich isotherm model**

Adsorption isotherms are commonly used to reflect the performance of adsorbents in adsorp‐ tion processes. To examine the relationship between the metal uptake (*q*e) and the concentra‐ tion of metal ions (*C*e) at equilibrium, adsorption isotherm models are widely used for fitting data.

The Langmuir model assumes monolayer adsorption on a surface given by the following equation:

$$\frac{C\_e}{q\_e} = \frac{C\_e}{q\_{\text{max}}} + \frac{1}{K\_\text{L} q\_{\text{max}}} \tag{2}$$

where *C*<sup>e</sup> is the concentration of U(VI) (or Pb2+) in a batch system at equilibrium (mg L−1), *q*e is the amount of U(VI) (or Pb2+) adsorption at equilibrium (mg g−1), *q*max is the maximum adsorp‐ tion capacity on the surface of activated carbon (or ZCHC) (mg g−1), and *K*<sup>L</sup> is the Langmuir adsorption constant (L mg−1) [27, 28]. A plot of *C*e/*q*e versus *C*e gives a straight line with a slope of 1/*q*max, and an intercept of 1/(*K*L*q*max).

The Freundlich equation is widely used in the field of environmental engineering and was applied based on the work by Dahiya et al. [1, 29]. Freundlich isotherm can also be used to explain adsorption phenomenon as given below:

$$
\log\_{10} q\_{\epsilon} = \log\_{10} \mathcal{K}\_{\text{F}} + (1/n) \log\_{10} \mathcal{C}\_{f} \tag{3}
$$

where *K*F and *n* are constants incorporating all factors affecting the adsorption capacity and an indication of the favorability of metal ion adsorption onto a biosorbent, respectively. It is shown that 1/*n* values between 0.1 and 1.0 correspond to beneficial adsorption. That is, *q*<sup>e</sup> versus *C*<sup>f</sup> in a log scale can be plotted to determine the values of 1/*n* and *K*F.

#### **2.4. Kinetic model**

temperature of 5–45 °C, contact time from 1 to 24 h, and an initial concentration from 0 to 200

Activated carbon was thoroughly mixed with 50 mL of a known amount of Pb2+ in a 200-mL conical flask, and the suspensions were shaken in a water bath at room temperature (25 ± 2 °C). Adsorption experiments were conducted in the pH range of 3–7, with an adsorbent dosage of 0.1–1.5 gL−1, contact time from 1 to 24 h, and an initial concentration from 20 to 200 mgL−1.

Each ZCHC was thoroughly mixed with 50 mL of a known amount of Pb2+ in a 200-mL conical flask, and the suspensions were shaken in a water bath at room temperature (25 ± 2 °C). Adsorption experiments were conducted in the pH range of 3–7, a contact time from 1 to 48 h, and an initial concentration from 20 to 200 mgL−1, with an adsorbent dosage of 1.0 gL−1.

Following each sorption experiment, the suspension containing modified carbon (or ZCHC) and heavy metal solution was filtered through a 0.45-μm membrane filter (Advantec Mixed Cellulose Ester, 47 mm) to remove heavy metals that have been adsorbed into the adsorbent, and the concentration of these metals in the filtrate was determined with ICP-MS (X2, Ther‐

The metal uptake by each adsorbent was calculated using the following equation:

( ) *C C* i e *q V m*

where *q* is the adsorption capacity of heavy metal with the adsorbent at equilibrium (mg g−1),

Adsorption isotherms are commonly used to reflect the performance of adsorbents in adsorp‐ tion processes. To examine the relationship between the metal uptake (*q*e) and the concentra‐ tion of metal ions (*C*e) at equilibrium, adsorption isotherm models are widely used for fitting

The Langmuir model assumes monolayer adsorption on a surface given by the following

e e

e max L max *C C* 1 *q q Kq*

 and *C*e are the initial and equilibrium concentrations of heavy metal in a batch system, respectively (mg L−1), *V* is the volume of the solution (*L*), and *m* is the weight of the adsorbent


= + (2)

*2.2.2. The Activated Carbon (AC) modified with potassium permanganate (KMnO4)*

*2.2.3. The zeolite/chitosan hybrid material (ZCHC)*

moFisher) or AAS (Z-5000, HITACHI).

**2.3. Langmuir and Freundlich isotherm model**

μgL−1.

58 Biomass Production and Uses

*C*i

(*g*).

data.

equation:

Kinetics of heavy metal uptake was modeled using the pseudo-first-order and pseudo-secondorder Lagergren equations [30, 31]. The pseudo-first-order reaction of Lagergren for sorption can be expressed as follows based on Bhat et al. [32]:

$$\mathbf{d} \, \mathbf{Q} / \, \mathbf{d}t = k\_1 (Qe - Qt) \tag{4}$$

where *Q*e and *Qt* are the amount of metal adsorbed per unit weight (μg/g) of sorbent at equilibrium and at any time *t* (min), respectively, and *k*1 is the rate constant of pseudo-firstorder sorption (min−1). The integrated form of the above equation after applying the boundary conditions, for *t* = 0, *qt* = 0, becomes

$$
\log(Qe - Qt) = \log(Qe) - (k\_1 / \text{2.303})t \tag{5}
$$

The value of the rate constant (*k*1) and *Q*e for the pseudo-first-order sorption reaction can be obtained by plotting log(*Q*e −*Qt*) versus *t*.

The pseudo-second-order rate of Lagergren can be expressed as follows:

$$\mathbf{DQ} / \det = k\_2(\mathbf{Q}e - \mathbf{Q}t)^2 \tag{6}$$

where *k*<sup>2</sup> (g/(μg min)) is the rate constant for the pseudo-second-order sorption. The integrated linear form of Eq. (9) can be represented as follows:

$$\text{At } / \text{Q} = 1 / k\_2 \text{Q}^2\_{\text{-}e} + (1 / Q\_{\text{e}})t \tag{7}$$

The pseudo-second-order rate constant (*k*2) and *Q*e can be calculated from the intercept and slope of the linear plot of *t*/*Qt* versus *t*.

The initial adsorption rate (*h*) can be determined from *k*2 and *Qe* values using

$$h = k\_2 \text{Qe}^2\tag{8}$$

## **3. Results and discussion**

## **3.1. The charcoal (Yukitsubaki carbon, YKC) modified with nitric acid (HNO3)**

#### *3.1.1. Characterization of YKC*

The morphologies of pristine and modified YKCs characterized by SEM (JCM-6000, JEOL) are shown in Figure 1. Moreover, the surface properties including specific surface areas of pristine and modified YKCs determined by N2-BET method (TriStar II 3020, Micromeritics) are shown in Table 1. The pore structures of all the samples are similar to each other. However, judging from the SEM image shown in Figure 1, the surface area of YKC30 (Figure 1C) seems to be slightly changed with the acid treatment, whereas that of YKC10 (Figure 1B) seems to be hardly varied. This is consistent with the data of the specific surface area shown in Table 1. The decrease in the specific surface area of YKC30 would be attributable to the excessive oxidation with a high concentration of nitric acid.

**Figure 1.** The SEM images of pristine and modified YKCs. (A: YKC0, B: YKC10, C: YKC30)

The FT-IR (FTIR-4200, Jasco) spectra of pristine and modified YKCs are shown in Figure 2. From the figure, a characteristic broadband, which may be due to graphite structure in YKCs,


**Table 1.** Surface properties of pristine and modified YKCs

2

The initial adsorption rate (*h*) can be determined from *k*2 and *Qe* values using

**3.1. The charcoal (Yukitsubaki carbon, YKC) modified with nitric acid (HNO3)**

A B C

The FT-IR (FTIR-4200, Jasco) spectra of pristine and modified YKCs are shown in Figure 2. From the figure, a characteristic broadband, which may be due to graphite structure in YKCs,

**Figure 1.** The SEM images of pristine and modified YKCs. (A: YKC0, B: YKC10, C: YKC30)

slope of the linear plot of *t*/*Qt* versus *t*.

60 Biomass Production and Uses

**3. Results and discussion**

*3.1.1. Characterization of YKC*

with a high concentration of nitric acid.

The pseudo-second-order rate constant (*k*2) and *Q*e can be calculated from the intercept and

2

The morphologies of pristine and modified YKCs characterized by SEM (JCM-6000, JEOL) are shown in Figure 1. Moreover, the surface properties including specific surface areas of pristine and modified YKCs determined by N2-BET method (TriStar II 3020, Micromeritics) are shown in Table 1. The pore structures of all the samples are similar to each other. However, judging from the SEM image shown in Figure 1, the surface area of YKC30 (Figure 1C) seems to be slightly changed with the acid treatment, whereas that of YKC10 (Figure 1B) seems to be hardly varied. This is consistent with the data of the specific surface area shown in Table 1. The decrease in the specific surface area of YKC30 would be attributable to the excessive oxidation

<sup>2</sup> / 1 / 1 /( ) *e e t Q kQ Q t* = + (7)

<sup>2</sup> *h kQ* = e (8)

is observed at around 1610 cm−1. The peak at 3300 cm−1 is related to hydroxyl groups (−OH), and 1120 cm−1 is related to carbonyl groups (−C=O), and then the peaks at 2920 and 2850 cm−1 are associated with C–H [33–36]. The results of FT-IR analysis show that some kinds of functional groups (such as carbonyl groups and hydroxyl groups) are introduced to YKC surfaces successfully by oxidation.

**Figure 2.** The FT-IR spectra of pristine and modified YKCs

#### *3.1.2. Influence of parameters on adsorption*

#### *3.1.2.1. Contact time*

The effect of contact time on U(VI) adsorption was investigated to study the adsorption rate of U(VI) removal. The percentage removal of U(VI) for the concentration of 100 μg/L with the adsorbent dosage of 30 mg at a pH of 3.0 is shown in Figure 3.

The adsorption equilibrium of U(VI) on YKC10 was reached within 30 min, much faster than that on YKC0 and YKC30. For the rest of the study, 480 min (8 h) was selected as the contact time.

**Figure 3.** Effect of contact time on U(VI) adsorption (pH = 3.0, *T* = 25 °C, and adsorbent dose of 30 mg)

**Figure 4.** Effect of pH on U(VI) adsorption (contact time of 8 h, *T* = 25 °C, and adsorbent dose of 30 mg)

The adsorption of U(VI) on the YKC as a function of pH is shown in Figure 4. From this figure, it can be seen that the amount of U(VI) uptake increased with increasing solution pH. The highest uptake was observed at a pH of 5–8 for all YKCs, and the uptake of U(VI) decreased slightly with increasing pH at a pH of >8. The difference in the pH dependence is not clearly found among YKCs, although the difference of adsorption capacities among YKCs was obviously found. It is known that U exists in different forms depending on the pH. U exists predominantly as monomeric species UO2 2+, and small amounts of UO2 (OH)+ at a pH of ≤4.3, and at a pH of ≥5, colloidal or oligomeric species, i.e., (UO2)2(OH)2 2+, (UO2)3(OH)5 + , (UO2)4(OH)7 + , and (UO2)3(OH)7 − , are formed [37–40].

That is, U usually exists as cationic species in solution at a pH of 5. Furthermore, from the results of the FT-IR spectra of YKC shown in Figure 2, hydroxyl groups (−OH) are introduced onto YKC.

From the above, it can be considered that the U(VI) adsorption occurred dominantly by the cation exchange reaction between the H+ of hydroxyl groups on YKC and the cationic species of U(VI).

Then, a pH of 5 was assumed for further experimental work, although the uptake of U(VI) maintains the highest level at a pH of 5–8.

#### *3.1.2.2. Adsorbent amount and adsorption temperature*

Under optimized conditions of pH (i.e., pH 5) and contact time (i.e., 8 h), the adsorption behavior at different adsorbent amounts (10–60 mg) or adsorption temperatures (5–45°C) has been studied in 50 mL of a 100-μg/L aqueous U(VI) solution in 100-mL flasks.

From the above experiment, the adsorbent amount was selected as 30 mg in our work in consideration of the economic cost, although the adsorption efficiency slightly increased with an increase in the adsorbent amount. On the other hand, 25 °C was selected because the highest uptake was observed at 25 °C, and there is no appreciable change in the uptake of U(VI) with increasing temperatures.

#### *3.1.3. Adsorption isotherm study*

0 500 1000 1500

time [min]

2 4 6 8 10

pH

**Figure 4.** Effect of pH on U(VI) adsorption (contact time of 8 h, *T* = 25 °C, and adsorbent dose of 30 mg)

**Figure 3.** Effect of contact time on U(VI) adsorption (pH = 3.0, *T* = 25 °C, and adsorbent dose of 30 mg)

 YKC30 YKC10 YKC0

> YKC30 YKC10 YKC0

0

0

20

40

U(VI) uptake percentage [%]

60

80

100

20

40

60

U(VI) uptake percentage [%]

80

100

62 Biomass Production and Uses

The Langmuir and Freundlich isotherms for the adsorption of U(VI) onto YKC are given in Figures 5 and 6.

The linear correlation coefficients (*R*<sup>2</sup> ) of Langmuir and Freundlich isotherms for U(VI) using pristine and modified YKCs are shown in Table 2 along with other parameters.

From this table, it is found that the *R*<sup>2</sup> value for each datum is comparatively large for both Langmuir and Freundlich isotherms. Furthermore, it is noted that *R*<sup>2</sup> values for these data are larger for the Langmuir isotherm than for the Freundlich isotherm. This result suggests that the adsorption on these samples mainly occurred by a monolayer reaction.

From the data of *Q*m in Table 2, it is also found that the adsorption capacity of U(VI) on modified YKC is much higher than that on pristine YKC. The results indicated that acid treatment is effective for enhancing the adsorption capacity of U(VI).

**Figure 5.** The Langmuir isotherm of U(VI) adsorption (pH = 5.0, *T* = 25 °C, and adsorbent dose of 30 mg)

**Figure 6.** The Freundlich isotherm of U(VI) adsorption (pH = 5.0, *T* = 25 °C, and adsorbent dose of 30 mg)


**Table 2.** Coefficients of Langmuir and Freundlich isotherm models

Here, *Q*<sup>m</sup> is the maximum adsorption capacity of the U(VI) (μg/g), *K*<sup>L</sup> is the Langmuir binding constant, which is related to the energy of adsorption (L/μg), and *K*F and *b*<sup>F</sup> are the Freundlich constants related to the adsorption capacity and intensity.

#### **3.2. The activated carbon modified with potassium permanganate (KMnO4)**

#### *3.2.1. Characterization of the modified activated carbon*

0 50 100 150 200

0.0 0.5 1.0 1.5 2.0 2.5

log Ce

**Figure 6.** The Freundlich isotherm of U(VI) adsorption (pH = 5.0, *T* = 25 °C, and adsorbent dose of 30 mg)

Ce [µg/L]

**Figure 5.** The Langmuir isotherm of U(VI) adsorption (pH = 5.0, *T* = 25 °C, and adsorbent dose of 30 mg)

YKC10 YKC0

 YKC30 YKC10 YKC0

0.0

2.2

2.3

2.4

log Qe

2.5

2.6 YKC30

0.1

0.2

0.3

0.4

Ce/Qe [g/L]

0.5

0.6

0.7

0.8

64 Biomass Production and Uses

The FT-IR spectra of the pristine and modified activated carbon (i.e., AC0, ACK1, and ACK3) are shown in Figure 7. The pristine and modified activated carbon displayed the characteristic bands of the graphite structure of carbon at 1615 cm−1 [8, 15, 41]. Moreover, an OH stretching band, one of the typical peaks of activated carbon, was found at 3300–3500 cm−1. The peak at 3433 cm−1 was related to the hydroxyl groups (−OH) stretch from deprotonated pristine and modified activated carbon. The wide peak at 1550–1750 cm−1 shows the asymmetric stretch of the carboxylate (−COO−) group [15].

**Figure 7.** FT-IR spectra of AC0, ACK1, and ACK3.

The surface properties of the activated carbon were investigated by N2 adsorption (TriStar II 3020 Micromeritics), and the analytical results for the adsorption/desorption isotherms are shown in Table 3.


**Table 3.** Textural characteristics of activated carbon

The pore volume was calculated from the amount of N2 adsorbed at the relative pressure of 0.99. The pore size was calculated from the adsorption average pore width (4V/A by BET) in this work. From Table 3, it is found that the pore volume and pore size as well as the specific surface area decreased significantly after modification with KMnO4. The isotherm showed a type H1 isotherm with a clear hysteretic loop, characteristic of disordered microporous materials. significantly after modification with KMnO4. The isotherm showed a type H1 isotherm with a clear hysteretic loop, characteristic of disordered microporous materials. The SEM micrographs of the activated carbon are shown in **Figure 8**. The modified AC (**Figures 8B and 8C**) seemed to exhibit a more compact stacking morphology than the pristine AC (**Figure 8A**),

The SEM micrographs of the activated carbon are shown in Figure 8. The modified AC (Figures 8B and 8C) seemed to exhibit a more compact stacking morphology than the pristine AC (Figure 8A), due to cohesive forces, which may be generated from the introduction of oxygencontaining functional groups. These results are consistent with those of the N2 adsorption– desorption experiment. The decrease of the pore volume and pore size may be related to the increase of acidic groups on the surface of activated carbon treated with KMnO4. due to cohesive forces, which may be generated from the introduction of oxygen-containing functional groups. These results are consistent with those of the N2 adsorption–desorption experiment. The decrease of the pore volume and pore size may be related to the increase of acidic groups on the surface of activated carbon treated with KMnO4.

 **Fig. 8** SEM micrographs of the surface of activated carbon: (A) unmodified; (B) modified with 0.01 mol/L KMnO4; (C) modified with 0.03 mol/L KMnO4 **Figure 8.** SEM micrographs of the surface of activated carbon: (A) unmodified; (B) modified with 0.01 mol/L KMnO4; (C) modified with 0.03 mol/L KMnO4

#### **3.2.2. Adsorption of lead on modified activated carbon**  *3.2.2. Adsorption of lead on modified activated carbon*

#### 3.2.2.1. Effect of pH *3.2.2.1. Effect of pH*

To investigate the effect of solution pH on Pb2+ adsorption efficiency, the pH of the solution was varied from 3 to 7, while the Pb2+ concentration was kept constant at 100 mg·L−<sup>1</sup> . The experimental To investigate the effect of solution pH on Pb2+ adsorption efficiency, the pH of the solution was varied from 3 to 7, while the Pb2+ concentration was kept constant at 100 mg L−1. The

results are presented in **Figure 9**. The Pb2+ adsorption efficiency was at pH 5 regardless of the kind of

adsorbent (**Figure 9**). The uptake of Pb2+ increased from 50.8% at pH 3 to 90.0% at pH 5, and at

higher pH values, it remained almost constant (or decreased only slightly). Notably, the adsorption

capacities decreased at low pH values due to the competition of protons with metal ions for active

binding. On the other hand, lead precipitated from the solution at higher pH values as lead hydroxide

[1]. From the FT-IR spectra of AC (**Figure 7**), it was clear that the hydroxyl groups (−OH) were

introduced onto AC. We hypothesized that the Pb2+ adsorption occurred predominantly by cation

14

experimental results are presented in Figure 9. The Pb2+ adsorption efficiency was at pH 5 regardless of the kind of adsorbent (Figure 9). The uptake of Pb2+ increased from 50.8% at pH 3 to 90.0% at pH 5, and at higher pH values, it remained almost constant (or decreased only slightly). Notably, the adsorption capacities decreased at low pH values due to the competition of protons with metal ions for active binding. On the other hand, lead precipitated from the solution at higher pH values as lead hydroxide [1]. From the FT-IR spectra of AC (Figure 7), it was clear that the hydroxyl groups (−OH) were introduced onto AC. We hypothesized that the Pb2+ adsorption occurred predominantly by cation exchange reaction between the H+ of the hydroxyl groups on modified AC and cationic Pb2+ species. However, it is possible that Pb2+ was removed to some extent via precipitation at higher pH values rather than by adsorp‐ tion on the modified AC. Hence, pH 5 was used for further experiments.

**Figure 9.** Effect of pH on the removal of Pb2+ (%) using modified activated carbon.

#### *3.2.2.2. Effect of contact time*

The surface properties of the activated carbon were investigated by N2 adsorption (TriStar II 3020 Micromeritics), and the analytical results for the adsorption/desorption isotherms are

AC0 381 0.402 4.23 ACK1 373 0.390 4.18 ACK3 346 0.348 4.03

The pore volume was calculated from the amount of N2 adsorbed at the relative pressure of 0.99. The pore size was calculated from the adsorption average pore width (4V/A by BET) in this work. From Table 3, it is found that the pore volume and pore size as well as the specific surface area decreased significantly after modification with KMnO4. The isotherm showed a type H1 isotherm with a clear hysteretic loop, characteristic of disordered microporous

significantly after modification with KMnO4. The isotherm showed a type H1 isotherm with a clear

The SEM micrographs of the activated carbon are shown in **Figure 8**. The modified AC (**Figures 8B** 

The SEM micrographs of the activated carbon are shown in Figure 8. The modified AC (Figures 8B and 8C) seemed to exhibit a more compact stacking morphology than the pristine AC (Figure 8A), due to cohesive forces, which may be generated from the introduction of oxygencontaining functional groups. These results are consistent with those of the N2 adsorption– desorption experiment. The decrease of the pore volume and pore size may be related to the

due to cohesive forces, which may be generated from the introduction of oxygen-containing

functional groups. These results are consistent with those of the N2 adsorption–desorption experiment.

The decrease of the pore volume and pore size may be related to the increase of acidic groups on the

(A) unmodified; (B) modified with 0.01 mol/L KMnO4; (C) modified with 0.03 mol/L KMnO4

**Figure 8.** SEM micrographs of the surface of activated carbon: (A) unmodified; (B) modified with 0.01 mol/L KMnO4;

To investigate the effect of solution pH on Pb2+ adsorption efficiency, the pH of the solution was

To investigate the effect of solution pH on Pb2+ adsorption efficiency, the pH of the solution was varied from 3 to 7, while the Pb2+ concentration was kept constant at 100 mg L−1. The

results are presented in **Figure 9**. The Pb2+ adsorption efficiency was at pH 5 regardless of the kind of

adsorbent (**Figure 9**). The uptake of Pb2+ increased from 50.8% at pH 3 to 90.0% at pH 5, and at

higher pH values, it remained almost constant (or decreased only slightly). Notably, the adsorption

capacities decreased at low pH values due to the competition of protons with metal ions for active

binding. On the other hand, lead precipitated from the solution at higher pH values as lead hydroxide

[1]. From the FT-IR spectra of AC (**Figure 7**), it was clear that the hydroxyl groups (−OH) were

introduced onto AC. We hypothesized that the Pb2+ adsorption occurred predominantly by cation

varied from 3 to 7, while the Pb2+ concentration was kept constant at 100 mg·L−<sup>1</sup>

**and 8C**) seemed to exhibit a more compact stacking morphology than the pristine AC (**Figure 8A**),

14

increase of acidic groups on the surface of activated carbon treated with KMnO4.

**·g−1) Pore volume (cm3**

**·g−1) Pore size (nm)**

. The experimental

shown in Table 3.

66 Biomass Production and Uses

materials.

**Adsorbent BET surface area (m2**

**Table 3.** Textural characteristics of activated carbon

surface of activated carbon treated with KMnO4.

hysteretic loop, characteristic of disordered microporous materials.

 **Fig. 8** SEM micrographs of the surface of activated carbon:

**3.2.2. Adsorption of lead on modified activated carbon** 

*3.2.2. Adsorption of lead on modified activated carbon*

3.2.2.1. Effect of pH

*3.2.2.1. Effect of pH*

(C) modified with 0.03 mol/L KMnO4

The effect of contact time on Pb2+ adsorption efficiency using 1.0 gL−1 ACK3 (100 mgL−1 of solution) was investigated at pH 5.

More than 80% Pb was removed within 1 h, and it gradually increased at 2 h. Approximately 90% of Pb was removed from the solution at the contact time of 2 h. After 2 h, there was no appreciable change. Therefore, 2 h was chosen as the optimized contact time for the rest of the experimental work.

**Figure 10.** Effect of sorbent dosage on percent removal of Pb2+ using modified activated carbon

#### *3.2.2.3. Effect of sorbent dosage*

Under the optimized pH conditions (i.e., pH 5) and contact time (i.e., 2 h), the adsorption behavior of ACK3 at different dosages (from 0.1 to 1.5 gL−1) was studied in 100 mgL−1 Pb2+ solution.

More than 90% of Pb2+ was removed with a dosage of 1.0 gL−1 (Figure 10). The removal increased remarkably with higher dosage rates, but no remarkable increase was observed at dosages greater than 1.0 gL−1. Therefore, 1.0 gL−1 was considered as the optimum dosage for the remainder of the study.

#### *3.2.2.4. Effect of competitive ions*

Competitive experiments were conducted under the optimized pH conditions (i.e., pH 5), contact time (i.e., time 2 h), and sorbent dosage (i.e., 1 g/L) using different concentrations of Na+ , K+ , Ca2+, or Mg2+ separately and combination of all four ions (i.e., 0, 10, 20, 50, 100, 200, and 500 mgL−1). The percent removal of Pb2+ decreased in the presence of Na+ , K+ , Ca2+, or Mg2+ with concentrations from 0 to 500 mgL−1 (Figure 11). A remarkable decrease in the adsorption capacity of Pb2+ was not observed, even with common ions at concentrations of 100 mgL−1 (i.e., more than 80% Pb2+ was removed; Figure 5). This implied that the activated carbon was an efficient adsorbent for Pb2+, although further investigations are required for the realization of practical application.

**Figure 11.** Effect of competitive ions on percent removal of Pb2+ using modified activated carbon.

**Figure 12.** Langmuir isotherm of Pb2+ adsorption on AC.

*3.2.2.3. Effect of sorbent dosage*

0

20

40

Percentage removal [%]

60

80

100

68 Biomass Production and Uses

the remainder of the study.

*3.2.2.4. Effect of competitive ions*

realization of practical application.

solution.

Na+ , K+

Under the optimized pH conditions (i.e., pH 5) and contact time (i.e., 2 h), the adsorption behavior of ACK3 at different dosages (from 0.1 to 1.5 gL−1) was studied in 100 mgL−1 Pb2+

0.0 0.5 1.0 1.5 2.0

 50 ppm 100 ppm

Activated carbon dose [ g L-1]

**Figure 10.** Effect of sorbent dosage on percent removal of Pb2+ using modified activated carbon

More than 90% of Pb2+ was removed with a dosage of 1.0 gL−1 (Figure 10). The removal increased remarkably with higher dosage rates, but no remarkable increase was observed at dosages greater than 1.0 gL−1. Therefore, 1.0 gL−1 was considered as the optimum dosage for

Competitive experiments were conducted under the optimized pH conditions (i.e., pH 5), contact time (i.e., time 2 h), and sorbent dosage (i.e., 1 g/L) using different concentrations of

Mg2+ with concentrations from 0 to 500 mgL−1 (Figure 11). A remarkable decrease in the adsorption capacity of Pb2+ was not observed, even with common ions at concentrations of 100 mgL−1 (i.e., more than 80% Pb2+ was removed; Figure 5). This implied that the activated carbon was an efficient adsorbent for Pb2+, although further investigations are required for the

and 500 mgL−1). The percent removal of Pb2+ decreased in the presence of Na+

, Ca2+, or Mg2+ separately and combination of all four ions (i.e., 0, 10, 20, 50, 100, 200,

, K+

, Ca2+, or

## *3.2.3. Adsorption isotherms*

Adsorption isotherms are commonly used to reflect the performance of adsorbents in adsorp‐ tion processes. The Langmuir adsorption isotherm was applied to the data obtained in this work. A plot of *C*e/*q*e versus *C*e based on the Langmuir model is shown in Figure 12.

From these data, the *q*max and *K*L of AC0, ACK1, and ACK3 were calculated and are shown in Table 4 along with the *R*<sup>2</sup> (correlation coefficient). The adsorption capacity of AC modified with 0.03 mol/L KMnO4 was about 4 times higher than that of pristine AC.


*q*max: the maximum adsorption capacity on the surface of activated carbon (mg g−1);

*K*L: the Langmuir adsorption constant (L mg−1);

*R*: the correlation coefficient.

**Table 4.** Coefficient of Langmuir isotherm for Pb2+ using AC

The *R*<sup>2</sup> value for each adsorbent was comparatively large (Table 4). Furthermore, the *R*<sup>2</sup> value for modified AC (i.e., ACK1 and ACK3) was particularly large. That is to say, a favorable adsorption of Pb2+ by activated carbon was apparent. This result suggested that the adsorption of these samples primarily occurred via a monolayer reaction.


**Table 5.** Comparison of Pb2+adsorption capacities of modified AC with other adsorbents

Next, the adsorption capacity of Pb2+ by modified AC was compared with other adsorbents under ambient conditions previously reported in the literature. As shown in Table 5, the maximum adsorption capacity of modified AC toward Pb2+ was higher than that of other adsorbents.

The adsorption capacity of activated carbon may be dependent on its surface properties ([7], [45]). Therefore, the modification with KMnO4 (an efficient oxidizer) could change the surface structure of activated carbon to enhance the adsorption capacity of AC toward metals.

#### *3.2.4. Kinetic studies*

*3.2.3. Adsorption isotherms*

70 Biomass Production and Uses

Table 4 along with the *R*<sup>2</sup>

**Adsorbent** *qmax (mg·g−1)*

**Table 4.** Coefficient of Langmuir isotherm for Pb2+ using AC

*K*L: the Langmuir adsorption constant (L mg−1);

Activated carbon prepared from apricot stone

Activated carbon modified with KMnO4

*R*: the correlation coefficient.

Adsorption isotherms are commonly used to reflect the performance of adsorbents in adsorp‐ tion processes. The Langmuir adsorption isotherm was applied to the data obtained in this

From these data, the *q*max and *K*L of AC0, ACK1, and ACK3 were calculated and are shown in

AC0 25.1 0.0452 0.983 ACK1 80.0 0.435 0.998 ACK3 101 4.60 1.00

The *R*<sup>2</sup> value for each adsorbent was comparatively large (Table 4). Furthermore, the *R*<sup>2</sup>

for modified AC (i.e., ACK1 and ACK3) was particularly large. That is to say, a favorable adsorption of Pb2+ by activated carbon was apparent. This result suggested that the adsorption

**Biosorbent Maximum adsorption capacity (mg·g−1) Reference**

22.9 [16]

101 Present study

Acidified MWCNTs 49.7 [42] Cicer arientinum biomass 27.8 [7] Sawdust of Meranti wood 37.2 [43]

Peanut husks carbon 70.0 [44]

Next, the adsorption capacity of Pb2+ by modified AC was compared with other adsorbents under ambient conditions previously reported in the literature. As shown in Table 5, the

(correlation coefficient). The adsorption capacity of AC modified

*KL* **(L·mg−1)**

*R*

value

work. A plot of *C*e/*q*e versus *C*e based on the Langmuir model is shown in Figure 12.

with 0.03 mol/L KMnO4 was about 4 times higher than that of pristine AC.

*q*max: the maximum adsorption capacity on the surface of activated carbon (mg g−1);

of these samples primarily occurred via a monolayer reaction.

**Table 5.** Comparison of Pb2+adsorption capacities of modified AC with other adsorbents

The linear plot of t/*qt* versus *t* for metal adsorption under the optimized experimental condi‐ tions is shown in Figure 13. The coefficient of determination was more than 0.996 of Pb2+ on modified AC. The pseudo-second-order rate constant (*k*) and the amount of adsorbed lead (*q*e) obtained from the intercept and slope of the plot of t/*qt* versus *t* are listed in Table 6 along with the regression coefficient (*R*<sup>2</sup> ).

**Figure 13.** The pseudo-second-order kinetic model for AC.

The adsorption kinetics based on the experimental values were in good agreement with the pseudo-second-order kinetic model. The intraparticle diffusion model indicated that the relationship between the concentration of Pb2+ and the square root of time was linear. This suggested that the adsorption process could be controlled by intraparticle diffusion. Moreover, the adsorption at higher temperatures became more dependent on intraparticle diffusion, the rate-determining step.


*q*e: the amount of adsorbed lead on the surface of activated carbon at equilibrium (mg g−1);

*k* : the rate constant of the pseudo-second-order adsorption (g mol−1 h−1);

*R*: the correlation coefficient.

**Table 6.** Kinetic coefficients for Pb2+ adsorption on AC

#### **3.3. The zeolite/chitosan hybrid material (ZCHC)**

#### *3.3.1. Characterization of ZCHC*

The surface properties of ZCHC before and after Pb2+ adsorption were investigated by N2 adsorption (TriStar II 3020, Micromeritics), and the analytical results for the adsorption/ desorption isotherms are shown in Table 7.


**Table 7.** Textural characteristics of ZCHC

The pore volume was calculated from the amount of N2 adsorbed at the relative pressure of 0.99. The pore size was calculated from the adsorption average pore width (4V/A by BET) in this work.

The SEM micrographs of the ZCHC before and after Pb2+ adsorption ZCHC are shown in Figure 14. From Figure 14, it is found that the morphology of ZCHC surface has hardly changed even after exposing Pb2+, although the SEM picture after Pb2+ adsorption slightly exhibits a more compact stacking morphology than that before adsorption. From the above observation, ZCHC should be predicted to withstand the repeated use, and hence it can be a good adsorbent for heavy metals such as Pb2+. These results are consistent with those of the N2 adsorption– desorption experiment.

**Table 7** Textural characteristics of ZCHC

(before adsorption) 9.25 0.0485 21.0

(after adsorption) 9.19 0.0479 20.7

The pore volume was calculated from the amount of N2 adsorbed at the relative pressure of 0.99. The

The SEM micrographs of the ZCHC before and after Pb2+ adsorption ZCHC are shown in **Figure 14**.

From **Figure 14**, it is found that the morphology of ZCHC surface has hardly changed even after

exposing Pb2+, although the SEM picture after Pb2+ adsorption slightly exhibits a more compact

stacking morphology than that before adsorption. From the above observation, ZCHC should be

Pb2+. These results are consistent with those of the N2 adsorption–desorption experiment.

pore size was calculated from the adsorption average pore width (4V/A by BET) in this work.

·g<sup>−</sup><sup>1</sup> ) Pore volume (cm3 ·g<sup>−</sup><sup>1</sup> )

Pore size (nm)

surface area (m2

Adsorbent BET

ZCHC

ZCHC

#### *3.3.2. Adsorption of lead on zeolite and ZCHC*

**Activated carbon** *qe*

**Table 6.** Kinetic coefficients for Pb2+ adsorption on AC

desorption isotherms are shown in Table 7.

**3.3. The zeolite/chitosan hybrid material (ZCHC)**

*R*: the correlation coefficient.

72 Biomass Production and Uses

*3.3.1. Characterization of ZCHC*

**Adsorbent**

ZCHC (before adsorption)

ZCHC (after adsorption)

desorption experiment.

this work.

**Table 7.** Textural characteristics of ZCHC

**(mg·g−1)**

*q*e: the amount of adsorbed lead on the surface of activated carbon at equilibrium (mg g−1);

*k* : the rate constant of the pseudo-second-order adsorption (g mol−1 h−1);

AC0 26.4 2.87 × 10−3 0.999 ACK1 81.8 1.16 × 10−2 0.999 ACK3 101 4.51 × 10−2 0.999

The surface properties of ZCHC before and after Pb2+ adsorption were investigated by N2 adsorption (TriStar II 3020, Micromeritics), and the analytical results for the adsorption/

**·g−1)**

The pore volume was calculated from the amount of N2 adsorbed at the relative pressure of 0.99. The pore size was calculated from the adsorption average pore width (4V/A by BET) in

The SEM micrographs of the ZCHC before and after Pb2+ adsorption ZCHC are shown in Figure 14. From Figure 14, it is found that the morphology of ZCHC surface has hardly changed even after exposing Pb2+, although the SEM picture after Pb2+ adsorption slightly exhibits a more compact stacking morphology than that before adsorption. From the above observation, ZCHC should be predicted to withstand the repeated use, and hence it can be a good adsorbent for heavy metals such as Pb2+. These results are consistent with those of the N2 adsorption–

**Pore volume (cm3 ·g−1)**

9.25 0.0485 21.0

9.19 0.0479 20.7

**BET surface area (m2**

*k* **(g·mol−1·h−1)**

*R*

**Pore size (nm)**

**3.3.2. Adsorption of lead on zeolite and ZCHC**  With the aim of obtaining the optimum conditions, the effects of pH value and contact time in the case of a fixed dosage of adsorbent (i.e., 1.0 g·L<sup>−</sup><sup>1</sup> ) on the removal of Pb2+ from the aqueous solution were investigated. The effect of pH on Pb2+ adsorption onto zeolite and ZCHC is shown in **Figure 15**. From **Figure 15**, the uptake of Pb2+ increased from 56.7% at pH 3 to 95.6% at pH 5, and at higher pH With the aim of obtaining the optimum conditions, the effects of pH value and contact time in the case of a fixed dosage of adsorbent (i.e., 1.0 g L−1) on the removal of Pb2+ from the aqueous solution were investigated. The effect of pH on Pb2+ adsorption onto zeolite and ZCHC is shown in Figure 15. From Figure 15, the uptake of Pb2+ increased from 56.7% at pH 3 to 95.6% at pH 5, and at higher pH value, it remained almost constant (or decreased slightly). Notably, the adsorption capacities decreased at low pH values due to the competition of protons with metal ions for active binding. On the other hand, lead precipitated from the solution at higher pH values as lead hydroxide. Hence, pH 5 was used for further experiments.

20 The effect of contact time on Pb2+ adsorption onto zeolite and ZCHC is shown in Figure 16. Approximately 90% of Pb was removed from the solution with ZCHC at the contact time of 1 h, and it gradually increased at 4 h as shown in Figure 16. More than 95% of Pb was removed from the solution at the contact time of 4 h. After 4 h, there was no appreciable change. On the other hand, a contact time of 8 h was needed to attain at equilibrium in case of zeolite. Therefore, 8 h was chosen as the optimized contact time for more certainty for these samples at the rest of the experimental work.

#### *3.3.3. Adsorption isotherms*

Adsorption isotherms are commonly used to reflect the performance of adsorbents in adsorp‐ tion processes. The Langmuir adsorption isotherm was applied to the data obtained in this work. A plot of *C*e/*q*e versus *C*e based on the Langmuir model is shown in Figure 17.

As shown in Table 8, *R* value for each adsorbent was comparatively large, and a favorable adsorption of Pb2+ by these samples was apparent. This result suggested that the adsorption of these samples primarily occurred via a monolayer reaction. Furthermore, the maximum adsorption capacity of ZCHC toward Pb2+ was higher than that of zeolite.

**Figure 15.** Effect of pH on the removal of Pb2+ removal of using zeolite and ZCHC. Pb2+ using zeolite and ZCHC.

**Figure 16.** Effect of contact time on the

**Figure 17.** Langmuir isotherm of Pb2+ adsorption on zeolite and ZCHC.


*q*max: the maximum adsorption capacity (mg g−1);

*K*L: the Langmuir adsorption constant (L mg−1);

*R*: the correlation coefficient.

3 4 5 6 7

pH

Zeolite ZCHC

0 4 8 12 16 20 24 28 32 36 40 44 48

t (h)

**Figure 15.** Effect of pH on the removal of Pb2+ removal of using zeolite and ZCHC. Pb2+ using zeolite and ZCHC.

20

40

**Figure 16.** Effect of contact time on the

50

60

70

Adsorption capacity (%)

80

90

100

40

60

Adsorption capacity (%)

80

100

74 Biomass Production and Uses

Zeolite ZCHC

**Table 8.** Coefficient of Langmuir isotherm for Pb2+ using zeolite and ZCHC

#### **4. Conclusion**

Yukitsubaki carbon (charcoal) modified with nitric acid (10% and 30%), activated carbon modified with potassium permanganate (KMnO4; 0.01 mol/L and 0.03 mol/L), and zeolite/ chitosan hybrid composite (ZCHC) prepared with sol–gel method were used as adsorbents for heavy metal ions in this work. Modified carbon treated with HNO3 or KMnO4 exhibits high ability of chemical adsorption and stronger chemical affinity than pristine carbon. ZCHC prepared in this work also could be suitable as sorbent materials for the removal of heavy metals from aqueous solutions. The adsorbent showed excellent adsorption capacity for Pb2+ under our experimental condition, even in the presence of diverse ions (Ca2+, Mg2+, Na+ , and

K+ ) up to the concentration of 100 mgdm−3. It is indicated that these materials used in this work could be effective adsorbents for practical use in the future.

It is also suggested that the removal of U and Pb from aqueous solutions by these materials is mainly due to monolayer sorption because of well fitting for Langmuir model. Furthermore, the best fit was obtained with a pseudo-second-order kinetic model while investigating the adsorption kinetic of carbon modified with HNO3 or KMnO4.

The data obtained and the method used in this work can be a useful tool from the viewpoint of environmental protection and resource recovery in future work.

## **Acknowledgements**

The present work was partially supported by a Grant-in-Aid for Scientific Research (Research Program (C), No. 25340083) of the Japan Society for the Promotion of Science. This research was also supported by a fund for the promotion of Niigata University KAAB Projects from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

The present work was partially supported by a Grant-in-Aid for Scientific Research of the Japan Society for the Promotion of Science. Yukitsubaki carbon samples were supplied by Mr. J. Sakai of the Facility of Engineering in Niigata University. The authors are also grateful to Mr. N. Saito, Mr. T. Nomoto, Prof. T. Tanaka, Mr. T. Hatamachi, Dr. M. Teraguchi and Prof. T. Aoki of the Facility of Engineering in Niigata University for permitting the use of SEM, Surface Area Analyzer, and FT-IR, and thanks to Dr. K. Fujii and Mr. M. Ohizumi of the Office for Envi‐ ronment and Safety in Niigata University for permitting the use of ICP-MS and for providing helpful advice in measurement.

## **Author details**

Naoki Kano\*

Address all correspondence to: kano@eng.niigata-u.ac.jp

Department of Chemistry and Chemical Engineering, Faculty of Engineering, Niigata University, Nishi-Ku, Niigata, Japan

## **References**

[1] Dahiya S., Tripathi R. M., Hegde A. G. (2008) Biosorption of Heavy Metals and Radi‐ onuclide from Aqueous Solutions by Pre-treated Arca Shell Biomass. J. Hazard. Ma‐ ter., 150: 376–386.

[2] Sachdeva S., Kumar A. (2009) Preparation of nanoporous composite carbon mem‐ brane for separation of rhodamine B dye. J. Membr. Sci., 329: 2–10.

K+

76 Biomass Production and Uses

**Acknowledgements**

helpful advice in measurement.

University, Nishi-Ku, Niigata, Japan

ter., 150: 376–386.

**Author details**

Naoki Kano\*

**References**

) up to the concentration of 100 mgdm−3. It is indicated that these materials used in this work

It is also suggested that the removal of U and Pb from aqueous solutions by these materials is mainly due to monolayer sorption because of well fitting for Langmuir model. Furthermore, the best fit was obtained with a pseudo-second-order kinetic model while investigating the

The data obtained and the method used in this work can be a useful tool from the viewpoint

The present work was partially supported by a Grant-in-Aid for Scientific Research (Research Program (C), No. 25340083) of the Japan Society for the Promotion of Science. This research was also supported by a fund for the promotion of Niigata University KAAB Projects from the

The present work was partially supported by a Grant-in-Aid for Scientific Research of the Japan Society for the Promotion of Science. Yukitsubaki carbon samples were supplied by Mr. J. Sakai of the Facility of Engineering in Niigata University. The authors are also grateful to Mr. N. Saito, Mr. T. Nomoto, Prof. T. Tanaka, Mr. T. Hatamachi, Dr. M. Teraguchi and Prof. T. Aoki of the Facility of Engineering in Niigata University for permitting the use of SEM, Surface Area Analyzer, and FT-IR, and thanks to Dr. K. Fujii and Mr. M. Ohizumi of the Office for Envi‐ ronment and Safety in Niigata University for permitting the use of ICP-MS and for providing

Department of Chemistry and Chemical Engineering, Faculty of Engineering, Niigata

[1] Dahiya S., Tripathi R. M., Hegde A. G. (2008) Biosorption of Heavy Metals and Radi‐ onuclide from Aqueous Solutions by Pre-treated Arca Shell Biomass. J. Hazard. Ma‐

could be effective adsorbents for practical use in the future.

adsorption kinetic of carbon modified with HNO3 or KMnO4.

of environmental protection and resource recovery in future work.

Ministry of Education, Culture, Sports, Science and Technology, Japan.

Address all correspondence to: kano@eng.niigata-u.ac.jp


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## **Microalgal Biorefineries**

Eduardo Jacob-Lopes, Luis Guillermo Ramírez Mérida, Maria Isabel Queiroz and Leila Q. Zepka

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59969

## **1. Introduction**

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80 Biomass Production and Uses

The world has been confronted with a food and energy crisis due to accelerated global population growth and the depletion of finite fossil fuel resources. The increase in nutritional problems along with rising fuel demands and environmental problems have necessitated the search for nutritional supplements and sustainable sources of energy. Currently, fossil fuel resources are not regarded as sustainable and their continued consumption is raising serious ecological, economic and environmental questions. However, while we move towards alternative sources of energy, there remains a need to replace fossil fuels with high energy density fuels. A highly contentious issue of great concern is the argument that emissions of carbon dioxide (CO2) from fossil fuel use, especially from coal combustion, are responsible for global climate change. As a result of studies during the past five decades, and most notably from the last 20 years, emissions of CO2 have become an important issue with respect to global climate change because atmospheric CO2 concentrations increased significantly in the last century and have continued to rise at an increasing rate [1].

The United Nations Kyoto Protocol of 1997 established regulations designed to control emissions of air pollutants with the objective of reducing greenhouse gases to the level of emissions in 1990, and more than 170 countries have ratified the protocol [2].

Various CO2 sequestration techniques have been developed and the various technologies for CO2 capture and storage need to be evaluated from the point of view of obtaining carbon credits, aimed at stabilizing emissions of the pollutant [2]. Of these techniques, CO2 capture by photosynthetic organisms such as microalgae shows good potential in view of the economic advantages it presents, rate of CO2 capture, and the speed with which the technology can be introduced to the industrial community.

© 2015 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.

Microalgae have great potential in generating energy from biotechnological processes using renewable sources and without compromising food security and agriculture. Microalgae have been of major interest in biofuel production as well as in the feed, chemical and pharmaceutical sectors [3]. Depending on the species and growth conditions, microalgae can be selected to produce a wide variety and abundance of lipids, proteins, carbohydrates, and feedstocks important for biofuel and production of nutraceuticals [3].

The rapid growth rate coupled with high productivity from a small area means that the production of microalgal biomass has a promising future [4].

Several investigations into the use of microalgae to obtain bioproducts have been successfully conducted. Upstream processing (USP) and downstream processing (DSP) are stages found in the processes of microalgae biorefineries (Fig. 1). USP involve four important areas: (i) microalgae strain, (ii) CO2 supply, (iii) nutrient source <nitrogen/phosphorus> and (iv) source of illumination [5].

Photobioreactors used for the culture of microalgae are of two basic designs — open or closed systems. Amongst the different types of open system design, the most popular is the raceway pond, while popular closed systems include flat-plate, vertical tubular, horizontal tubular and hybrid type photobioreactors. Growth of microalgae in photobioreac‐ tors occurs due to the use of CO2 rich gas as a means of mixing, as well as being a source of carbon. Generally, in this type of reactor, the agitation, mass transfer, efficient provi‐ sion of light, removal of photosynthetically generated oxygen, understanding of hydrody‐ namic aspects, and scalable photobioreactor technology are aspects that should be taken into account to achieve good yields [6,7].

Conventional DSP includes all unit processes that follow the process taking place within the photobioreactor. They involve biomass harvesting and biorefinery techniques which facilitate the integration of the biomass conversion processes and equipment for the production of several fractions of interest through the use of mild separation technology. Biorefining involves assessment and use of different technologies to obtain different types of bioproducts from biomass, which can be marketed and used to solve specific problems in many different areas. Finally, there must be the safe and inexpensive disposal of all waste products generated during the process. Therefore, a portion of the residual biomass can go to an anaerobic digester to generate biogas, and the rest can be used as nutrients to feed the photobioreactor again.

As such, the aim of this chapter is to present an overview of the potential uses of the technology in the transformation of carbon dioxide into biomolecules, and to describe the processes involved in the biological conversion of CO2 in photobioreactors as well as biorefinery techniques suitable for the treatment of microalgal biomass and the produc‐ tion of biomolecules.

## **2. Carbon dioxide emissions and mitigation**

Climate change occurs mainly due to increased levels of CO2 in the atmosphere. During the twentieth century an increase in CO2 concentration of 30% was observed. This rate of increase

biorefinery techniques suitable for the treatment of microalgal biomass and the production of

Photobioreactors used for the culture of microalgae are of two basic designs open or closed systems. Amongst the different types of open system design, the most popular is the raceway pond, while popular closed systems include flat-plate, vertical tubular, horizontal tubular and hybrid type photobioreactors. Growth of microalgae in photobioreactors occurs due to the use of CO2 rich gas as a means of mixing, as well as being a source of carbon. Generally, in this type of reactor, the agitation, mass transfer, efficient provision of light, removal of photosynthetically generated oxygen, understanding of hydrodynamic aspects, and scalable photobioreactor technology are aspects that should be

Conventional DSP includes all unit processes that follow the process taking place within the photobioreactor. They involve biomass harvesting and biorefinery techniques which facilitate the integration of the biomass conversion processes and equipment for the production of several fractions of interest through the use of mild separation technology. Biorefining involves assessment and use of different technologies to obtain different types of bioproducts from biomass, which can be marketed and used to solve specific problems in many different areas. Finally, there must be the safe and inexpensive disposal of all waste products generated during the process. Therefore, a portion of the residual biomass can go to an anaerobic digester to generate biogas, and the rest can be used as nutrients to feed

**Figure 1.** Outline of the formation process of microalgal biomass and bioproducts.

taken into account to achieve good yields [6,7].

the photobioreactor again.

biomolecules.

Microalgae have great potential in generating energy from biotechnological processes using renewable sources and without compromising food security and agriculture. Microalgae have been of major interest in biofuel production as well as in the feed, chemical and pharmaceutical sectors [3]. Depending on the species and growth conditions, microalgae can be selected to produce a wide variety and abundance of lipids, proteins, carbohydrates, and feedstocks

The rapid growth rate coupled with high productivity from a small area means that the

Several investigations into the use of microalgae to obtain bioproducts have been successfully conducted. Upstream processing (USP) and downstream processing (DSP) are stages found in the processes of microalgae biorefineries (Fig. 1). USP involve four important areas: (i) microalgae strain, (ii) CO2 supply, (iii) nutrient source <nitrogen/phosphorus> and (iv) source

Photobioreactors used for the culture of microalgae are of two basic designs — open or closed systems. Amongst the different types of open system design, the most popular is the raceway pond, while popular closed systems include flat-plate, vertical tubular, horizontal tubular and hybrid type photobioreactors. Growth of microalgae in photobioreac‐ tors occurs due to the use of CO2 rich gas as a means of mixing, as well as being a source of carbon. Generally, in this type of reactor, the agitation, mass transfer, efficient provi‐ sion of light, removal of photosynthetically generated oxygen, understanding of hydrody‐ namic aspects, and scalable photobioreactor technology are aspects that should be taken

Conventional DSP includes all unit processes that follow the process taking place within the photobioreactor. They involve biomass harvesting and biorefinery techniques which facilitate the integration of the biomass conversion processes and equipment for the production of several fractions of interest through the use of mild separation technology. Biorefining involves assessment and use of different technologies to obtain different types of bioproducts from biomass, which can be marketed and used to solve specific problems in many different areas. Finally, there must be the safe and inexpensive disposal of all waste products generated during the process. Therefore, a portion of the residual biomass can go to an anaerobic digester to generate biogas, and the rest can be used as nutrients to feed the photobioreactor again.

As such, the aim of this chapter is to present an overview of the potential uses of the technology in the transformation of carbon dioxide into biomolecules, and to describe the processes involved in the biological conversion of CO2 in photobioreactors as well as biorefinery techniques suitable for the treatment of microalgal biomass and the produc‐

Climate change occurs mainly due to increased levels of CO2 in the atmosphere. During the twentieth century an increase in CO2 concentration of 30% was observed. This rate of increase

important for biofuel and production of nutraceuticals [3].

production of microalgal biomass has a promising future [4].

of illumination [5].

82 Biomass Production and Uses

tion of biomolecules.

into account to achieve good yields [6,7].

**2. Carbon dioxide emissions and mitigation**

will lead to an increase in CO2 levels of 49% by the end of this century [8]. CO2 emissions from fossil fuel combustion saw an increase of 41% between 1990 and 2008 [9]. Three potential sources of CO2 can be found: stationary, mobile and natural. CO2 is the primary greenhouse gas emitted through human activities. Stationary sources contribute the highest percentage of CO2 emissions of these, and are anthropogenic in origin, such as from industrial or domestic processes. The industrial processes contributing to increasing atmospheric CO2 concentrations consist of hydrogen and ammonia production plants, power stations, cement companies, ethanol companies, and chemical factories. Flue gases from power plants are responsible for more than 8% of the world's total CO2 emissions. Mobile sources are those from transport, while natural sources include volcanoes and elements of human or animal decomposition [10].

A number of research and development efforts have been directed at reducing CO2 emissions. Many of these studies use different microalgae strains or new photobioreactors with geometric configurations that may be a fundamental step forward for the consolida‐ tion of this technology [11-14].

Biological methods for CO2 mitigation can be carried out by photosynthetic microorganisms such as microalgae and plants, the latter with an estimate for CO2 capture of only 3–6% of fossil fuel emissions [15]. Biomitigation using microalgae as a method of decreasing CO2 emissions by fixing CO2 through photosynthesis is considered one of the most effective. Generally, microalgae have higher growth rates, a higher CO2 fixation efficiency and a larger production of biomass enabling the subsequent development of quantities of bioproducts with high added value [16].

However, much the flue gas of industrial origin, in addition to contributing to CO2 emissions, produces other compounds, including oxygen (O2), water vapour, carbon monoxide (CO), nitrogen oxides (NOx), sulfur oxides (SOx), hydrochloric acid, heavy metals, and particulate matter [17]. These compounds have a toxic and/or inhibitory effect on microalgal growth. Tolerance of microalgae to elements of flue gas is dependent on the strain. NOx present in flue gas can be taken up and could become an alternative nitrogen source for the growth of microalgae growth. The main impact is due to SOx which reacts with water to form sulfurous acids. This can be prevented by buffering or active pH control [18].

Besides high amounts of NOx and SOx, the high temperature influences the growth of microalgae. Industrial plants discharge flue gas at a temperature between 70–120 °C [19]. Therefore, to complete the CO2 capture process, it is necessary either to install a post-cooling system, or to use thermophilic species [18]. Additionally, oxidant compounds found in flue gas can cause damage to proteins and pigments, and compromise the integrity of cell mem‐ branes [20].

Besides the use of CO2 from industrial flue gas, other alternative sources are known such as ethanol production facilities, winegrowing, ammonia and hydrogen production or gasprocessing plants. The capture of CO2 from the fermentation process is relatively simple and cheap due to the higher state of purity in which it is present [21].


**Table 1.** Global companies with CO2 sequestering technology for algae culture.

Table 1 shows some global companies which employ CO2 sequestering technologies for the production of biofuel and/or bioproducts from algal cultures. Furthermore, many companies and research centers worldwide are investigating the upstream and/or downstream process.

## **3. Microalgae strains and photosynthetic metabolism**

nitrogen oxides (NOx), sulfur oxides (SOx), hydrochloric acid, heavy metals, and particulate matter [17]. These compounds have a toxic and/or inhibitory effect on microalgal growth. Tolerance of microalgae to elements of flue gas is dependent on the strain. NOx present in flue gas can be taken up and could become an alternative nitrogen source for the growth of microalgae growth. The main impact is due to SOx which reacts with water to form sulfurous

Besides high amounts of NOx and SOx, the high temperature influences the growth of microalgae. Industrial plants discharge flue gas at a temperature between 70–120 °C [19]. Therefore, to complete the CO2 capture process, it is necessary either to install a post-cooling system, or to use thermophilic species [18]. Additionally, oxidant compounds found in flue gas can cause damage to proteins and pigments, and compromise the integrity of cell mem‐

Besides the use of CO2 from industrial flue gas, other alternative sources are known such as ethanol production facilities, winegrowing, ammonia and hydrogen production or gasprocessing plants. The capture of CO2 from the fermentation process is relatively simple and

**Algae companies Country Description Ref**

Founded in 2010. This company uses algae that are fed by nutrients recovered from wastewater treatment plants, electricity generation, and sunlight. Biodiesel, the main product produced in these biorefineries is cost competitive with petroleum products.

Uses CO2 sequestered from industrial facilities and power plants for conversion into renewable fuels and other valuable products such as food additives.

Founded in 2003. The company aims to develop microalgae biomass for the production of food additives and biofuel using flue gas from coal burning power stations.

This company develops biomass production methods with CO2 capture from winegrowing for production of oil, nutraceuticals, food additives and biochemical compounds.

Founded in 1960. IGV Biotech develops microalgae biotechnology processes for the production of several products such as food, pharmaceuticals and chemicals. This company uses advanced technology for the cultivation of photosynthetic microorganisms and CO2 capture.

Founded in 2006. The company uses CO2 and seawater as a culture medium for bioethanol. Nitrogen fixing technology is used to reduce production costs of fertilizers by cyanobacteria.

[22]

[23]

[24]

[25]

[26]

[27]

acids. This can be prevented by buffering or active pH control [18].

cheap due to the higher state of purity in which it is present [21].

AFS BioOil Co. San Francisco, USA

AFS Biofarm™ San Francisco, USA

Seambiotic Ltd. Tel Aviv, Israel

IGV Biotech Nuthetal, Germany

Algenol Biofuels Florida, USA

**Table 1.** Global companies with CO2 sequestering technology for algae culture.

Aeon Biogroup Chile

branes [20].

84 Biomass Production and Uses

Microalgae are fast growing photosynthetic microorganisms that produce valuable com‐ pounds, are easy to harvest, exhibit a unicellular or simple multicellular structure, and a large surface-to-volume body ratio. Eukaryotic microalgae such as green algae (*Chlorophyta*) and diatoms (*Bacillariophyta*) as well as prokaryotes like cyanobacteria species (*Cyanophyceae*) use oxygenic photosynthesis to fix CO2 like macroalgae and plants [18].

CO2 fixation, biomass production and bioproduct diversity vary with microalgal species, although the data may not be strictly comparable as the microalgae may have different biological behavior or may have been cultured in different conditions. The general chemical composition of different microalgal species varies, with some species having greater potential for the production of certain bioproducts [28]. Microalgae have a varied biochemical profile (Table 2). The high protein content of microalgae species is notable. These proteins, mainly amino acids, provide nutritional elements that can meet food requirements in humans and animals.

Consequently, a successful and economically viable microalgae industry producing bioprod‐ ucts mainly depends on the selection of appropriate microalgae strains.

Microalgae comprise a diversity of species characterized by a variety of phenotypes dependent on their pigments and cell structure. Chlorophyll-*a* and phycobiliproteins may be present and are involved in harvesting light energy for photosynthesis. They are, therefore, a good choice for the generation of biomass. In addition to photosynthesis, some species show an ability to adapt to different environments and metabolisms such as respiration and nitrogen fixation, chromatic adaptation and the ability to form symbiotic associations with yeast, fungi, bacteria and plants [31]. The part of the photosynthetic process in which CO2 is converted into carbohydrates is catalyzed by the carboxylase activity of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), this step is called the Calvin cycle [32]. The Calvin cycle is the metabolic mechanism for CO2 fixation in microalgae and the process comprises three phases: carboxylation, reduction and regeneration. The photosystem II (PSII) complex is the starting point of photosynthesis, where via the electron transport chain, an electron is trans‐ ferred to cytochrome b6f and PSI. A proton-motive force is created due to the pumping of electrons in opposite directions, creating a difference in charge across the membrane. This is used for ATP synthesis and the formation of ferredoxin and NADPH. The electron is donated by water and oxygen is formed as a waste product [33,34]. To generate one molecule of carbohydrate (CH2O), O2 and H2, at least eight (8) photons are needed. The mean energy content for photosynthetically active radiation (PAR) photons is close to 220 kJ/mol and the total potential light energy captured by photosynthesis is 1744 kJ/mol of CH2O. The theoretical


**Table 2.** Chemical composition of different microalgae expressed on a dry matter basis (%).

maximum efficiency for the conversion of light to ATP is approximately 27%. However, only 42.3% of PAR can be utilized [35]. Furthermore, light intensity and light quality also play a key role in the growth rate of the cell [36].Inorganic and organic carbon represent one of the main sources of nutrition for microalgae. Different microalgae species can be maintained at various concentrations of CO2 due to a mechanism called the carbon concentration mechanism (CCM), which accumulates inorganic carbon, concentrating it in the CO2 in RuBisCO [37].

Microalgae can assimilate carbon through three routes: (i) direct absorption of CO2 by the cell membrane; (ii) the use of bicarbonate by inducing the enzyme carbonic anhydrase and (iii) transport of HCO3 by the cell membrane. The enzyme carbonic anhydrase catalyzes the reaction converting HCO3 into CO2, moreover RuBisCO uses CO2 as the substrate on which it forms phosphoglycerate. Limitations to CO2 production can occur, slowing the rate of reaction. Thus, carbonic anhydrase is a very efficient enzyme that can generate high concentrations of CO2 [38–40].

## **4. Requirements of microalgae biorefineries**

In addition to CO2, other nutrients such as phosphorus, nitrogen and trace metals are important for the production of microalgae. These provides the necessary conditions for microalgae to carry out the metabolic reactions necessary for growth and so generate biomass or primary metabolites [41]. Most microalgae species can utilize inorganic and organic nitrogen sources. Ammonium salts, ammonium sulfate, diammonium hydrogen phosphate and ammonia are supplied as inorganic nitrogen sources, and urea is supplied as an organic nitrogen source [42– 43]. Phosphorus is supplied as hydrogen phosphate and dihydric phosphate in small amounts. Normally, sufficient quantities of minerals such as cobalt, copper, iron, molybdenum, man‐ ganese and zinc are present in the water supply, or they may be added as specific salts [44]. An adequate supply of nutrients is a prerequisite for high production rates. The introduction of certain nutrient stresses may affect the biochemistry. For example, nitrogen stress is important for carotenogenesis in *Dunaliella salina* [45] and increased lipid production in *Chlorella vulgaris* [46].

The most important factor in CO2 fixation and microalgae biomass production is light intensity. Light sources can be divided into natural sunlight, which is applied in both open and closed cultivation, and artificial cold light that is mainly applied in closed cultivation. Various studies have been performed on the use of artificial light. Many lamps are available commercially such as light emitting diodes (LED), fluorescent tubes, halogen, tungsten, and high intensity discharge lamps (HID), and optical fibers. The investment costs, shelf life and stability of light intensity are important factors to consider when choosing a lighting source [47]. Recommend‐ ed light sources for microalgae cultivation include the following: In laboratory research fluorescent tubes exhibiting a PAR efficiency of 1.25 μmol-ph s−1 W−1 are used, while HID with a PAR efficiency of 1.87 μmol-ph s−1W−1 are the most commonly employed in horticulture, along with LED lamps with a PAR efficiency of 1.91 μmol-phs−1W−1 [47]. The same type of lamp can emit different wavelengths, with blue LED and red LED having an adsorption at around 450–470 nm and 645–665 nm respectively [48]. Wang et al. [49] found the highest biomass yield using red LED in *Spirulina platensis* cultivation [49]. An analysis of Table 3 demonstrates a variety of possible sources of illumination for use in microalgae production.

The rate of photosynthesis is proportional to light intensity. When irradiance is increased, microalgal growth rate accelerates, but exposure of cells to long periods of high light intensity causes photoinhibition. Microalgae can only utilize the energy available in the 400–700 nm wavelength range, represented by PAR [50]. Moreover, several studies reported that the optimal wavelength varied from species to species [51]. Light intensities of 100 and 200 μE/m2 /s are frequently used [52].

maximum efficiency for the conversion of light to ATP is approximately 27%. However, only 42.3% of PAR can be utilized [35]. Furthermore, light intensity and light quality also play a key role in the growth rate of the cell [36].Inorganic and organic carbon represent one of the main sources of nutrition for microalgae. Different microalgae species can be maintained at various concentrations of CO2 due to a mechanism called the carbon concentration mechanism (CCM),

**Microalgae species Protein lipid Carbohydrate Nucleic acid Ref.** *Anabaena cylindrical* 43–56 4–7 25–30 – [28]

*Arthrospira maxima* 60–71 6–7 13–16 – [28] *Chlamydomonas rheinhardii* 48 21 17 – [28] *Chlorella pyrenoidosa* 57 2 26 4–5 [28] *Chlorella vulgaris* 51–58 14–22 12–17 4–5 [28] *Dunaliella bioculata* 49 8 4 – [30] *Dunaliella salina* 57 6 32 – [30] *Euglena gracilis* 39–61 14–20 14–18 – [28] *Porphyridium cruentum* 28–39 9–14 40–57 – [30] *Prymnesium parvum* 28–45 22–38 25–33 1–2 [30] *Scenedesmus dimorphus* 8–18 16–40 21–52 3–6 [28] *Scenedesmus obliquus* 50–56 12–14 10–17 3–6 [28] *Spirogyra* sp*.* 6–20 11–21 33–64 – [28] *Spirulina maxima* 60–71 6–7 13–16 2–5 [28] *Spirulina platensis* 46–63 4–9 8–14 2–5 [28] *Synechoccus* sp*.* 63 11 15 5 [30]

41–49 8–9 13–18 3–4 [29]

Microalgae can assimilate carbon through three routes: (i) direct absorption of CO2 by the cell membrane; (ii) the use of bicarbonate by inducing the enzyme carbonic anhydrase and (iii)

forms phosphoglycerate. Limitations to CO2 production can occur, slowing the rate of reaction. Thus, carbonic anhydrase is a very efficient enzyme that can generate high concentrations of

In addition to CO2, other nutrients such as phosphorus, nitrogen and trace metals are important for the production of microalgae. These provides the necessary conditions for microalgae to carry out the metabolic reactions necessary for growth and so generate biomass or primary metabolites [41]. Most microalgae species can utilize inorganic and organic nitrogen sources.

by the cell membrane. The enzyme carbonic anhydrase catalyzes the

into CO2, moreover RuBisCO uses CO2 as the substrate on which it

which accumulates inorganic carbon, concentrating it in the CO2 in RuBisCO [37].

**Table 2.** Chemical composition of different microalgae expressed on a dry matter basis (%).

transport of HCO3

*Aphanothece microscopica*

86 Biomass Production and Uses

*Nägeli*

CO2 [38–40].

reaction converting HCO3



**4. Requirements of microalgae biorefineries**

Besides light intensity, it was found that light–dark cycles could also significantly influence microalgal growth by avoiding sustained exposure to high photon flux density and providing dark time for microalgae to repair photo-induced damage [53].

The light–dark periods for most microalgal cultivation are 24h:0h,16h:8h and 12h:12h. This varies with microalgal species. Experiments carried out with *Aphanothece microscopica Nägeli* in photoperiods of (22:2), showed that growth rate is not conditional on incident lighting over 22 h. This provides evidence that *Aphanothece microscopica Nägeli* have the possibility of storing energy for their biochemical processes, without affecting the rate of photosynthet‐ ic metabolism [54].

On the one hand, the light intensity of natural sunlight is cheaper, but the light cycle depends on weather and latitude, which often preclude higher biomass production. At the same time, artificial illumination is generally expensive, but the control of light intensity afforded is excellent, allowing for greater flexibility and constant biomass production [55].


**Table 3.** Characteristics and power consumption for different sources of artificial light

A photobioreactor is a device consisting of an illuminated culture vessel designed for the controlled bioconversion of CO2 into biomass and bioproducts. The two basic types of photobioreactors used for the large scale culture of microalgae are open or closed systems. Open systems can be built more easily, are more economical and relatively simple to control in relation to closed systems. Most open systems are natural lakes or open ponds. Two types of open systems are known: (i) circular ponds stirred with a rotating arm and (ii) raceway ponds, which are shallow artificial ponds divided into a rectangular grid with paddle wheels for culture mixing. Raceway ponds are the most popular open system design [58].

The reactor surface is illuminated with natural light and the intensity of illumination affects the microalgae culture. The depth of this type of reactor may not exceed 35–40 cm so that it does not prevent the passage of light to the bottom of reactor. The reactor performance declines with increasing depth due to the fact that diminishing amounts of light energy are available. Moreover, the use of an open system for the sole purpose of CO2 sequestration is mitigated by the very low residence time of gas in the culture, which therefore offers a short time in which the fixing of CO2 from flue gases by the microalgae can occur. Open systems produce low yields of products with high added value due to contamination problems [55]. Closed systems support high yields of microalgae biomass and they have certain advantages with regard to minimizing contamination, allowing axenic microalgal cultivation, providing a control system for various parameters such as pH, temperature, light, and CO2 concentration. They also reduce CO2 losses, prevent water evaporation, allow for a greater control of biomass growth, and permit the production of complex biomolecules. Closed systems are currently being assessed for microalgae cultivation in configurations such as flat-plate, vertical tubular, horizontal tubular and hybrid type photobioreactors [59].

artificial illumination is generally expensive, but the control of light intensity afforded is

**Electrical**

**consumption Stability**

**Investment cost**

High High Low Moderate [47]

Low High Low High [47,56]

Low High Low Moderate [47]

High Moderate High High [57]

Absent Low Low High [55]

**Weather durability**

**Ref.**

excellent, allowing for greater flexibility and constant biomass production [55].

**Light source Commentary**

LED lamp Low heat generation, Greater

High productivity of biomass, large area lighting, generation of high temperature

resistance to on/off cycles

Generation of high temperature, high efficiency. However, losses from trapped light in protective covers and lenses, inefficient ballasts

Small space requirements for installation, good light distribution, uniformity of illumination, low risk of contamination

Variable biomass productivity depending on weather conditions, good lighting area, economic and adequate light distribution

**Table 3.** Characteristics and power consumption for different sources of artificial light

A photobioreactor is a device consisting of an illuminated culture vessel designed for the controlled bioconversion of CO2 into biomass and bioproducts. The two basic types of photobioreactors used for the large scale culture of microalgae are open or closed systems. Open systems can be built more easily, are more economical and relatively simple to control in relation to closed systems. Most open systems are natural lakes or open ponds. Two types of open systems are known: (i) circular ponds stirred with a rotating arm and (ii) raceway ponds, which are shallow artificial ponds divided into a rectangular grid with paddle wheels

The reactor surface is illuminated with natural light and the intensity of illumination affects the microalgae culture. The depth of this type of reactor may not exceed 35–40 cm so that it does not prevent the passage of light to the bottom of reactor. The reactor performance declines with increasing depth due to the fact that diminishing amounts of light energy are available.

for culture mixing. Raceway ponds are the most popular open system design [58].

Conventional lamp (halogen, tungsten, fluorescent)

88 Biomass Production and Uses

HID lamp

Optical fiber excited by lamps

> Natural sunlight

Flat-plate and tubular photobioreactors are the commonest types used for cultivation of microalgae in the laboratory and on a pilot scale. These photobioreactors are based on the same principles of a large surface-to-volume (S/V) ratio, optimal use of CO2 and suitable mixing. Tubular photobioreactors (airlift or bubble column) seem the most suitable for CO2 seques‐ tration due to their homogeneous mixture, greater gas transfer, smaller hydrodynamic stress, ease of construct and high productive output. Flat-plate photobioreactors are very expensive to build, which makes them unfeasible for industrial use. A hybrid photobioreactor is a combination of at least two types of different photobioreactor. Usually, integrating a horizontal tubular photobioreactor with a vertical tubular photobioreactor will compensate for the drawbacks in scale-up and the enhanced S/V ratio of vertical tubular photobioreactors. There are many configurations that have been studied, producing good results [55].

Photobioreactor development is perhaps one of the major steps that should be undertaken for the efficient large-scale cultivation of microalgae and bioproduct formation. Shape consider‐ ations must be taken into account when installing a system to produce bioproducts. Closed reactors are best for production of compounds of high added value.

There is a complex CO2 transfer process in a photobioreactor. In the gas aerating method, mass transfer performance and biochemical reaction rate depend of the type and size of the photobioreactor, the range of operational conditions, the influence of physicochemical properties on hydrodynamics due to the high viscosity of the liquid, its rheological behavior, the measuring method used, bubble size, gas hold-up, the gas/liquid contact area, and CO2 concentration and gas/liquid ratio [55].

In terms of solubility, oxygen is less soluble in water than CO2. However, both gases are poorly soluble in aqueous solution so there is a need for the provision of these elements throughout the process. CO2 bubbling in solution alone does not produce complete dissolution, since a fraction of the CO2 injected is lost in the gas outlet. The chemical reactivity of CO2 in the water forms H2CO3. The pH decreases with increasing insolubility and carbonic acid formation. The H2CO3 dissociates to HCO3 and CO3 2-. Consequently, the total inorganic carbon concentration is represented by the totality of the compounds CO3 2-, HCO3 and CO2 [4].

The volumetric mass transfer coefficient (KLa) is the property of the photobioreactor that determines the appropriate conditions that will ensure cell growth in the reactor. KLa repre‐ sents a function of microalgal characteristics and operating conditions. Efficiency of CO2 transfer is necessary to increase the KLa of CO2 allowing for improved transfer of gas to the liquid phase. Photobioreactors require an efficient CO2 transfer system [4].

During microalgae growth in the photobioreactor, the accumulation of O2 can occur. The water dissociation activity of PSII is responsible for the oxygen produced during photosynthesis. The increase of O2 in the culture medium is a hard problem to solve. The level of dissolved O2 in the culture medium causes photoinhibition reducing photosynthetic efficiency. Accumulation of O2 becomes a complicated problem in a closed photobioreactor when the reactor configu‐ ration does not provide an interface between the culture and the surrounding atmosphere, in contrast to horizontal tubular reactors or a flat panel configuration. The solutions proposed to date rely on the installation of a degasser. In photobioreactors, degassing is only necessary when O2 production (due to basal activity of PSII) is higher than respiration [60].

## **5. Microalgal biomass harvesting technologies**

When the biochemical process in the photobioreactor have finished, the upstream processing ends and gives way to downstream processing and harvesting of the biomass and refining of the bioproducts in the biorefinery. As a result, most of the production costs in microalgae biorefineries are influenced by DSP. The microalgal harvest and reduction of its water content does not depend on a single method. Efficient and profitable harvesting methods are required to process the biomass and bioproducts economically [61]. The microalgae recovery techniques represent between 20–30% of total production costs. Of harvesting techniques, the most commonly used are flocculation, filtration, centrifugation, gravity sedimentation and flotation [62]. Some factors influencing the choice of harvesting technique are morphology, density and size of the microalgae, as well as the type and quality of product to be obtained [35].

Flocculation is a process in which particles are dispersed from the medium by using chemicals to aggregate the microalgal cells. Flocculants stimulate flocculation by causing colloids and other suspended particles in solution to form flocs. Chemical flocculants regularly used to harvest microalgae include ferric chloride, aluminum sulfate, alum, ferric sulfate, polyferric sulfate, as well as cationic polymers (polyelectrolytes), and organic flocculants (chitosan). Researchers have developed a process of cell autoflocculation, through the adjustment of pH in the microalgae culture [63].

Filtration operated under pressure or in a vacuum is satisfactory for recovering relatively large (>70mm) and/or filamentous microalgae, but is less effective in separating microalgae species with dimensions close to those of prokaryotes. Membrane microfiltration and ultrafiltration processes may be an option for the recovery of microalgae biomass under 30 mm [63]. Petrusevski [64] recovered 70–89% of microalgae using cross flow filtration with the advantage of maintaining the integrity of the microalgae biomass. On the other hand, use of a chambermembrane filter press could achieve a concentration factor of 245 times the original concen‐ tration for *Coelastrum proboscideum* and produce a sludge with 27% solids [65]. Filtration is an expensive process due to membrane exchange and pumping. At larger scales of production (>20 m3 per day) other methods can be cheaper. For the processing of small volumes (e.g., < 2 m3 per day) it can be more cost effective when compared with centrifugation [66].

sents a function of microalgal characteristics and operating conditions. Efficiency of CO2 transfer is necessary to increase the KLa of CO2 allowing for improved transfer of gas to the

During microalgae growth in the photobioreactor, the accumulation of O2 can occur. The water dissociation activity of PSII is responsible for the oxygen produced during photosynthesis. The increase of O2 in the culture medium is a hard problem to solve. The level of dissolved O2 in the culture medium causes photoinhibition reducing photosynthetic efficiency. Accumulation of O2 becomes a complicated problem in a closed photobioreactor when the reactor configu‐ ration does not provide an interface between the culture and the surrounding atmosphere, in contrast to horizontal tubular reactors or a flat panel configuration. The solutions proposed to date rely on the installation of a degasser. In photobioreactors, degassing is only necessary

When the biochemical process in the photobioreactor have finished, the upstream processing ends and gives way to downstream processing and harvesting of the biomass and refining of the bioproducts in the biorefinery. As a result, most of the production costs in microalgae biorefineries are influenced by DSP. The microalgal harvest and reduction of its water content does not depend on a single method. Efficient and profitable harvesting methods are required to process the biomass and bioproducts economically [61]. The microalgae recovery techniques represent between 20–30% of total production costs. Of harvesting techniques, the most commonly used are flocculation, filtration, centrifugation, gravity sedimentation and flotation [62]. Some factors influencing the choice of harvesting technique are morphology, density and

size of the microalgae, as well as the type and quality of product to be obtained [35].

Flocculation is a process in which particles are dispersed from the medium by using chemicals to aggregate the microalgal cells. Flocculants stimulate flocculation by causing colloids and other suspended particles in solution to form flocs. Chemical flocculants regularly used to harvest microalgae include ferric chloride, aluminum sulfate, alum, ferric sulfate, polyferric sulfate, as well as cationic polymers (polyelectrolytes), and organic flocculants (chitosan). Researchers have developed a process of cell autoflocculation, through the adjustment of pH

Filtration operated under pressure or in a vacuum is satisfactory for recovering relatively large (>70mm) and/or filamentous microalgae, but is less effective in separating microalgae species with dimensions close to those of prokaryotes. Membrane microfiltration and ultrafiltration processes may be an option for the recovery of microalgae biomass under 30 mm [63]. Petrusevski [64] recovered 70–89% of microalgae using cross flow filtration with the advantage of maintaining the integrity of the microalgae biomass. On the other hand, use of a chambermembrane filter press could achieve a concentration factor of 245 times the original concen‐ tration for *Coelastrum proboscideum* and produce a sludge with 27% solids [65]. Filtration is an expensive process due to membrane exchange and pumping. At larger scales of production

liquid phase. Photobioreactors require an efficient CO2 transfer system [4].

when O2 production (due to basal activity of PSII) is higher than respiration [60].

**5. Microalgal biomass harvesting technologies**

in the microalgae culture [63].

90 Biomass Production and Uses

Centrifugation is a methodology that includes the use of centrifugal acceleration for the sedimentation of microalgae in heterogeneous mixtures. The size and density of the structure determines the centrifugal separation of element. The supernatant is a liquid located in the upper layer of the centrifuge tube and the microalgae concentrate is represented by the remnant solid. The fast and intensive process depends on the sedimentation of cells for biomass recovery, as well as the amount of time of the cell suspension is in the centrifuge, and settling depth [63]. This method can lead to cell injury due to the gravitational and shear forces encountered [67]. Many researchers have recommended this method for the reliable recovery of microalgae [68–70]. Recovery by centrifugation is an efficient method when used with small volumes of fluid and high energy consumption. The drawbacks of centrifugation are the high initial investment costs, the noise generated during operation, and the cost of the electricity used [71].

Gravitational sedimentation is widely used to separate microalgae in aqueous solution and for wastewater treatment. The sedimentation rates of microalgae are influenced by the rate of sedimentation of solids and are determined by the density and area of the microalgae cells [35]. Gravitational sedimentation, preceded by flocculation, is one of the most widely-used techniques for the harvesting of microalgae biomass. Disadvantages of the method are that is very slow (0.1 to 2.6 m h-1) and the biomass may suffer decomposition under conditions of high temperature. Furthermore, the technique is suited for use with large microalgae or those with a filamentous morphology [72].

Flotation methods are based on the binding of microalgae cells using air bubbles. The resulting flocs rise to the surface of the liquid and are recovered by either physical or chemical proce‐ dures. Particles as small as 500 μm can be recovered by flotation. Some strains have gas vacuoles and float at the surface of the water [72]. The incorporation of air bubbles depends on several aspects such as the contact angle of air, solid, and aqueous phases. According to the method of bubble production, flotation techniques can be divided into dissolved air flotation (DAF), dispersed flotation and electrolytic flotation. DAF is the most used method in the treatment of industrial wastewater. Microalgae cells are recovered by dissolving air in the water under pressure and are then released into a reservoir at atmospheric pressure thus producing small bubbles. Chemical flocculation has been used with DAF to separate micro‐ algae [73]. Garg et al. [74] evaluated the effects of froth flotation in different microalgae strains and found that *Chlorella* sp. showed a good response of floatability due to its high hydropho‐ bicity. This method represents a promising choice for the industrial scale harvesting of microalgae and represents a very versatile technique for the separation of small particles. Microalgae with low surface hydrophobicity are difficult to harvest by flotation separation. Surface hydrophobicity and bubble size are the key factors affecting algae flotation, and a stepwise optimization can lead to effective separation by flotation of difficult-to-harvest microalgae [64]. Although flotation has been widely used by researchers as a harvesting method, there are feasibility and economic limitations.

## **6. Biorefinery**

Biorefinery comprises a number of specialized methods used to extract the most out of primary and/or secondary metabolic products. Microalgae biorefineries must use methods and technology for isolating compounds and obtaining principal constituents from biomass, without damaging one or more of the product fractions, thereby adding value to the bioprod‐ uct formed [75].

The main focus when obtaining the products should be the dehydration or drying of the biomass when there is a requirement for its immediate use, otherwise the harvested biomass suspension must be processed rapidly. Methods used for drying microalgae include lyophi‐ lization, spray drying, drum drying and sun drying. The next step is cell disruption as some target products are intracellular and therefore cell disruption is required in order to release the products and ready them for extraction. Several methods can be used depending on the metabolites of interest [76].

The product fractions obtained from microalgae can be transformed into high-value molecules, antioxidants, anti-inflammatories, natural pigments, biofuels, and food supplements for human and animal feed. Microalgae biorefinery is, therefore, a process of great industrial impact and must be undertaken properly (Figure 2).

Methods of drying microalgal biomass are used with the purpose of increasing the longevity of cells. Drying methods may include sun drying, lyophilization, drum drying, spray drying, and fluidized bed drying. Sun drying is cheap but is very slow. Spray drying is the method chosen for obtaining bioproducts with a high added value, though this procedure is not recommended for extraction of microalgae pigments as it may affect the pigments' molecular structure. Lyophilization is widely used in scientific research procedures, but is very expensive for use on a large scale. It is useful with respect to some enzymes and pharmaceuticals. This method eliminates thermal and osmotic damage and preserves the cell constituents microalgae [77].

By comparing different drying techniques (sun drying, lyophilization) for the effective extraction of lipids from *Scenedesmus* sp. grown in a raceway reactor Guldhe et al. [77] showed that drying methods are critical for effective downstream processing in the synthesis of microalgal biodiesel. No statistically significant difference was found in the drying methods used for the extraction of lipids

The extraction of intracellular components requires the breakdown of the microalgal cell wall. Various disruption methods involving chemical treatments (solvents, acids), mechanical treatments (ultrasound, high-pressure homogenization, bead beating and blending), auto‐ claving, freezing–thawing sequences and supercritical fluids have been used. Some of the extraction and fractionation techniques will be described briefly below. Microalgae biorefi‐ neries seek to apply these methods at an industrial scale and at low cost. Their high function‐ ality with low concentrated streams is advantageous. Choosing the most appropriate method depends of various biological factors and the energy required. The integration of cell disrup‐

**Figure 2.** General outline of microalgae biorefinery.

**6. Biorefinery**

92 Biomass Production and Uses

uct formed [75].

metabolites of interest [76].

cell constituents microalgae [77].

used for the extraction of lipids

impact and must be undertaken properly (Figure 2).

Biorefinery comprises a number of specialized methods used to extract the most out of primary and/or secondary metabolic products. Microalgae biorefineries must use methods and technology for isolating compounds and obtaining principal constituents from biomass, without damaging one or more of the product fractions, thereby adding value to the bioprod‐

The main focus when obtaining the products should be the dehydration or drying of the biomass when there is a requirement for its immediate use, otherwise the harvested biomass suspension must be processed rapidly. Methods used for drying microalgae include lyophi‐ lization, spray drying, drum drying and sun drying. The next step is cell disruption as some target products are intracellular and therefore cell disruption is required in order to release the products and ready them for extraction. Several methods can be used depending on the

The product fractions obtained from microalgae can be transformed into high-value molecules, antioxidants, anti-inflammatories, natural pigments, biofuels, and food supplements for human and animal feed. Microalgae biorefinery is, therefore, a process of great industrial

Methods of drying microalgal biomass are used with the purpose of increasing the longevity of cells. Drying methods may include sun drying, lyophilization, drum drying, spray drying, and fluidized bed drying. Sun drying is cheap but is very slow. Spray drying is the method chosen for obtaining bioproducts with a high added value, though this procedure is not recommended for extraction of microalgae pigments as it may affect the pigments' molecular structure. Lyophilization is widely used in scientific research procedures, but is very expensive for use on a large scale. It is useful with respect to some enzymes and pharmaceuticals. This method eliminates thermal and osmotic damage and preserves the

By comparing different drying techniques (sun drying, lyophilization) for the effective extraction of lipids from *Scenedesmus* sp. grown in a raceway reactor Guldhe et al. [77] showed that drying methods are critical for effective downstream processing in the synthesis of microalgal biodiesel. No statistically significant difference was found in the drying methods

The extraction of intracellular components requires the breakdown of the microalgal cell wall. Various disruption methods involving chemical treatments (solvents, acids), mechanical treatments (ultrasound, high-pressure homogenization, bead beating and blending), auto‐ claving, freezing–thawing sequences and supercritical fluids have been used. Some of the extraction and fractionation techniques will be described briefly below. Microalgae biorefi‐ neries seek to apply these methods at an industrial scale and at low cost. Their high function‐ ality with low concentrated streams is advantageous. Choosing the most appropriate method depends of various biological factors and the energy required. The integration of cell disrup‐ tion into downstream processing has to be easy and should not have a negative impact on subsequent processing steps [78].

Pulsed electric field procedure can be a promising alternative to conventional cell disintegra‐ tion methods. The procedure is based on the dosing of short electrical pulses in high intensity electric fields. These alter the structure of the cell membrane, which as a result loses its barrier function and becomes permeable — a phenomenon often referred to as electroporation [79].

Goettel et al. [78] evaluated the application of the pulsed electric field procedure for the disruption of *Auxenochlorella protothecoides* cells. For all pulse parameters applied, there was evidence that cell disintegration resulted in the release of soluble intracellular matter into the suspension. The efficiency of cell disruption improved with increasing treatment energy, whereas the field strength had no major influence. Thus, the investigation proposed the use of the pulsed electric field procedure of cell disruption and selective two-step extraction. As an initial step, the pulsed electric field procedure allows separation of water soluble intracel‐ lular substances. In a subsequent step, lipids can be very efficiently extracted by solvents.

The ultrasound method is based on the incorporation of high frequency sound waves in microalgae cells so that pressure variation can disrupt the cell wall [80].

Ehimen et al. [81] successfully used ultrasound to improve methods of oil extraction in *Chlorella* biomass samples. The results of the study showed that it is feasible to reduce volumes of methanol used in the trans-esterification process. The combination of an ultrasonic process and solvent use demonstrated the potential for recovery of greater yields of fatty acid methyl esters (FAME) for biodiesel production.

Enzymatic degradation is another method of cell wall disruption. This method is used on a laboratory scale since high costs limits its use on a larger, industrial scale. The advantage is that the enzyme may be inactivated, removed, recovered and reused. It has a high specificity without interfering in the recovery of bioproducts. Enzymatic disruption of microalgae cell walls can be performed with a mixture of β-glucanases and lysozyme. Studies of enzymatic hydrolysis of *Chlorella* cell walls have demonstrated the high specificity of the disruption so that mechanical degradation can be performed with low energy costs [82].

Chemical treatments using acid are performed by immersing diluted microalgae biomass in strong acid followed by a strong base, at high temperature for a specific time. One disadvantage of the method is the toxicity of the acid, and as a result the method is not widely used [83]. Sathish and Sims [83] demonstrated a method of extracting transesterifiable lipids using acid and base hydrolysis for *Chlorella* sp. and *Scenedesmus* sp. with 84% of moisture. On average, 60% of lipids were extracted and converted to biodiesel by transesterification. This was achieved without drying the recovered biomass and the use of a smaller volume of organic solvent was evident.

The choice of cell disruption method is dependent on the bioproduct, the strain of microalgae used, and the costs and efficiency of the process

## **7. Extraction and purification of microalgae metabolites**

Solvent extraction systems are extensively used to extract microalgae metabolites from processed biomass. Solvents such as ethanol, chloroform, diethyl ether, hexane and methanol are commonly used. These can extract carbohydrates, amino acids, salts, hydrophobic proteins, lipids and pigments. The disadvantages of solvent extraction are that: (i) the process requires high capital investments; (ii) the energy requirements are high; (iii) the solvent is highly flammable; and (iv) the difficulty of recovering the solvent [84].

Different process for the extraction of fatty acid from *Aphanothece microscopica Nageli*, *Phaeo‐ dactylum tricornutum*, *Isochrysis galbana* have been described [29,85]. Extraction using aqueous buffers is employed to obtain phycobiliproteins from *P. cruentum* and lutein from three *Chlorella* species [86].

The regeneration of solvent for subsequent operation is difficult, further decreasing the efficiency of extraction. A method that can recover the solvent for reuse would be ideal from an economic point of view. This phase splitting could be induced by changing the nature of the solvent [87].

Du et al [87] studied the extraction of oil from *Desmodesmus* sp. by CO2-switchable solvents. In this research, the secondary amines dipropylamine and ethylbutylamine were able to extract lipid from a liquid medium without damaging microalgae cells. These solvents allow the process of quick and efficient lipid extraction in the presence of water induced by the presence of ambient temperatures and atmospheric CO2. These solvent systems provide a potential for reuse and recovery leading to decreased costs and provide an efficient method of microalgae lipid extraction [87]. Properties of the cell membrane are of great importance in the solvent extraction process. Therefore, disruption of the cell wall is critical [88].

Crude extracts are generally filtered and purified by several chromatographic methods in order to obtain the metabolite of interest. In choosing a chromatographic technique certain considerations should kept in mind. These include molecular weight, isoelectric point, hydrophobicity and biological affinity. Supercritical fluid extraction has been shown to be an efficient technique for extracting carotenoids from microalgae *Scenedesmus* sp. [89] and fatty acids, and of the three microalgae strains evaluated, *S. obliquus* is the best source of α-linolenic acid [90].

that the enzyme may be inactivated, removed, recovered and reused. It has a high specificity without interfering in the recovery of bioproducts. Enzymatic disruption of microalgae cell walls can be performed with a mixture of β-glucanases and lysozyme. Studies of enzymatic hydrolysis of *Chlorella* cell walls have demonstrated the high specificity of the disruption so

Chemical treatments using acid are performed by immersing diluted microalgae biomass in strong acid followed by a strong base, at high temperature for a specific time. One disadvantage of the method is the toxicity of the acid, and as a result the method is not widely used [83]. Sathish and Sims [83] demonstrated a method of extracting transesterifiable lipids using acid and base hydrolysis for *Chlorella* sp. and *Scenedesmus* sp. with 84% of moisture. On average, 60% of lipids were extracted and converted to biodiesel by transesterification. This was achieved without drying the recovered biomass and the use of a smaller volume of organic

The choice of cell disruption method is dependent on the bioproduct, the strain of microalgae

Solvent extraction systems are extensively used to extract microalgae metabolites from processed biomass. Solvents such as ethanol, chloroform, diethyl ether, hexane and methanol are commonly used. These can extract carbohydrates, amino acids, salts, hydrophobic proteins, lipids and pigments. The disadvantages of solvent extraction are that: (i) the process requires high capital investments; (ii) the energy requirements are high; (iii) the solvent is highly

Different process for the extraction of fatty acid from *Aphanothece microscopica Nageli*, *Phaeo‐ dactylum tricornutum*, *Isochrysis galbana* have been described [29,85]. Extraction using aqueous buffers is employed to obtain phycobiliproteins from *P. cruentum* and lutein from three

The regeneration of solvent for subsequent operation is difficult, further decreasing the efficiency of extraction. A method that can recover the solvent for reuse would be ideal from an economic point of view. This phase splitting could be induced by changing the nature of

Du et al [87] studied the extraction of oil from *Desmodesmus* sp. by CO2-switchable solvents. In this research, the secondary amines dipropylamine and ethylbutylamine were able to extract lipid from a liquid medium without damaging microalgae cells. These solvents allow the process of quick and efficient lipid extraction in the presence of water induced by the presence of ambient temperatures and atmospheric CO2. These solvent systems provide a potential for reuse and recovery leading to decreased costs and provide an efficient method of microalgae lipid extraction [87]. Properties of the cell membrane are of great importance in the solvent

that mechanical degradation can be performed with low energy costs [82].

**7. Extraction and purification of microalgae metabolites**

flammable; and (iv) the difficulty of recovering the solvent [84].

extraction process. Therefore, disruption of the cell wall is critical [88].

solvent was evident.

94 Biomass Production and Uses

*Chlorella* species [86].

the solvent [87].

used, and the costs and efficiency of the process

Some other chromatographic methods included reverse phase chromatography, silica gel adsorption chromatography, and ion exchange chromatography (for proteins). Chromato‐ graphic techniques are usually employed for higher-value products. An economical evaluation could be useful to help calculate the optimum conditions for industrial applications [91].

## **8. Potential uses for bioproducts obtained from microalgal biorefineries**

Microalgae have massive potential to produce biomolecules due to the low cost of energy and nutrient sources used, as well as fast growth rates and the capacity to accumulate or secrete metabolites. Microalgal biorefineries allow the transformation of biomass into the production of fuels, food, feed, chemicals, polymers and value-added ingredients [92].

Thus, the use of these microorganisms in carbon sequestration processes combines the treatment of polluting compounds with the production of consumables in a variety of forms. Table 4 shows some potential uses for the bioproducts obtained from microalgal biorefineries and formation by the biological conversion of CO2 in photobioreactors.


**Table 4.** Potential uses for bioproducts obtained from microalgal biorefineries

Microalgae possess a versatile metabolic capacity that can be transformed into valuable products through various processing routes. Some microalgae species as *Chlorella*, *Chlamydo‐ monas*, *Dunaliella*, *Scenedesmus*, and *Tetraselmis* have a high carbohydrate content (37–55%) that mainly comes from starch in chloroplasts and cellulose cell walls [101]. Carbohydrate-rich microalgal biomass were evaluated for bioethanol production and were found to provide good yields [102].

The lipid profile of microalgae shows values of 2–77% depending on species and growth conditions. Microalgae lipids are classified into two groups, one for transformation in biofuel and one for food supplements, with carbon numbers of between 14–20 and 20 carbons respectively. Microalgae have a promising future, with production of eicosapentaenoic acid and docosahexaenoic acid as the main product. The species of microalgae producing omega-3 polyunsaturated fatty acids are mainly *Bacillariophyta, Chlorophyta, Cryptophyta, Haptophyta, Heterokontophyta* and *Rhodophyta* [103,104].

Proteins are among the main constituents of microalgae, at proportions of 50–70% depending on species, and they are an important product of microalgae biorefineries. Microalgal proteins can be used in human or animal nutrition (from aquaculture to farm animals). However, some microalgae contain toxic proteins, so analytical analyses need to be performed [105]. Nutri‐ tional and toxicological evaluations have demonstrated that microalgal biomass offers a valuable feed supplement or substitute for conventional animal feed sources [106].

Microalgae are known to be a good source of pigments and bioactive compounds. Chloro‐ phylls, phycobilins and carotenoids are molecules with a high added value that can be obtained from *Porphyridium cruentum, Synechococcus* sp. and *Chlorella* and used in the chemical industry. Rodrigues et al. [107] showed that *Phormidium autumnale* has potential for the production of carotenoids. Sensitivity analysis showed the possibility of obtaining 107,902.5 kg/year of total carotenoids at the industrial scale. Symplostatin and curacin A have been isolated from the cyanobacteria *Symploca hydnoides* and *Lyngbya majuscula* respectively. These compounds exhibited cytotoxicity against a human carcinoma cell line [108].

The microalgae biorefineries industry promises much from the economic point of view. Global annual sales of beta-1,3-glucan from *Chlorella* sp. are in excess of USD\$38 billion [105]. Moreover, phycobiliproteins present in cyanobacteria and some algae used to develop compounds for the pharmaceutical industry, represented a market of about USD\$6–11 million with prices that varied from USD\$3–25 mg-1 [105]. Considering that Kenekar and Deodhar [109] reported a phycocyanin yield of 0.071 gL-1 in *Geitlerinema sulphureum* culture, a photo‐ bioreactor with 100 L could generate a profit of approximately USD\$177,500 [109]. Microalgae biomass produces more than 5,000 tons of dried mass/year with an annual revenue greater than USD\$1.25 billion, not including processed products, demonstrating the potential of this type of biotechnological process [99]. Despite the promising conditions for the production of microalgae biomass and bioproducts, the industrial-scale development is currently a long way from the high profits available in theory. This is due to the lack of methods and photobior‐ eactors that can produce large enough quantities to supply the market [12].

Finally, microalgae cells can produce methane. Sialve et al. [110] showed methane production values from anaerobic digestion in microalgae biomass in the range of 0.09–0.54 L CH4/g volatile solids [110]. Furthermore, compounds such as non-methane hydrocarbon, organohal‐ ogens, and aldehydes are continuously being formed and released from the liquid phase of photobioreactors. The production of renewable polymers is an emerging industrial field [4].

Therefore, microalgal biotechnology can be seen as a promising scientific tool in the near future and microalgae biorefineries have the potential to solve some of the environmental, nutritional and pharmaceutical problems afflicting society.

## **9. Final considerations**

Microalgae possess a versatile metabolic capacity that can be transformed into valuable products through various processing routes. Some microalgae species as *Chlorella*, *Chlamydo‐ monas*, *Dunaliella*, *Scenedesmus*, and *Tetraselmis* have a high carbohydrate content (37–55%) that mainly comes from starch in chloroplasts and cellulose cell walls [101]. Carbohydrate-rich microalgal biomass were evaluated for bioethanol production and were found to provide good

The lipid profile of microalgae shows values of 2–77% depending on species and growth conditions. Microalgae lipids are classified into two groups, one for transformation in biofuel and one for food supplements, with carbon numbers of between 14–20 and 20 carbons respectively. Microalgae have a promising future, with production of eicosapentaenoic acid and docosahexaenoic acid as the main product. The species of microalgae producing omega-3 polyunsaturated fatty acids are mainly *Bacillariophyta, Chlorophyta, Cryptophyta, Haptophyta,*

Proteins are among the main constituents of microalgae, at proportions of 50–70% depending on species, and they are an important product of microalgae biorefineries. Microalgal proteins can be used in human or animal nutrition (from aquaculture to farm animals). However, some microalgae contain toxic proteins, so analytical analyses need to be performed [105]. Nutri‐ tional and toxicological evaluations have demonstrated that microalgal biomass offers a

Microalgae are known to be a good source of pigments and bioactive compounds. Chloro‐ phylls, phycobilins and carotenoids are molecules with a high added value that can be obtained from *Porphyridium cruentum, Synechococcus* sp. and *Chlorella* and used in the chemical industry. Rodrigues et al. [107] showed that *Phormidium autumnale* has potential for the production of carotenoids. Sensitivity analysis showed the possibility of obtaining 107,902.5 kg/year of total carotenoids at the industrial scale. Symplostatin and curacin A have been isolated from the cyanobacteria *Symploca hydnoides* and *Lyngbya majuscula* respectively. These compounds

The microalgae biorefineries industry promises much from the economic point of view. Global annual sales of beta-1,3-glucan from *Chlorella* sp. are in excess of USD\$38 billion [105]. Moreover, phycobiliproteins present in cyanobacteria and some algae used to develop compounds for the pharmaceutical industry, represented a market of about USD\$6–11 million with prices that varied from USD\$3–25 mg-1 [105]. Considering that Kenekar and Deodhar [109] reported a phycocyanin yield of 0.071 gL-1 in *Geitlerinema sulphureum* culture, a photo‐ bioreactor with 100 L could generate a profit of approximately USD\$177,500 [109]. Microalgae biomass produces more than 5,000 tons of dried mass/year with an annual revenue greater than USD\$1.25 billion, not including processed products, demonstrating the potential of this type of biotechnological process [99]. Despite the promising conditions for the production of microalgae biomass and bioproducts, the industrial-scale development is currently a long way from the high profits available in theory. This is due to the lack of methods and photobior‐

valuable feed supplement or substitute for conventional animal feed sources [106].

exhibited cytotoxicity against a human carcinoma cell line [108].

eactors that can produce large enough quantities to supply the market [12].

yields [102].

96 Biomass Production and Uses

*Heterokontophyta* and *Rhodophyta* [103,104].

Most research into microalgae biorefineries has been undertaken at the laboratory or pilot scale, and the number of full-scale studies is limited. Large-scale microalgae processes have been developed mainly using open photobioreactors. Some successful initiatives have been carried out in closed systems, but the closed systems need to operate at a large scale, to overcome the many drawbacks. At a large scale, algal growth conditions need to be closely controlled. The processes can be economical when using inexpensive sources of CO2 from flue gas emissions, wastewaters, and/or with the extraction of bioproducts for industrial use.

Finally, many companies are investing in biotechnology, increasing spending on the produc‐ tion systems in order to obtain microalgal biofuel and high value-added bioproducts. Al‐ though at present there is no consensus on the criteria for the large scale development of photobioreactors for microalgae cultivation. Conventional configurations of closed systems, and hybrid photobioreactors are being employed and constantly improved for use at the industrial scale.

## **Author details**

Eduardo Jacob-Lopes1\*, Luis Guillermo Ramírez Mérida2 , Maria Isabel Queiroz3 and Leila Q. Zepka1

\*Address all correspondence to: jacoblopes@pq.cnpq.br

1 Food Science and Technology Department, Federal University of Santa Maria, UFSM, San‐ ta Maria, RS, Brazil

2 Department of Biology, University of Carabobo, UC, Avenue Universidad, Valencia, Edo. Carabobo, Venezuela

3 School of Chemistry and Food, Federal University of Rio Grande, FURG, Eng. Alfredo Huch, CEP Rio Grande, RS, Brazil

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