Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture

*Selvan Ravindran, Pooja Singh, Sanjay Nene, Vinay Rale, Nutan Mhetras and Anuradha Vaidya*

#### **Abstract**

Screening for novel producer strains and enhanced therapeutic production at reduced cost has been the focus of most of the biopharmaceutical industries. The obligation to carry out prolonged intensive pilot scale experiments gave birth to micro-scale bioreactor systems. Screening large number of microorganisms using shake flasks and benchtop bioreactors is tedious and consumes resources. Microbioreactors that mimic benchtop bioreactors are capable not only of high throughput screening of producer strains, but also aid in optimizing the growth kinetics and expression of proteins. Modern technology has enabled the collection of precise online data for variables such as optical density (OD), pH, temperature, dissolved oxygen (DO), and adjusting in mixing inside microreactors. Microbioreactors have become an irreplaceable tool for biochemical engineers and biotechnologists to perform a large number of experiments simultaneously. Another aspect that is vital to any industry is the product yield and subsequent downstream processing. Perfusion bioreactors are one of the upcoming advances in bioreactor systems that have the potential to revolutionize biologics production. This chapter intends to take a review of different aspects of microbioreactors and perfusion bioreactors including their potential in high throughput pilot studies and microbial and mammalian cell cultivation technologies.

**Keywords:** microbioreactors, biopharmaceuticals, bioprocess, perfusion microbioreactors, microorganisms

#### **1. Introduction**

#### **1.1 Need for microbioreactors in bioprocess development**

Microbioreactor systems are an integral part of bioprocess engineering. In the past few years, microbioreactors have extensively been used for high throughput screening [1]. In addition to this, extensive bioprocess experiments were monitored and controlled. Industries involved in bulk production of pharmaceuticals, chemicals, enzymes for feed and food from microbial cell factories are in need of microbioreactors [2–4]. Advanced shaker microliter cultivation devices or down-scaled stirred tank reactors are two basic microbioreactors. Primary and secondary screening experiments based on microbial library are conducted in shake flasks and micro liter plates. This screening process aids in selecting the microbial strain candidates that are promising. Then,

selected microbial candidates are subjected to lab-scale experimental conditions for better bioprocess control. Bioprocess method developed by microbioreactors is followed by successful lab-scale testing, and it is transferred to pilot scale. Pilot scale experiments are essential to understand the bioreactor inhomogeneities which can ultimately be addressed using simulators that can scale down the process of bioreactors [5, 6].

Traditional microbiorector experiments start from primary screening; followed by secondary screening using shake flasks; then, process development; and, finally, optimization leading to process validation and pilot scale. It is worth mentioning that microbioreactors reduce the number of steps involved in traditional bioreactors. Microbioreactors start with primary screening, followed by accelerated bioprocess development for secondary screening, resulting in process validation and pilot scale at a faster rate. Introduction of bioreactors has reduced the number of steps involved in traditional bioreactors to scale up the bioprocesses. Hence, microbioreactors have proved to me more economical as they reduce the usage of secondary screening by shake flasks, thus making the process development and optimization more efficient. Ecology and environmental concerns arising during traditional bioprocess development have also been addressed by microbioreactors. Therefore, the focus of this chapter is on new developments in microbioreactors and their impact on bioprocesses.

#### **1.2 Advantages and expected outcome of microbioreactors**

Microbioreactors have numerous advantages over traditional bioreactors during bioprocess development. Microbioreactors are necessary for high throughput quantitative microbial phenotyping under controlled experimental conditions. Microbioreactors reduce the time involved in the traditional bioprocess development by replacing the shake flasks and large lab-scale bioreactors (**Figure 1**). Microbioreactors are more than necessary with specifications for efficient and economical bioprocess development. Expected outcomes of microbioreactors are as follows: (a) reduction in volume for each cultivation experiment; (b) time conservation during experiments; (c) simple, friendly, and operational without

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*Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture*

fail; (d) automated operation with minimal or no supervision; (e) examination of bioprocess variables with high resolution; (f) temperature, pH, and feeding profiles for controlled cultivation process; (h) culture accessibility for sampling and dosing; (i) cultivation by fed-batch, batch, and continuous modes; (j) robotic systems with advanced hardware and software; and (k) scalability is compared to laboratory

For realizing all the above-mentioned desired properties of microbioreactors, researchers are emphasizing on applied and basic research. Extensive research has resulted in various microbioreactors with different technologies and applications.

Commercially available microbioreactors are based on their applications, specifications, and capabilities. Different types of microbioreactors are born out of

Biolector (m2p-labs.com), a commercial microbioreactor manufacturer, had developed microbioreactors for organism phenotyping, screening the strains, toxicity screening, and optimization of feed and growth parameters. These bioreactors support single-use 48-well plates that can hold culture volume of 0.8–2.4 ml. Biomass formation can be monitored via fluorescence and by optodes pH and dissolved oxygen (DO). This system is integrated with liquid handlers for pH adjustment; feeding; sampling; and control over temperature, gas, and humidity [8]. Microbioreactors (32 plate and 48 plate) [9] from Biolector were used for optimization of feeding rate, media screening, and fermentation parameters for anaerobic and microaerophyllic organisms. Few other applications that are possible with biolector microbioreactors are growth characterization, high throughput protein characterization, enzyme and cell activity tests, functional genomics and proteomic

RoboLector [10] is another microbioreactor where microbioreactor system from biolector is interfaced with liquid handling robot. These microbioreactors with the aid of 48 or 96 parallel cultivations through microplates provide fermentation data repeatedly for every 5–15 minutes. In addition to this, a robotic system also controls nutrient feeding, adjusts pH by adding acid or base, and does sampling based on user definition. Another commercial manufacturer Micro-24 (pall.com) [11] developed a microbioreactor for screening strains and cell lines besides optimization of growth and feed parameters. These microbioreactors support single-use cassettes with 24 columns with culture volume ranging from 3 to 7 ml and regulate pH using ammo-

Microbioreactors to study biotransformation, phenotyping, strain and toxicity screening were developed by commercial manufacturer Bioscreen C (bioscreen. fi). These microbioreactors consist of two parallel 100-well plates with a capacity to hold culture volume of 0.4 ml. Optical density was used for quasi-continuous

Growth profiler (enzyscreen.com), Applikon (applikonbio.com), SensorDish Reader (presens.de), ambr 15 (tapbiosystems.com), bioReactor (emag.de), and Sartorius (Sartorius.com) are other manufacturers of microbioreactors for various

During recombinant protein expression, *E. coli* synthesizes proteins at a faster

rate with low multiplication rate. In traditional microbioreactors, fed-batch

*DOI: http://dx.doi.org/10.5772/intechopen.83825*

**2. Types of microbioreactors**

extensive basic and applied research.

studies, inhibition and toxicity studies, and quality control.

nia, carbon dioxide, and control over temperature.

applications as per the requirements of clients.

**2.1 Microbioreactors for fed-batch cultivation of** *Escherichia coli*

monitoring of biomass.

bioreactors [7].

**Figure 1.** *Comparison of workflow for traditional bioreactors and microbioreactors.*

*Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture DOI: http://dx.doi.org/10.5772/intechopen.83825*

fail; (d) automated operation with minimal or no supervision; (e) examination of bioprocess variables with high resolution; (f) temperature, pH, and feeding profiles for controlled cultivation process; (h) culture accessibility for sampling and dosing; (i) cultivation by fed-batch, batch, and continuous modes; (j) robotic systems with advanced hardware and software; and (k) scalability is compared to laboratory bioreactors [7].

For realizing all the above-mentioned desired properties of microbioreactors, researchers are emphasizing on applied and basic research. Extensive research has resulted in various microbioreactors with different technologies and applications.

#### **2. Types of microbioreactors**

*Biotechnology and Bioengineering*

selected microbial candidates are subjected to lab-scale experimental conditions for better bioprocess control. Bioprocess method developed by microbioreactors is followed by successful lab-scale testing, and it is transferred to pilot scale. Pilot scale experiments are essential to understand the bioreactor inhomogeneities which can ultimately be addressed using simulators that can scale down the process of bioreactors [5, 6].

Traditional microbiorector experiments start from primary screening; followed by secondary screening using shake flasks; then, process development; and, finally, optimization leading to process validation and pilot scale. It is worth mentioning that microbioreactors reduce the number of steps involved in traditional bioreactors. Microbioreactors start with primary screening, followed by accelerated bioprocess development for secondary screening, resulting in process validation and pilot scale at a faster rate. Introduction of bioreactors has reduced the number of steps involved in traditional bioreactors to scale up the bioprocesses. Hence, microbioreactors have proved to me more economical as they reduce the usage of secondary screening by shake flasks, thus making the process development and optimization more efficient. Ecology and environmental concerns arising during traditional bioprocess development have also been addressed by microbioreactors. Therefore, the focus of this chapter is on new developments in microbioreactors and their impact on bioprocesses.

Microbioreactors have numerous advantages over traditional bioreactors during bioprocess development. Microbioreactors are necessary for high throughput quantitative microbial phenotyping under controlled experimental conditions. Microbioreactors reduce the time involved in the traditional bioprocess development by replacing the shake flasks and large lab-scale bioreactors (**Figure 1**). Microbioreactors are more than necessary with specifications for efficient and economical bioprocess development. Expected outcomes of microbioreactors are as follows: (a) reduction in volume for each cultivation experiment; (b) time conservation during experiments; (c) simple, friendly, and operational without

**1.2 Advantages and expected outcome of microbioreactors**

**92**

**Figure 1.**

*Comparison of workflow for traditional bioreactors and microbioreactors.*

Commercially available microbioreactors are based on their applications, specifications, and capabilities. Different types of microbioreactors are born out of extensive basic and applied research.

Biolector (m2p-labs.com), a commercial microbioreactor manufacturer, had developed microbioreactors for organism phenotyping, screening the strains, toxicity screening, and optimization of feed and growth parameters. These bioreactors support single-use 48-well plates that can hold culture volume of 0.8–2.4 ml. Biomass formation can be monitored via fluorescence and by optodes pH and dissolved oxygen (DO). This system is integrated with liquid handlers for pH adjustment; feeding; sampling; and control over temperature, gas, and humidity [8].

Microbioreactors (32 plate and 48 plate) [9] from Biolector were used for optimization of feeding rate, media screening, and fermentation parameters for anaerobic and microaerophyllic organisms. Few other applications that are possible with biolector microbioreactors are growth characterization, high throughput protein characterization, enzyme and cell activity tests, functional genomics and proteomic studies, inhibition and toxicity studies, and quality control.

RoboLector [10] is another microbioreactor where microbioreactor system from biolector is interfaced with liquid handling robot. These microbioreactors with the aid of 48 or 96 parallel cultivations through microplates provide fermentation data repeatedly for every 5–15 minutes. In addition to this, a robotic system also controls nutrient feeding, adjusts pH by adding acid or base, and does sampling based on user definition.

Another commercial manufacturer Micro-24 (pall.com) [11] developed a microbioreactor for screening strains and cell lines besides optimization of growth and feed parameters. These microbioreactors support single-use cassettes with 24 columns with culture volume ranging from 3 to 7 ml and regulate pH using ammonia, carbon dioxide, and control over temperature.

Microbioreactors to study biotransformation, phenotyping, strain and toxicity screening were developed by commercial manufacturer Bioscreen C (bioscreen. fi). These microbioreactors consist of two parallel 100-well plates with a capacity to hold culture volume of 0.4 ml. Optical density was used for quasi-continuous monitoring of biomass.

Growth profiler (enzyscreen.com), Applikon (applikonbio.com), SensorDish Reader (presens.de), ambr 15 (tapbiosystems.com), bioReactor (emag.de), and Sartorius (Sartorius.com) are other manufacturers of microbioreactors for various applications as per the requirements of clients.

#### **2.1 Microbioreactors for fed-batch cultivation of** *Escherichia coli*

During recombinant protein expression, *E. coli* synthesizes proteins at a faster rate with low multiplication rate. In traditional microbioreactors, fed-batch

cultivations of *E. coli* were performed by stirred tank reactors [1]. Carbon source, magnesium, and ammonium were used as feeding solutions to match the nutritional requirements. It is worth mentioning that two fed-batch cultivations can be performed simultaneously with different nutrient compositions in the RoboLector systems. For example, feeding solution of one set of reaction is constituted with 1 M sodium hydroxide, 400 g/l glycerol, 100 g/l ammonium phosphate, and 1 g/l of magnesium sulfate 7H2O whereas that of another set is composed of 200 g/l glycerol, 100 g/l ammonium phosphate, 2 g/l of magnesium sulfate 7H2O, and 1 M NaOH. Cultivations were performed at a shaking frequency of 1100 rpm with diameter 3 mm and temperature was set at 30°. Modified medium with 10 g/l glycerol and 100 mM MOPS with minimum salt was used. Online-monitored DO measurements were used to control the repeated additions of feeding solutions and both the feeding methods resulted in high cell densities of approximately 80 OD besides pH stabilization in between 6.5 and 7 for favorable growth of *E. coli*. Both the cultivations, one with high volume of glycerol and another with low volume of the same, exhibit high biomass concentration. Traditional bioreactors need huge amounts of energy for mixing, heating, and cooling during scale-up process but RoboLector microbioreactors are efficient and precise to hasten the development of bioprocesses using microbial cells [1].

#### **2.2 Cultivation of** *Pichia pastoris* **using microbioreactors**

Secreted proteins during bioprocesses undergo proteolysis and fragmentation; therefore, samples are generally removed from fermentation broth to obtain a kinetic growth curve. The purpose is to identify the optimum cultivation setup to reach maximum activity for the recombinant enzyme. RoboLectors are used to understand the kinetics of *Pichia pastoris* fed-batch cultivation [1]. Kinetics was monitored online by drawing 20 μl automatically and the concentrations of secreted enzymes were plotted against time to identify the space-time yield. During the automatic pipetting process, robotic tips immersed in the solution without any shaking, thus preventing artifacts of sedimentation.

To increase the productivity, softwares are programmed to provide outline of the experiments as per the input parameters. Dosing volume and variations in the concentrations of glycerol, ammonium hydroxide, and methanol can be given in the inputs. In 48 parallel fed-batch cultivations, a factorial design play gives a value, 24 + 3 = 19; these different possibilities can be performed in one single analysis. Once the experiment is completed, the system automatically generates a contour plot and summarizes the activity profile of the recombinant enzymes with respect to the dosing volume and feeding solutions. For a particular recombinant product from *Pichia pastoris,* following factors were summarized at the completion of the experiment: 35% v/v methanol, 25% w/w ammonium hydroxide, and 150 g/l as feed composition for a dosing volume of 5 μl. The above-mentioned experiment is repeated for a particular design to identify and confirm the variable factors and increase the productivity [1].

#### **2.3 Microbioreactors to produce monoclonal antibodies**

Monoclonal antibodies are one of the major products among biotherapeutics. Organisms such as Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells are utilized to produce monoclonal antibodies, and several bioprocess parameters need to be optimized [12, 13] for their production. Composition of culture media, cell growth rate, and antibody growth rate need favorable physicochemical parameters such as pH, temperature, and dissolved oxygen. Physicochemical parameters are controlled to initiate and sustain recombinant

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*Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture*

sumes enormous time during research and development (R&D).

technical functions of bioreactors becomes essential.

the specifications based upon the above requirements.

maximum production of monoclonal antibodies [15].

**2.4 Microbioreactors in drug discovery**

(b) oxygen transfer value >100 h<sup>−</sup><sup>1</sup>

expression in the CHO cell culture and to provide nutrients and relevant growth factors to bioreactors. During the production of monoclonal antibodies, possibilities of forming various variants of immunoglobulin (IgG) molecule and glycosylated IgG forms can be seen. Similar to monoclonal antibodies, other protein therapeutics also pass through issues such as oxygenation, amination, and degraded product molecules. Each of these above-mentioned processes is lengthy and con-

Microbioreactors, due to their miniature size and parallel testing, are needed to accelerate research and development (R&D) at a faster pace. Microbioreactors encompassed with sensors [14] that can hold volume in the range of 1–20 μl are ideal to monitor the bioprocess parameters. Microbioreactors need to be designed as per the needs of users. For example, for users to develop bioprocess procedures for mammalian cell cultures, the list of biological functions of mammalian cells and

Biological functions and requirements include: (a) nature of the cells (CHO or HEK cells) with concentration ranging from 10,000 to 10,000,000, (b) expression of proteins such as IgGs, (c) serum-free medium, and (d) culture time of 7–14 days [15]. Technical information parameters must include: (a) shaking for better mixing,

a large-scale process, (c) oxygen permeability <1%, (d) surface hydrophobicity (10°) along with a confocal microscope installed in situ without disturbing the reaction, (e) cell culture volume, (f) transportation of culture media to contained culture, and (g) measurement of chemical and physical conditions of culture and media [15].

Information function, namely online or offline information, is also essential: (a) online information about sensors to control and detect pH, temperature, and pO2 is significant and (b) offline information such as monomers in culture media, forms of IgGs, excreted metabolites, products formed, and residual nutrients is needed for analysis of analytes. It is important that microbioreactors should be designed as per

These specifications also cater to the requirements of microbioreactors in laboratories with 2–5-liter capacity. Therefore, such processes could be scaled up to laboratory level with effortless ease at reduced cost. CHO cell culture microbioreactors used to optimize the production of monoclonal antibodies are schematically shown in **Figure 2**. This is a part of Hubka-Eder map [16] focused on biological and technical functions. Hubka-Eder map shows the importance of integration among the subsystems, that is, the expression systems, cell line, medium with various technical and information functions for the design of the microbioreactors for the

Organ-on-a-chip [17] is an ongoing research to hasten the process of drug discovery and development. In vitro drug screening and safety testing [18–20] are essential to minimize extensive studies on laboratory animals. Microbioreactors designed as per the need of the study will be highly beneficial to understand the potency and toxicity of the developed drug molecules. Drug molecules upon absorption get distributed to various organs such as lung, liver, gut, intestine, etc. These organs majorly consist of enzymes that can metabolize the drug molecules to metabolites [21] which can either be potent or toxic. After metabolism, the drug and its metabolites need to reach the target site for a particular time and get eliminated from the circulating system [22]. Kidneys play an important role in excreting drugs and its metabolites. Hence, developing various organs on a chip, programmed through a microbioreactor will be very helpful to understand the safety of drugs and metabolites.

to aerate the culture so that it can be extended to

*DOI: http://dx.doi.org/10.5772/intechopen.83825*

#### *Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture DOI: http://dx.doi.org/10.5772/intechopen.83825*

expression in the CHO cell culture and to provide nutrients and relevant growth factors to bioreactors. During the production of monoclonal antibodies, possibilities of forming various variants of immunoglobulin (IgG) molecule and glycosylated IgG forms can be seen. Similar to monoclonal antibodies, other protein therapeutics also pass through issues such as oxygenation, amination, and degraded product molecules. Each of these above-mentioned processes is lengthy and consumes enormous time during research and development (R&D).

Microbioreactors, due to their miniature size and parallel testing, are needed to accelerate research and development (R&D) at a faster pace. Microbioreactors encompassed with sensors [14] that can hold volume in the range of 1–20 μl are ideal to monitor the bioprocess parameters. Microbioreactors need to be designed as per the needs of users. For example, for users to develop bioprocess procedures for mammalian cell cultures, the list of biological functions of mammalian cells and technical functions of bioreactors becomes essential.

Biological functions and requirements include: (a) nature of the cells (CHO or HEK cells) with concentration ranging from 10,000 to 10,000,000, (b) expression of proteins such as IgGs, (c) serum-free medium, and (d) culture time of 7–14 days [15].

Technical information parameters must include: (a) shaking for better mixing, (b) oxygen transfer value >100 h<sup>−</sup><sup>1</sup> to aerate the culture so that it can be extended to a large-scale process, (c) oxygen permeability <1%, (d) surface hydrophobicity (10°) along with a confocal microscope installed in situ without disturbing the reaction, (e) cell culture volume, (f) transportation of culture media to contained culture, and (g) measurement of chemical and physical conditions of culture and media [15].

Information function, namely online or offline information, is also essential: (a) online information about sensors to control and detect pH, temperature, and pO2 is significant and (b) offline information such as monomers in culture media, forms of IgGs, excreted metabolites, products formed, and residual nutrients is needed for analysis of analytes. It is important that microbioreactors should be designed as per the specifications based upon the above requirements.

These specifications also cater to the requirements of microbioreactors in laboratories with 2–5-liter capacity. Therefore, such processes could be scaled up to laboratory level with effortless ease at reduced cost. CHO cell culture microbioreactors used to optimize the production of monoclonal antibodies are schematically shown in **Figure 2**. This is a part of Hubka-Eder map [16] focused on biological and technical functions. Hubka-Eder map shows the importance of integration among the subsystems, that is, the expression systems, cell line, medium with various technical and information functions for the design of the microbioreactors for the maximum production of monoclonal antibodies [15].

#### **2.4 Microbioreactors in drug discovery**

Organ-on-a-chip [17] is an ongoing research to hasten the process of drug discovery and development. In vitro drug screening and safety testing [18–20] are essential to minimize extensive studies on laboratory animals. Microbioreactors designed as per the need of the study will be highly beneficial to understand the potency and toxicity of the developed drug molecules. Drug molecules upon absorption get distributed to various organs such as lung, liver, gut, intestine, etc. These organs majorly consist of enzymes that can metabolize the drug molecules to metabolites [21] which can either be potent or toxic. After metabolism, the drug and its metabolites need to reach the target site for a particular time and get eliminated from the circulating system [22]. Kidneys play an important role in excreting drugs and its metabolites. Hence, developing various organs on a chip, programmed through a microbioreactor will be very helpful to understand the safety of drugs and metabolites.

*Biotechnology and Bioengineering*

bioprocesses using microbial cells [1].

**2.2 Cultivation of** *Pichia pastoris* **using microbioreactors**

shaking, thus preventing artifacts of sedimentation.

cultivations of *E. coli* were performed by stirred tank reactors [1]. Carbon source, magnesium, and ammonium were used as feeding solutions to match the nutritional requirements. It is worth mentioning that two fed-batch cultivations can be performed simultaneously with different nutrient compositions in the RoboLector systems. For example, feeding solution of one set of reaction is constituted with 1 M sodium hydroxide, 400 g/l glycerol, 100 g/l ammonium phosphate, and 1 g/l of magnesium sulfate 7H2O whereas that of another set is composed of 200 g/l glycerol, 100 g/l ammonium phosphate, 2 g/l of magnesium sulfate 7H2O, and 1 M NaOH. Cultivations were performed at a shaking frequency of 1100 rpm with diameter 3 mm and temperature was set at 30°. Modified medium with 10 g/l glycerol and 100 mM MOPS with minimum salt was used. Online-monitored DO measurements were used to control the repeated additions of feeding solutions and both the feeding methods resulted in high cell densities of approximately 80 OD besides pH stabilization in between 6.5 and 7 for favorable growth of *E. coli*. Both the cultivations, one with high volume of glycerol and another with low volume of the same, exhibit high biomass concentration. Traditional bioreactors need huge amounts of energy for mixing, heating, and cooling during scale-up process but RoboLector microbioreactors are efficient and precise to hasten the development of

Secreted proteins during bioprocesses undergo proteolysis and fragmentation; therefore, samples are generally removed from fermentation broth to obtain a kinetic growth curve. The purpose is to identify the optimum cultivation setup to reach maximum activity for the recombinant enzyme. RoboLectors are used to understand the kinetics of *Pichia pastoris* fed-batch cultivation [1]. Kinetics was monitored online by drawing 20 μl automatically and the concentrations of secreted enzymes were plotted against time to identify the space-time yield. During the automatic pipetting process, robotic tips immersed in the solution without any

To increase the productivity, softwares are programmed to provide outline of the experiments as per the input parameters. Dosing volume and variations in the concentrations of glycerol, ammonium hydroxide, and methanol can be given in the inputs.

these different possibilities can be performed in one single analysis. Once the experiment is completed, the system automatically generates a contour plot and summarizes the activity profile of the recombinant enzymes with respect to the dosing volume and feeding solutions. For a particular recombinant product from *Pichia pastoris,* following factors were summarized at the completion of the experiment: 35% v/v methanol, 25% w/w ammonium hydroxide, and 150 g/l as feed composition for a dosing volume of 5 μl. The above-mentioned experiment is repeated for a particular design to identify

Monoclonal antibodies are one of the major products among biotherapeutics. Organisms such as Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells are utilized to produce monoclonal antibodies, and several bioprocess parameters need to be optimized [12, 13] for their production. Composition of culture media, cell growth rate, and antibody growth rate need favorable physicochemical parameters such as pH, temperature, and dissolved oxygen. Physicochemical parameters are controlled to initiate and sustain recombinant

+ 3 = 19;

In 48 parallel fed-batch cultivations, a factorial design play gives a value, 24

and confirm the variable factors and increase the productivity [1].

**2.3 Microbioreactors to produce monoclonal antibodies**

**94**

Heart-on-a-chip is one such device which is developed to understand the efficacy of drug molecules with cardiac cells. Studies have proved that these devices have the capability to lend a helping hand in drug discovery and development process.

**Figure 2.** *Bioprocess optimization of a CHO cell culture to produce monoclonal antibodies.*

### **3. Perfusion Bioreactors**

#### **3.1 Perfusion technology for biopharmaceutical production**

The leading focus of R&D of any biopharma-based industry is to develop production process at reduced cost of production or rather work toward a minimal Cost of Goods (CoG) for cell culture-based products. Fed-batch mode of production has been the most tried and tested one, documented and dominantly followed methodology for production of biologics or biopharmaceuticals from mammalian cell culture. Due to the presence of expertise on this process within the industry, it has been proved to be the efficient process with judicious use of media coupled with a hassle-free downstream process. However, fed-batch mode has a severe limitation of inhibition of product formation and cell growth due to accumulation of inhibitory products during culture, especially ammonium ions, lactates, and proteases [23]. This leads to loss of key nutrients and incurs massive financial and resource loss to the production setup. This is especially significant if the product is a sensitive one. An alternative to this technology that came up in the early 1990s is perfusion technology. It involves addition of media or key media constituents at regular intervals along with retention of cells in the reactor and harvesting of formed products. However, in the initial phase, due to less advancement in media formulation and process development as well as dearth of efficient expression systems, not much advantage was offered by perfusion-based systems as compared to the wellestablished fed-batch systems and, hence, perfusion technology faced many failures and could not progress significantly. Many companies involved in manufacturing of perfusion-based systems especially for hybridoma-based monoclonal antibodies,

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*Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture*

like Endotronics, Cellex, and Biosyn, also folded up due to low market for perfusion bioreactors [24]. However, the pharma sector was on a constant lookout for newer or more effective production processes and hence advancements were continuously being made in perfusion technology. Now, with major advancements in media formulation, multiple technological options are available for cell retention and a rising trend has been witnessed in pharmaceutical industry for investment in adoption of new technologies and the cost effectiveness of perfusion-based processes, and their product yield increased significantly in comparison to conventional fed-batch processes [25]. Hence, this technology is on a comeback for the last few years and numerous life science companies are advocating the use of perfusion for biologicals

Factor VIII (ReFacto®) and IgG (Remicade® by Jansen Biotech and Simulect® by Novartis) are two of the leading products being produced commercially using perfusion technology [26, 27]. Apart from these, other products include interferon β-1a (Rebif from merck Sereno) and Ab Golimulab (Simponi® by Jansen Biotech), Factor VIII (Kogenate from Bayer Schering); anti-platelet MAb (ReoPro) and tumor necrosis factor MAb (Remicade from Centocor/J&J); CD52 a MAb (Campath from Genzyme/Sanofi); and a modified Factor VIII (Xyntha/ReFacto from Wyeth/ Pfizer) among others. This substantiates the wide application of perfusion-based technologies for a variety of biologicals. The growing need of the pharma sector for reduced cost and enhanced productivity led to the resurgence of perfusion-based production technologies which have the key to revolutionize biopharmaceutical

Perfusion-based bioreactors are one of the upcoming reactor technologies based on continuous bioprocessing that offers the ease of continuous culturing of cells without nuisance of filter clogging or low throughput. In addition, there are less possibilities of waste accumulation and, hence, minimized chances of any product inhibition, especially while dealing with proteins prone to instability. Since nutrients are continuously exchanged and product harvest is maintained throughout along with cell retention, the availability of key media constituents is maintained consistently by providing host cells a stable environment leading to a high cell density and higher productivity with respect to desired compound. Typically,

cells/ml is the titer achieved using perfusion as compared to 5–25 × 106

in fed-batch cultures. Besides that, cost of goods in the 10,000–20,000-l fed-batch reactor is equivalent to that achieved using 1000 -l perfusion bioreactor [28]. Since the cells are subjected to a more stable and consistent environment, the recombinant proteins and other molecules produced are more like native compounds with similar glycosylation pattern and biological activity. This further increases the stability of the product and gives a high product yield. In a recent economical comparison between fed-batch and perfusion mode for the production of a glycosylated protein, CoG was analyzed and compared for both the processes based on BioSolve software. Continuous perfusion was calculated and found to be the most productive technology giving product at the rate of 265 kg/year as compared to 130 kg/year in fed-batch mode. Perfusion was also found to be the most cost-effective mode with the lowest overall CoG of \$87/g as compared to \$118/g for the continuous fed-batch process [29]. Also, there are less possibilities of failure and economic loss. Even if a problem is encountered, only the part being processed would need to be discarded, saving the rest for further processing. In a comparative study on production by both perfusion and fed-batch modes, CMC Biologics reported yield of 425 mg/l/day from perfusion bioreactor as compared to a yield of only 55 mg/l/day from fed-batch

*DOI: http://dx.doi.org/10.5772/intechopen.83825*

production process from mammalian cells.

**3.2 Why perfusion technology?**

production.

3–10 × 107

*Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture DOI: http://dx.doi.org/10.5772/intechopen.83825*

like Endotronics, Cellex, and Biosyn, also folded up due to low market for perfusion bioreactors [24]. However, the pharma sector was on a constant lookout for newer or more effective production processes and hence advancements were continuously being made in perfusion technology. Now, with major advancements in media formulation, multiple technological options are available for cell retention and a rising trend has been witnessed in pharmaceutical industry for investment in adoption of new technologies and the cost effectiveness of perfusion-based processes, and their product yield increased significantly in comparison to conventional fed-batch processes [25]. Hence, this technology is on a comeback for the last few years and numerous life science companies are advocating the use of perfusion for biologicals production.

Factor VIII (ReFacto®) and IgG (Remicade® by Jansen Biotech and Simulect® by Novartis) are two of the leading products being produced commercially using perfusion technology [26, 27]. Apart from these, other products include interferon β-1a (Rebif from merck Sereno) and Ab Golimulab (Simponi® by Jansen Biotech), Factor VIII (Kogenate from Bayer Schering); anti-platelet MAb (ReoPro) and tumor necrosis factor MAb (Remicade from Centocor/J&J); CD52 a MAb (Campath from Genzyme/Sanofi); and a modified Factor VIII (Xyntha/ReFacto from Wyeth/ Pfizer) among others. This substantiates the wide application of perfusion-based technologies for a variety of biologicals. The growing need of the pharma sector for reduced cost and enhanced productivity led to the resurgence of perfusion-based production technologies which have the key to revolutionize biopharmaceutical production process from mammalian cells.

#### **3.2 Why perfusion technology?**

*Biotechnology and Bioengineering*

**3. Perfusion Bioreactors**

**Figure 2.**

**3.1 Perfusion technology for biopharmaceutical production**

*Bioprocess optimization of a CHO cell culture to produce monoclonal antibodies.*

The leading focus of R&D of any biopharma-based industry is to develop production process at reduced cost of production or rather work toward a minimal Cost of Goods (CoG) for cell culture-based products. Fed-batch mode of production has been the most tried and tested one, documented and dominantly followed methodology for production of biologics or biopharmaceuticals from mammalian cell culture. Due to the presence of expertise on this process within the industry, it has been proved to be the efficient process with judicious use of media coupled with a hassle-free downstream process. However, fed-batch mode has a severe limitation of inhibition of product formation and cell growth due to accumulation of inhibitory products during culture, especially ammonium ions, lactates, and proteases [23]. This leads to loss of key nutrients and incurs massive financial and resource loss to the production setup. This is especially significant if the product is a sensitive one. An alternative to this technology that came up in the early 1990s is perfusion technology. It involves addition of media or key media constituents at regular intervals along with retention of cells in the reactor and harvesting of formed products. However, in the initial phase, due to less advancement in media formulation and process development as well as dearth of efficient expression systems, not much advantage was offered by perfusion-based systems as compared to the wellestablished fed-batch systems and, hence, perfusion technology faced many failures and could not progress significantly. Many companies involved in manufacturing of perfusion-based systems especially for hybridoma-based monoclonal antibodies,

Heart-on-a-chip is one such device which is developed to understand the efficacy of drug molecules with cardiac cells. Studies have proved that these devices have the capability to lend a helping hand in drug discovery and development process.

**96**

Perfusion-based bioreactors are one of the upcoming reactor technologies based on continuous bioprocessing that offers the ease of continuous culturing of cells without nuisance of filter clogging or low throughput. In addition, there are less possibilities of waste accumulation and, hence, minimized chances of any product inhibition, especially while dealing with proteins prone to instability. Since nutrients are continuously exchanged and product harvest is maintained throughout along with cell retention, the availability of key media constituents is maintained consistently by providing host cells a stable environment leading to a high cell density and higher productivity with respect to desired compound. Typically, 3–10 × 107 cells/ml is the titer achieved using perfusion as compared to 5–25 × 106 in fed-batch cultures. Besides that, cost of goods in the 10,000–20,000-l fed-batch reactor is equivalent to that achieved using 1000 -l perfusion bioreactor [28]. Since the cells are subjected to a more stable and consistent environment, the recombinant proteins and other molecules produced are more like native compounds with similar glycosylation pattern and biological activity. This further increases the stability of the product and gives a high product yield. In a recent economical comparison between fed-batch and perfusion mode for the production of a glycosylated protein, CoG was analyzed and compared for both the processes based on BioSolve software. Continuous perfusion was calculated and found to be the most productive technology giving product at the rate of 265 kg/year as compared to 130 kg/year in fed-batch mode. Perfusion was also found to be the most cost-effective mode with the lowest overall CoG of \$87/g as compared to \$118/g for the continuous fed-batch process [29]. Also, there are less possibilities of failure and economic loss. Even if a problem is encountered, only the part being processed would need to be discarded, saving the rest for further processing. In a comparative study on production by both perfusion and fed-batch modes, CMC Biologics reported yield of 425 mg/l/day from perfusion bioreactor as compared to a yield of only 55 mg/l/day from fed-batch

system for the same period of time [30]. Since most of the pharma companies thrive on economic profits, perfusion technology offers a lucrative mode of production especially as it beats the conventional fed-batch system in terms of productivity, efficiency, and capital investments.

#### **3.3 Cell retention in perfusion**

The prominent aspect which makes perfusion systems different and more valuable than fed-batch systems is the ability to yield a high cell mass due to the presence of cell retention devices. There are various ways through which cell retention is achieved [31]. Cells can be retained by making them grow inside bioreactor on hollow capillary fibers, flat plates, sponge-like materials, microcarrier particles, or other membranes. It can also be done by use of various cell separation devices like gravity-based cell settlers, spin filters, centrifuges, cross-flow filters, alternating tangential-flow filters, vortex-flow filters, acoustic settlers (sonoperfusion), and hydrocyclones [32]. Spin filter was one of the earliest available devices for cell retention which used a two-dimensional screen to retain the cells. However, it had limited scale-up potential especially in the scenario where rapid feed rate is needed. Gravity-based cell settlers are cost-effective but are marked by inefficient cell separation and significant cell loss, which lowers output and increases cost. Centrifuges have been known to give good performance but increase the production cost. Alternating tangential-flow filters (TFFs) have emerged as the most effective and practical means of high-density cell retention in a perfusion bioreactor [33]. The alternating tangential-flow action in these filters and location of diaphragm in the system prevent clogging as well as ensure a faster return of cells back to the reactor, bringing complete clarification. However, what need to be worked upon are other reactor specifications for handling large cell load at reduced volume and culture time. Also, scalability complications are a deterrent for many manufacturers. Many companies are targeting advancements in ATF system to handle increased cell load at smaller reactor volume. In a recent report on biologics development and manufacturing, the advancement in perfusion and its leading incorporation in manufacturing processes by leading biologics-based companies was attributed mostly to the advancements made in ATF systems which enhance the cell titers by multiple folds over extended periods of time, leading to higher volumetric productivity [34]. Acoustic wave separation (AWS) is another technology used by many companies for cell separation. Applikon Biotechnology and Pall Life Sciences are two such manufacturers advocating the use of acoustic waves to clump and settle down cells leading to their eventual separation. Sigma Aldrich Co. LLC (Merck & Co. Inc.), FiberCell Systems Inc., Zellwerk GmbH (Glen Mills in the United States), Cell Culture Company, ATMI Incorporated, PBS Biotech, Inc., GE Healthcare Life Sciences, Applikon Biotechnology, WAVE Life Sciences Biovest, AmProtein, Xcellerex, etc. are few of the leading manufacturers of perfusion bioreactors [35]. These reactors are revolutionizing the biopharmaceutical production industry and have established their presence in this sector preferably to stay for many years to come.

#### **3.4 Perfusion and microbioreactors**

Integration of perfusion technology with microbioreactors enhances the advantages associated with microbioreactors effectively. It further minimizes the losses associated with batch failure due to contamination. Even if contamination occurs earlier in the process, lesser media and other consumables would be wasted. However, the compatibility of the setup is amenable to technology

**99**

opment costs [39].

**3.5 Future of perfusion technology**

Healthcare, and many others.

*Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture*

development, scale-up, optimization, parameter sensitivity studies, and validation. Advancements in cell retention systems at microfluidic levels continue to be made. In a recent report, a novel microfluidic cell retention device based on inertial sorting was tested positively for retention of IgG1-producing Chinese hamster ovary (CHO) cell line. Parameters tested were cell retention efficiency, biocompatibility, and scalability. This was a spiral membrane-less system accomplishing cell retention based on hydrodynamic forces. The device was fabricated with polydimethylsiloxane (PDMS) and connected to spinner vessel-containing cells. There was also the flexibility of configuring the device to separate different-sized cells with a specific input flow rate. This gave an added advantage of flushing out non-viable cells, thus creating a healthier environment for the productive cell batch, leading to an increased productivity and product specificity [36]. In a first-of-its-kind study on feasibility of growth of human embryonic stem cells (hESCs), in continuousflow microbioreactors, key cellular behavioral factors were assessed for robust cell growth under a range of flow rates. This would provide an assay platform for screening multiple cell lines for their capability to function in perfusion culture conditions as well as would aid in identifying optimum flow rates for their application in other microfluidic cell culture systems. Such microfluidic reactor setups would help in better understanding of stem cells maintenance and differentiation under varying stimulation conditions [37]. Perfusion microfluidic systems can also be used for growth and expression of proteins from bacterial cells. Growth of suspended cultures of *Pichia pastoris* in microbioreactor, integrated with perfusion devices, established the feasibility of use of such integrated systems for suspended bacterial growth and expression. Expression of recombinant human growth hormone (rhGH) or recombinant interferon alfa-2b (rIFNα-2b) by *Pichia pastoris* was found to be maintained after 11 days of growth which is close to the duration of many industrial processes with duration lasting from 7 to 30 days. Success of this study establishes the feasibility of use of perfusion-capable bioreactors to study impact of perfusion culture on expression and process optimization of many other microbial systems [38] at microfluidic levels, thus saving the initial process devel-

Despite being in the sector for many years, perfusion technology did not gain much usage. However, with new methods for cell retention in the market and growing need for high throughput mammalian cell-based production systems, there is a renewed interest in the use of perfusion bioreactors. With a low capital and start-up cost, smaller setup, a smaller requirement for upstream and downstream processes, and reduced cost of failures, more and more compounds are being commercially produced using this technology. From leading biological manufacturers like Pfizer to upcoming ones like CMC Biologics, many companies are propagating the use of this technology with setup ranging from 250 to 2000 liters. Current research, however, would focus on scalability of the technology, creating a robust cell retention system, high-yield cell lines used in single-use technologies, cutting down on the initial establishment cost and creating the availability of expertise in perfusion-based devices and also the ease of bioprocessing modeling during continuous mode of operation. Also of relevance and on the rise is the use of single-use perfusion-based reactors for production. There are many manufacturers dealing with development of single-use bioreactors, sensors, and other systems to simplify setup and operation of perfusion cell culture. These include among Pall Life Sciences, Applikon Biotechnology, Sartorius, AcuSyst, GE

*DOI: http://dx.doi.org/10.5772/intechopen.83825*

*Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture DOI: http://dx.doi.org/10.5772/intechopen.83825*

development, scale-up, optimization, parameter sensitivity studies, and validation. Advancements in cell retention systems at microfluidic levels continue to be made. In a recent report, a novel microfluidic cell retention device based on inertial sorting was tested positively for retention of IgG1-producing Chinese hamster ovary (CHO) cell line. Parameters tested were cell retention efficiency, biocompatibility, and scalability. This was a spiral membrane-less system accomplishing cell retention based on hydrodynamic forces. The device was fabricated with polydimethylsiloxane (PDMS) and connected to spinner vessel-containing cells. There was also the flexibility of configuring the device to separate different-sized cells with a specific input flow rate. This gave an added advantage of flushing out non-viable cells, thus creating a healthier environment for the productive cell batch, leading to an increased productivity and product specificity [36]. In a first-of-its-kind study on feasibility of growth of human embryonic stem cells (hESCs), in continuousflow microbioreactors, key cellular behavioral factors were assessed for robust cell growth under a range of flow rates. This would provide an assay platform for screening multiple cell lines for their capability to function in perfusion culture conditions as well as would aid in identifying optimum flow rates for their application in other microfluidic cell culture systems. Such microfluidic reactor setups would help in better understanding of stem cells maintenance and differentiation under varying stimulation conditions [37]. Perfusion microfluidic systems can also be used for growth and expression of proteins from bacterial cells. Growth of suspended cultures of *Pichia pastoris* in microbioreactor, integrated with perfusion devices, established the feasibility of use of such integrated systems for suspended bacterial growth and expression. Expression of recombinant human growth hormone (rhGH) or recombinant interferon alfa-2b (rIFNα-2b) by *Pichia pastoris* was found to be maintained after 11 days of growth which is close to the duration of many industrial processes with duration lasting from 7 to 30 days. Success of this study establishes the feasibility of use of perfusion-capable bioreactors to study impact of perfusion culture on expression and process optimization of many other microbial systems [38] at microfluidic levels, thus saving the initial process development costs [39].

#### **3.5 Future of perfusion technology**

Despite being in the sector for many years, perfusion technology did not gain much usage. However, with new methods for cell retention in the market and growing need for high throughput mammalian cell-based production systems, there is a renewed interest in the use of perfusion bioreactors. With a low capital and start-up cost, smaller setup, a smaller requirement for upstream and downstream processes, and reduced cost of failures, more and more compounds are being commercially produced using this technology. From leading biological manufacturers like Pfizer to upcoming ones like CMC Biologics, many companies are propagating the use of this technology with setup ranging from 250 to 2000 liters. Current research, however, would focus on scalability of the technology, creating a robust cell retention system, high-yield cell lines used in single-use technologies, cutting down on the initial establishment cost and creating the availability of expertise in perfusion-based devices and also the ease of bioprocessing modeling during continuous mode of operation. Also of relevance and on the rise is the use of single-use perfusion-based reactors for production. There are many manufacturers dealing with development of single-use bioreactors, sensors, and other systems to simplify setup and operation of perfusion cell culture. These include among Pall Life Sciences, Applikon Biotechnology, Sartorius, AcuSyst, GE Healthcare, and many others.

*Biotechnology and Bioengineering*

efficiency, and capital investments.

**3.3 Cell retention in perfusion**

system for the same period of time [30]. Since most of the pharma companies thrive on economic profits, perfusion technology offers a lucrative mode of production especially as it beats the conventional fed-batch system in terms of productivity,

The prominent aspect which makes perfusion systems different and more valuable than fed-batch systems is the ability to yield a high cell mass due to the presence of cell retention devices. There are various ways through which cell retention is achieved [31]. Cells can be retained by making them grow inside bioreactor on hollow capillary fibers, flat plates, sponge-like materials, microcarrier particles, or other membranes. It can also be done by use of various cell separation devices like gravity-based cell settlers, spin filters, centrifuges, cross-flow filters, alternating tangential-flow filters, vortex-flow filters, acoustic settlers (sonoperfusion), and hydrocyclones [32]. Spin filter was one of the earliest available devices for cell retention which used a two-dimensional screen to retain the cells. However, it had limited scale-up potential especially in the scenario where rapid feed rate is needed. Gravity-based cell settlers are cost-effective but are marked by inefficient cell separation and significant cell loss, which lowers output and increases cost. Centrifuges have been known to give good performance but increase the production cost. Alternating tangential-flow filters (TFFs) have emerged as the most effective and practical means of high-density cell retention in a perfusion bioreactor [33]. The alternating tangential-flow action in these filters and location of diaphragm in the system prevent clogging as well as ensure a faster return of cells back to the reactor, bringing complete clarification. However, what need to be worked upon are other reactor specifications for handling large cell load at reduced volume and culture time. Also, scalability complications are a deterrent for many manufacturers. Many companies are targeting advancements in ATF system to handle increased cell load at smaller reactor volume. In a recent report on biologics development and manufacturing, the advancement in perfusion and its leading incorporation in manufacturing processes by leading biologics-based companies was attributed mostly to the advancements made in ATF systems which enhance the cell titers by multiple folds over extended periods of time, leading to higher volumetric productivity [34]. Acoustic wave separation (AWS) is another technology used by many companies for cell separation. Applikon Biotechnology and Pall Life Sciences are two such manufacturers advocating the use of acoustic waves to clump and settle down cells leading to their eventual separation. Sigma Aldrich Co. LLC (Merck & Co. Inc.), FiberCell Systems Inc., Zellwerk GmbH (Glen Mills in the United States), Cell Culture Company, ATMI Incorporated, PBS Biotech, Inc., GE Healthcare Life Sciences, Applikon Biotechnology, WAVE Life Sciences Biovest, AmProtein, Xcellerex, etc. are few of the leading manufacturers of perfusion bioreactors [35]. These reactors are revolutionizing the biopharmaceutical production industry and have established their presence in this sector preferably to stay

**98**

for many years to come.

**3.4 Perfusion and microbioreactors**

Integration of perfusion technology with microbioreactors enhances the advantages associated with microbioreactors effectively. It further minimizes the losses associated with batch failure due to contamination. Even if contamination occurs earlier in the process, lesser media and other consumables would be wasted. However, the compatibility of the setup is amenable to technology

### **4. Conclusion**

Microbioreactors are essential for high throughput screening of various strains and optimize the bioprocess development in industries. Microbioreactors assist in identifying the appropriate experimental conditions to scale up the production process. Production of biopharmaceuticals, enzymes, proteins, and medicinally important chemical compounds from organisms was attempted successfully. Microbioreactors have been demonstrated as a suitable technology to cultivate *Pichia pastoris*, *Escherichia coli*, monoclonal antibodies, etc., at large scale that are important for biopharmaceutical industries. Incorporation of microbioreactors in drug discovery and development will reduce the cost and time for developing therapeutic molecules. The growing need for a high production process and efficient production strategy has also championed the cause of perfusion-based technology interfaced with microbioreactors. Perfusion microfluidic bioreactors have been extremely beneficial in maintaining stem cells, expression of proteins, growth of cells, and microbial cultures. Thus, microbioreactors aid in developing high-quality products at affordable cost with minimal resources.

### **Acknowledgements**

Authors thank Symbiosis International (Deemed University) for funding and research facility.

#### **Conflict of interest**

None declared.

#### **Author details**

Selvan Ravindran1 \*, Pooja Singh1 , Sanjay Nene2 , Vinay Rale1 , Nutan Mhetras1 and Anuradha Vaidya1

1 Symbiosis School of Biological Sciences, Symbiosis International (Deemed) University, Pune, India

2 Innovation Biologicals Private Limited, Pune, India

\*Address all correspondence to: selvan\_ravindran@yahoo.com

© 2019 The Author(s). Licensee IntechOpen. 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.

**101**

*Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture*

labs.com

2014;**82**:105-116

2015;**31**:623-632

note, 1-6. Available from: www.m2p-

[10] Muhlman M, Kunze M, Ribeiro J, Geinitz B, Lehmann C, Schwaneberg U, et al. Cellulolytic robolector-towards an automated high-throughput screening platform for recombinant cellulose expression. Journal of Biological Engineering. 2017;**11**(1):1-18

[11] Betts JPJ, Warr SRC, Finka GB, Uden M, Town M, Janda JM, et al. Impact of aeration strategies on fed-batch cell culture kinetics in a single-use 24-well miniature bioreactor. Biochemical Engineering Journal.

[12] Janakiraman V, Kwiatkowski C, Kshirsagar R, Ryll T, Huang YM. Application of high-throughput mini-bioreactor system for systematic

scale-down modeling, process characterization and control strategy development. Biotechnology Progress.

and typical manufacturing

2010;**26**:1400-1410

2008;**99**:884-892

[13] Huang YM, Hu WW, Rustandi E, Chang K, Makagianser YK, Ryll T. Maximizing productivity of CHO cell based fed-batch culture using chemically defined media conditions

equipment. Biotechnology Progress.

[14] Kommenhoek EE, Van Leeuwen M, Gardeniers H, Van Gulik WM, Van den Berg A, Li X, et al. Lab-scale fermentation tests of microchip with integrated electrochemical sensors for pH, temperature, dissolved oxygen and viable biomass concentration. Biotechnology and Bioengineering.

[15] Mandenius CF. Conceptual design of micro-bioreactors and organ-onchips for studies of cell cultures. Bioengineering. 2018;**5**(56):1-22

*DOI: http://dx.doi.org/10.5772/intechopen.83825*

[1] Kensy F, Hemmerich J. Automation of microbioreactors—operating 48 parallel fed-batch fermentations at microscale. Bioprocess International.

[2] Adrio JL, Demain AL. Microbial enzymes: Tools for Biotechnological

[3] Hemmerich J, Kensy F. Automated

pharmaceutical bioprocessing: Profiling of seeding and induction conditions in high throughput fermentations. Pharmaceutical Bioprocessing.

[4] Erickson B, Winters PN. Perspective

**References**

2013;**11**(8):68-76

2014;**2**(3):227-235

2012;**7**(2):176-185

2018;**13**:e1700141

processes. Biomolecules. 2014;**4**(1):117-139

microbioreactor systems for

on opportunities in industrial biotechnology in renewable chemicals. Biotechnology Journal.

[5] Unthan S, Radek A, Wiechert W, Oldiges M, Noack S. Bioprocess automation on a mini pilot plant enables fast quantitative microbial phenotyping. Microbial Cell Factories. 2015;**14**:1-11

[6] Noack S, Wiechert W. Quantitative metabolomics: A platform? Trends in Biotechnology. 2014;**32**(5):238-244

[7] Hemmerich J, Noack S, Weichert W, Oldiges M. Microbioreactor systems for accelerated bioprocess development. Biotechnology Journal.

[8] Toeroek C, Puschmann MC, Bayer K, Striedner G. Fed-batch like cultivation in a micro-bioreactor: Screening conditions relevant for *Escherichia coli* based production processes. SpringerPlus. 2015;**4**(490):1-10

[9] 32 parallel microbioreactors pH control continuous feeding online monitoring scalability, Application *Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture DOI: http://dx.doi.org/10.5772/intechopen.83825*

#### **References**

*Biotechnology and Bioengineering*

products at affordable cost with minimal resources.

\*, Pooja Singh1

2 Innovation Biologicals Private Limited, Pune, India

\*Address all correspondence to: selvan\_ravindran@yahoo.com

**4. Conclusion**

**Acknowledgements**

**Conflict of interest**

None declared.

**Author details**

Selvan Ravindran1

and Anuradha Vaidya1

University, Pune, India

research facility.

**100**

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. 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,

, Sanjay Nene2

1 Symbiosis School of Biological Sciences, Symbiosis International (Deemed)

, Vinay Rale1

, Nutan Mhetras1

Microbioreactors are essential for high throughput screening of various strains and optimize the bioprocess development in industries. Microbioreactors assist in identifying the appropriate experimental conditions to scale up the production process. Production of biopharmaceuticals, enzymes, proteins, and medicinally important chemical compounds from organisms was attempted successfully. Microbioreactors have been demonstrated as a suitable technology to cultivate *Pichia pastoris*, *Escherichia coli*, monoclonal antibodies, etc., at large scale that are important for biopharmaceutical industries. Incorporation of microbioreactors in drug discovery and development will reduce the cost and time for developing therapeutic molecules. The growing need for a high production process and efficient production strategy has also championed the cause of perfusion-based technology interfaced with microbioreactors. Perfusion microfluidic bioreactors have been extremely beneficial in maintaining stem cells, expression of proteins, growth of cells, and microbial cultures. Thus, microbioreactors aid in developing high-quality

Authors thank Symbiosis International (Deemed University) for funding and

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[3] Hemmerich J, Kensy F. Automated microbioreactor systems for pharmaceutical bioprocessing: Profiling of seeding and induction conditions in high throughput fermentations. Pharmaceutical Bioprocessing. 2014;**2**(3):227-235

[4] Erickson B, Winters PN. Perspective on opportunities in industrial biotechnology in renewable chemicals. Biotechnology Journal. 2012;**7**(2):176-185

[5] Unthan S, Radek A, Wiechert W, Oldiges M, Noack S. Bioprocess automation on a mini pilot plant enables fast quantitative microbial phenotyping. Microbial Cell Factories. 2015;**14**:1-11

[6] Noack S, Wiechert W. Quantitative metabolomics: A platform? Trends in Biotechnology. 2014;**32**(5):238-244

[7] Hemmerich J, Noack S, Weichert W, Oldiges M. Microbioreactor systems for accelerated bioprocess development. Biotechnology Journal. 2018;**13**:e1700141

[8] Toeroek C, Puschmann MC, Bayer K, Striedner G. Fed-batch like cultivation in a micro-bioreactor: Screening conditions relevant for *Escherichia coli* based production processes. SpringerPlus. 2015;**4**(490):1-10

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[11] Betts JPJ, Warr SRC, Finka GB, Uden M, Town M, Janda JM, et al. Impact of aeration strategies on fed-batch cell culture kinetics in a single-use 24-well miniature bioreactor. Biochemical Engineering Journal. 2014;**82**:105-116

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[36] Kwon T, Prentice H, De Oliveira J, Madziva N, Warkiani ME, Hamel JFP, et al. Microfluidic cell retention device for perfusion of mammalian suspension culture. Scientific Reports.

2017;**7**(1):6703. DOI: 10.1038/

[37] Titmarsh D, Hidalgo A, Turner J, Wolvetang E, Cooper-White J. Optimization of flowrate for expansion of human embryonic stem cells in perfusion microbioreactors. Biotechnology and Bioengineering.

[38] Rokade R, Ravindran S, Singh p SJ. Microbial biotransformation for the production of steroid medicament. In: Vijayakumar R, Raja SSS, editors. Secondary Metabolites—Sources and Applications. London, United Kingdom:

InTech Open; 2018. pp. 115-124

[39] Mozdzierz NJ, Love KR, Lee KS, Lee HL, Shah KA, Ram RJ, et al. A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production. Lab on

a Chip. 2015;**15**(14):2918-2922

s41598-017-06949-8

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[Accessed: 21-11-2018]

*Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture DOI: http://dx.doi.org/10.5772/intechopen.83825*

[33] Carter JB and Shevitz J. A Brief History of Perfusion Biomanufacturing. 2011. Available from: https://pdfs. semanticscholar.org/0131/5d5a12ed 010e8ee20882cfa8dfbf6546484f.pdf [Accessed: 21-11-2018]

[34] Mahler G. Advancing Biologics development and Manufacturing. 2016. Available from: http://www. agcbio.com/resource-center/news/ advancing-biologics-development-andmanufacturing [Accessed: 26-11-2018]

[35] Perfusion Bioreactors Market: Global Industry Analysis and Forecast 2017-2025. Available from: https:// www.persistencemarketresearch.com/ market-research/perfusion-bioreactorsmarket.asp [Assessed: 27-11-2018]

[36] Kwon T, Prentice H, De Oliveira J, Madziva N, Warkiani ME, Hamel JFP, et al. Microfluidic cell retention device for perfusion of mammalian suspension culture. Scientific Reports. 2017;**7**(1):6703. DOI: 10.1038/ s41598-017-06949-8

[37] Titmarsh D, Hidalgo A, Turner J, Wolvetang E, Cooper-White J. Optimization of flowrate for expansion of human embryonic stem cells in perfusion microbioreactors. Biotechnology and Bioengineering. 2011;**108**(12):2894-2904

[38] Rokade R, Ravindran S, Singh p SJ. Microbial biotransformation for the production of steroid medicament. In: Vijayakumar R, Raja SSS, editors. Secondary Metabolites—Sources and Applications. London, United Kingdom: InTech Open; 2018. pp. 115-124

[39] Mozdzierz NJ, Love KR, Lee KS, Lee HL, Shah KA, Ram RJ, et al. A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production. Lab on a Chip. 2015;**15**(14):2918-2922

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2014;**28**:1547-1553

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[24] Langer ES. Trends in perfusion bioreactors. Bioprocess International.

[25] Langer ES, Rader RA. Continuous bioprocessing and perfusion: Wider adoption coming as bioprocessing matures. Bioprocessing Journal.

[26] Farid SS. Established bioprocesses for producing antibodies as a basis for future planning. In: Scheper T, editor. Advances in Biochemical Engineering/ Biotechnology. Vol. 2006. Heidelberg:

[27] Kelley B, Jankowski M, Booth J. An improved manufacturing process for Xyntha/ReFacto AF. Haemophilia.

[28] Compton BJ. Use of Perfusion Technology on the Rise. 2007. Available from: https://www.genengnews.com/ magazine/use-of-perfusion-technologyon-the-rise/ [Accessed: 21-11-2018]

[29] Lim J, Sinclair A, Shevitz J, Bonham-Carter J. An economic comparison of three cell culture techniques. BioPharm

[30] Carstens JN, Clarke HRG and Jensen JP. Perfusion! Jeopardy or the Ultimate Advantage? CMC Biologics. September, 2009. Available from: http://www. cmcbio.com/ Portals/0/CMC/docs/ perfusion.pdf [Accessed: 21-11-2018]

[31] Voisard D, Meuwly F, Ruffieux PA, Baer G, Kadouri A. Potential of cell retention techniques for largescale high-density perfusion culture of suspended mammalian cells. Biotechnology and Bioengineering.

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[18] Ravindran S, Rokade R, Suthar J, Singh P, Deshpande P, Khambadkar R, et al. *In vitro* biotransformation in drug discovery. In: Boparalla V, editor. Drug Discovery—Concepts to Market. London, United Kingdom: InTech Open;

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characterize and quantitate metabolites in drug discovery and development. Biomedical Chromatography.

[20] Surve P, Ravindran S, Acharjee A, Rastogi H, Basu S, Honrao P. Metabolite characterization of anti-cancer agent Gefitinib in human hepatocytes. Drug Metabolism Letters. 2013;**7**:126-136

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mammalian cell culture for recombinant

**105**

**Chapter 8**

**Abstract**

biocatalysis

**1. Introduction**

Bioreactors

are presented for both applications.

(environmental-friendly processes) [2–4].

electrodialysis (ED), etc., will be presented.

inserted into bioprocesses.

Integration of Membranes and

Combined application of bioreactors and membrane separations are considered as membrane bioreactors (MBRs). Examples for the application of MBRs are given in this chapter both for large scale (wastewater treatments) and in other areas in smaller scale. Wastewater treatments are the majority of the large-scale applications, where biological degradation is coupled with membrane filtration (microfiltration and ultrafiltration). Other types of MBRs include integration of biotransformations and bioconversions by microorganisms and enzymes with membrane separation processes, not only with filtration but also with pervaporation, electrodialysis, and gas separation. These MBRs provide significant advantages compared to the conventional batch bioprocesses. In this chapter, several examples

**Keywords:** ultrafiltration, pervaporation, electrodialysis, gas separation,

"As per definition, the bioreactor is the designed space where biochemical reactions take place" [1]. If some compounds should be removed from the bioreactor, a separation step can be connected to the reactor. Among separation processes, membrane techniques are especially attractive since they operate under mild conditions, they are easy to combine and vary, the scale-up is simple due to the modular construction, and they do not need and produce hazardous materials

Application of membranes integrated in bioreactions is often considered as membrane bioreactors (MBRs). In the literature [5–8] the term *MBRs* itself is referred almost exclusively for various wastewater treatments. Concerning fullscale applications, it seems correct. In these technologies only pressure-driven membrane processes such as microfiltration (MF), ultrafiltration (MF), nanofiltration (NF), and reverse osmosis (RO) have been used, although there are numerous other membrane separation techniques that are available nowadays, which can be

In this work the first section is going to summarize the "conventional" applications of MBRs, i.e., wastewater treatments, while in the second part, some other types of MBRs will be presented where—beyond pressure-driven processes—some more membrane separation methods, like pervaporation (PV), gas separation (GS),

*Katalin Belafi-Bako and Peter Bakonyi*

#### **Chapter 8**

## Integration of Membranes and Bioreactors

*Katalin Belafi-Bako and Peter Bakonyi*

#### **Abstract**

Combined application of bioreactors and membrane separations are considered as membrane bioreactors (MBRs). Examples for the application of MBRs are given in this chapter both for large scale (wastewater treatments) and in other areas in smaller scale. Wastewater treatments are the majority of the large-scale applications, where biological degradation is coupled with membrane filtration (microfiltration and ultrafiltration). Other types of MBRs include integration of biotransformations and bioconversions by microorganisms and enzymes with membrane separation processes, not only with filtration but also with pervaporation, electrodialysis, and gas separation. These MBRs provide significant advantages compared to the conventional batch bioprocesses. In this chapter, several examples are presented for both applications.

**Keywords:** ultrafiltration, pervaporation, electrodialysis, gas separation, biocatalysis

#### **1. Introduction**

"As per definition, the bioreactor is the designed space where biochemical reactions take place" [1]. If some compounds should be removed from the bioreactor, a separation step can be connected to the reactor. Among separation processes, membrane techniques are especially attractive since they operate under mild conditions, they are easy to combine and vary, the scale-up is simple due to the modular construction, and they do not need and produce hazardous materials (environmental-friendly processes) [2–4].

Application of membranes integrated in bioreactions is often considered as membrane bioreactors (MBRs). In the literature [5–8] the term *MBRs* itself is referred almost exclusively for various wastewater treatments. Concerning fullscale applications, it seems correct. In these technologies only pressure-driven membrane processes such as microfiltration (MF), ultrafiltration (MF), nanofiltration (NF), and reverse osmosis (RO) have been used, although there are numerous other membrane separation techniques that are available nowadays, which can be inserted into bioprocesses.

In this work the first section is going to summarize the "conventional" applications of MBRs, i.e., wastewater treatments, while in the second part, some other types of MBRs will be presented where—beyond pressure-driven processes—some more membrane separation methods, like pervaporation (PV), gas separation (GS), electrodialysis (ED), etc., will be presented.

#### **2. Large-scale applications**

The usage of membrane bioreactors means a well-established technology for several types of wastewater treatments. The recently worldwide opened (of will opened soon) large-scale—over 100,000 m3 /d capacity—MBR plants for municipal wastewater treatment are summarized in [8]. Other main application areas are as follows [6, 8]:


In these applications MBRs are considered as complex systems integrating biological degradation of waste products with membrane filtration [6]. Thus an MBR is composed of two parts: a biological unit and a membrane module. According to the location of the membrane module (from the aspect of architecture), the MBRs can be classified into two groups: internal and external systems. The internal system (frequently called submerged MBR) of the membrane module is placed in the bioreactor [6]. Outer skin membranes are applied, and the permeate side is under suction (vacuum); moreover, aeration and mixing can be easily achieved, as well. In the external MBR, the membrane is located outside the bioreactor, and the treated wastewater is recirculated through the primary side of the membrane module. The driving force is provided by the pressure from the high cross flow volumetric rate along the membrane surface [6].

Regarding configuration there have been numerous types of modules (plate and frame, tubular, rotary disk, hollow fiber) and membranes (cellulose acetate, polyethylene, polysulfone, polyolefin, metallic, ceramic—mainly in ultrafiltration and microfiltration range) applied.

MBRs are widely considered as an effective technology in removing both organic and inorganic contaminants in wastewater, have a good control of biological activity—the effluent is generally free of bacteria and pathogens—need smaller plant size, and provide higher organic loading rates [5, 6]. Beyond the benefits, however, there are some serious drawbacks and issues which should be enhanced, like membrane fouling and energy consumption. To solve these problems and to widen the application opportunities, several special techniques and methods have been investigated and suggested, where unusual environments (e.g., varying the level of oxygen, flow pattern, airlift) and unique procedures (e.g., applying electric power, magnetic effects, illumination, vibration, osmosis) are added and/or applied for the MBR systems. Some of them are listed in **Table 1**, together with their abbreviations.

*Anaerobic* degradation of organic wastes [10, 11]—similar to the classical aerobic wastewater treatments—can be carried out by microbial consortia. The technology results in gas products ("biogas"). It consists usually of methane, CO2, and H2S mainly. To separate these compounds, membranes can be applied, as well, but these membranes have selectivity toward one of the compounds in the gaseous mixture [12, 15]. The process is called membrane gas separation, and its driving force is mainly the pressure difference, similar to UF and MF.

Recently biogas plants are built in connection with wastewater plants to complete the degradation process and to obtain energy which may cover the energy demand of the process; moreover other types of wastes (slaughter wastes,

**107**

*Integration of Membranes and Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.84513*

agricultural wastes) can be degraded, as well. In these complex plants, MBRs can be applied in both aerobic (coupled with pressure-driven membrane techniques) and

Membrane photobioreactor MPBR [8] Osmotic membrane bioreactor OMBR [7] Reciprocation membrane bioreactor rMBR [8] Single fiber membrane gradostat reactor SFMGR [14]

**Name Abbr. Ref**

Anaerobic fluidized bed membrane bioreactor AFMBR [9] Anaerobic membrane bioreactor AnMBR [6–11] Airlift oxidation ditch membrane bioreactor AOXMBR [8] Bioelectrochemical membrane reactor BEMR [8] Batch granulation membrane bioreactor BG-MBR [8] Baffled membrane bioreactor BMBR [10] Electrochemical membrane bioreactor EMBR [8] Gas separation—membrane bioreactor GS-MBR [12] Hybrid-growth membrane bioreactor HG-MBR [9] Immersed hollow fiber membrane bioreactor IHFMB [13] Membrane electro-bioreactor MEBR [8] Membrane gradostat reactor MGR [14]

O MBR [8]

MMV-MBR [8]

Anaerobic-anoxic-oxic membrane bioreactor A2

Integrated systems where membranes are combined with the bioreactions other than wastewater degradation—can be classified similarly: external and integral setups. However, it is difficult to connect certain types of membrane processes (e.g., pervaporation, electrodialysis) to the bioreactor *externally*; thus they are used as *internal* systems. **Figure 1** presents an example for internal MBR, where the configuration is illustrated. These systems have the advantage to handle (e.g., disconnect easily) the membrane module independently from the bioreactor. On the other hand, there are successful examples for external (immersed membranes) MBR systems, which can be applied in special cases, e.g., for manufacture of pharmaceutical intermediaries. Loh and colleagues reported [13] that the biotransformation of indene to cis-indandiol was achieved in an IHFMB system

MBR systems can be distinguished according to the membrane process integrated [16, 17]. Beyond pressure-driven methods (microfiltration, ultrafiltration, nanofiltration), other techniques like pervaporation, electrodialysis, and gas

anaerobic (coupled with membrane gas separation) systems.

**3. Novel applications in developing stage**

Magnetically induced membrane vibration membrane

bioreactor

**Table 1.**

**3.1 Types of MBRs and classifications**

*Various unusual membrane bioreactors.*

resulting in higher effectiveness.


#### **Table 1.**

*Biotechnology and Bioengineering*

**2. Large-scale applications**

follows [6, 8]:

opened soon) large-scale—over 100,000 m3

• Industrial wastewater treatment

• Treatment of human excrement

• Landfill leachate treatment

along the membrane surface [6].

and microfiltration range) applied.

mainly the pressure difference, similar to UF and MF.

Recently biogas plants are built in connection with wastewater plants to complete the degradation process and to obtain energy which may cover the energy demand of the process; moreover other types of wastes (slaughter wastes,

• Sludge digestion

The usage of membrane bioreactors means a well-established technology for several types of wastewater treatments. The recently worldwide opened (of will

wastewater treatment are summarized in [8]. Other main application areas are as

In these applications MBRs are considered as complex systems integrating biological degradation of waste products with membrane filtration [6]. Thus an MBR is composed of two parts: a biological unit and a membrane module. According to the location of the membrane module (from the aspect of architecture), the MBRs can be classified into two groups: internal and external systems. The internal system (frequently called submerged MBR) of the membrane module is placed in the bioreactor [6]. Outer skin membranes are applied, and the permeate side is under suction (vacuum); moreover, aeration and mixing can be easily achieved, as well. In the external MBR, the membrane is located outside the bioreactor, and the treated wastewater is recirculated through the primary side of the membrane module. The driving force is provided by the pressure from the high cross flow volumetric rate

Regarding configuration there have been numerous types of modules (plate and frame, tubular, rotary disk, hollow fiber) and membranes (cellulose acetate, polyethylene, polysulfone, polyolefin, metallic, ceramic—mainly in ultrafiltration

MBRs are widely considered as an effective technology in removing both organic and inorganic contaminants in wastewater, have a good control of biological activity—the effluent is generally free of bacteria and pathogens—need smaller plant size, and provide higher organic loading rates [5, 6]. Beyond the benefits, however, there are some serious drawbacks and issues which should be enhanced, like membrane fouling and energy consumption. To solve these problems and to widen the application opportunities, several special techniques and methods have been investigated and suggested, where unusual environments (e.g., varying the level of oxygen, flow pattern, airlift) and unique procedures (e.g., applying electric power, magnetic effects, illumination, vibration, osmosis) are added and/or applied for the MBR systems. Some of them are listed in **Table 1**, together with their abbreviations. *Anaerobic* degradation of organic wastes [10, 11]—similar to the classical aerobic wastewater treatments—can be carried out by microbial consortia. The technology results in gas products ("biogas"). It consists usually of methane, CO2, and H2S mainly. To separate these compounds, membranes can be applied, as well, but these membranes have selectivity toward one of the compounds in the gaseous mixture [12, 15]. The process is called membrane gas separation, and its driving force is

/d capacity—MBR plants for municipal

**106**

*Various unusual membrane bioreactors.*

agricultural wastes) can be degraded, as well. In these complex plants, MBRs can be applied in both aerobic (coupled with pressure-driven membrane techniques) and anaerobic (coupled with membrane gas separation) systems.

#### **3. Novel applications in developing stage**

#### **3.1 Types of MBRs and classifications**

Integrated systems where membranes are combined with the bioreactions other than wastewater degradation—can be classified similarly: external and integral setups. However, it is difficult to connect certain types of membrane processes (e.g., pervaporation, electrodialysis) to the bioreactor *externally*; thus they are used as *internal* systems. **Figure 1** presents an example for internal MBR, where the configuration is illustrated. These systems have the advantage to handle (e.g., disconnect easily) the membrane module independently from the bioreactor.

On the other hand, there are successful examples for external (immersed membranes) MBR systems, which can be applied in special cases, e.g., for manufacture of pharmaceutical intermediaries. Loh and colleagues reported [13] that the biotransformation of indene to cis-indandiol was achieved in an IHFMB system resulting in higher effectiveness.

MBR systems can be distinguished according to the membrane process integrated [16, 17]. Beyond pressure-driven methods (microfiltration, ultrafiltration, nanofiltration), other techniques like pervaporation, electrodialysis, and gas

**Figure 1.** *Outline of an internal (integrated) system.*

separation can be applied. To connect these membrane processes to the bioreaction, careful design and optimization should be accomplished before starting the operation of MBRs. Plate and frame, tubular, as well as hollow fiber modules can be used in the MBRs.

The novel MBR systems can be further categorized regarding the biocatalyst used: enzymes as well as microbes (moreover microbial consortia) can be applied. The biocatalysts can be further classified concerning the state of the biocatalysts, as well (free or immobilized); moreover they can be immobilized onto the membrane (which is considered as *catalytic active membrane* [18]) or on other supports. Some examples are presented here for all the classes.

#### **3.2 Enzymatic MBRs**

Enzymatic MBRs have been used mainly in hydrolytic reactions where the substrates are, e.g., triacylglycerols and polysaccharides (starch, cellulose, pectin), but some other reactions occur, as well, like esterification. **Table 2** summarizes the important features of these systems.

Hydrolysis of triacylglycerols (fats and oils) results in glycerol and fatty acids (long chain carboxylic acids). The higher demand of fatty acids in various industrial sectors (e.g., production of cosmetics, detergents) made the hydrolysis an important process recently. The main difficulty of the process is that the two reactants, triacylglycerols and water, are not miscible. When the reaction is carried out by enzymatic catalysis [17], numerous advantages are provided: it takes place


**109**

*Integration of Membranes and Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.84513*

are separated by the membrane.

continuous operation.

for the hydrolysis of various oils and fats [20, 21].

degradation of starch effectively and continuously.

enzyme reaction was accomplished.

under mild conditions, instead of high temperature and pressure, implying energy saving and better quality products. Accomplishing the hydrolysis in a MBR, further benefits are added to the process: the two phases remain separated during the reaction and there is no emulsion formed and no separation needed, since the products

The enzyme suitable for the hydrolysis is called lipase. It can be immobilized onto the membrane. During the reaction, the pure triacylglycerol substrate (no solvent is present) and water are flown separately into the two sides of the membrane. Both hydrophilic and hydrophobic membranes can be applied [19]. In case of hydrophilic membrane, the substrate should be circulated on the enzyme side of the membrane, while water is on the other side. Thus fatty acids formed are remaining in the oily phase (and accumulated there), while glycerol is passing through the hydrophilic membrane into the water phase. Applying hydrophobic membrane the arrangement is the other way around: the substrate is flown on the nonenzyme side of the membrane. In our experiments lipase from *Candida rugosa* (Sigma) and hydrophilic cellulose acetate membrane (cut off 3000) was used [19] successfully

In hydrolytic degradation of polymers (polysaccharides and proteins), the smallsized products (e.g., glucose) often have inhibitory effect, and thus their removal is beneficial for the reaction. MBRs provide a simple solution, since the membrane applied serves for rejection of the long polymer chains as well as the biocatalysts (enzymes or cells), while the product can easily pass through the membrane.

Hydrolysis of various proteins—e.g., from milk, whey, plasma—is an important step in industrial processes, and it is realized by protease enzymes. The MBRs provide significant advantages for the process [18] including retention and reuse of the biocatalysts, avoiding product inhibition—a possibility for

Regarding enzymatic hydrolysis of polysaccharides, starch, cellulose, and pectin are considered. Hydrolytic products of starch are utilized widely in food industry (e.g., maltodextrin, dextrose, and high fructose syrups). The reaction is catalyzed by amylase enzymes. When the hydrolysis is carried out in MBRs, higher effectivity can be achieved [22, 23]. In our work hollow fiber cellulose acetate (UF) membrane module (jacketed) was used, and the experimental results confirmed that not only purified amylase preparations but a crude fermentation filtrate, an enzyme complex solution (mainly glucoamylase) produced by *Aspergillus awamori*, was capable for

Cellulose is a long-chain polysaccharide containing glucose (monomer) similarly to starch. Enzymatic hydrolysis of cellulose, however, is more difficult [24]. The reaction can be carried out in MBRs to overcome some of the problems. A special tubular membrane module was used to carry out the reaction, where a fine, hairy surface membrane was built in [24]. The hydrolysis was catalyzed by a Celluclast preparation (Novozymes), and it was found that the special MBR had a positive effect on the process and enhanced conversion and productivity of the

Pectin is a polysaccharide occurring in the cell walls of certain plants, mainly in the fruits like apple, orange, some berried, etc. It is important to use pectolytic enzymes in processing of fruits (production of fruit juices) [25, 26] to enhance yields and improve liquefaction and clarification. As a result of the hydrolytic degradation of pectin, galacturonic acid (monomer) is formed. It was assumed that the hydrolysis was inhibited by the monomer; therefore MBRs seemed a promising reactor type to realize the process effectively. Regenerated cellulose UF membrane (30 kD) and polygalacturonase enzyme preparation (Sigma) from *Aspergillus niger* were used in the experiments, and it was found that the MBR worked reliably with

#### *Integration of Membranes and Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.84513*

*Biotechnology and Bioengineering*

*Outline of an internal (integrated) system.*

in the MBRs.

**Figure 1.**

**3.2 Enzymatic MBRs**

examples are presented here for all the classes.

important features of these systems.

Esterification Acids and

*Examples for enzymatic MBRs.*

alcohols

separation can be applied. To connect these membrane processes to the bioreaction, careful design and optimization should be accomplished before starting the operation of MBRs. Plate and frame, tubular, as well as hollow fiber modules can be used

The novel MBR systems can be further categorized regarding the biocatalyst used: enzymes as well as microbes (moreover microbial consortia) can be applied. The biocatalysts can be further classified concerning the state of the biocatalysts, as well (free or immobilized); moreover they can be immobilized onto the membrane (which is considered as *catalytic active membrane* [18]) or on other supports. Some

Enzymatic MBRs have been used mainly in hydrolytic reactions where the substrates are, e.g., triacylglycerols and polysaccharides (starch, cellulose, pectin), but some other reactions occur, as well, like esterification. **Table 2** summarizes the

Hydrolysis of triacylglycerols (fats and oils) results in glycerol and fatty acids (long chain carboxylic acids). The higher demand of fatty acids in various industrial sectors (e.g., production of cosmetics, detergents) made the hydrolysis an important process recently. The main difficulty of the process is that the two reactants, triacylglycerols and water, are not miscible. When the reaction is carried out by enzymatic catalysis [17], numerous advantages are provided: it takes place

**Reaction Substrate Enzyme State Membrane Ref.** Hydrolysis Triacylglycerol Lipase Immobilized UF [19–21]

Hydrolysis Starch Amylases Free UF [22, 23] Hydrolysis Cellulose Cellulases Immobilized UF [24]

immobilized

UF [18]

[25–27] [28–31]

ED

Lipases Immobilized PV [32–35]

Hydrolysis Protein Protease Free and

Hydrolysis Pectin Pectinases Free UF

**108**

**Table 2.**

under mild conditions, instead of high temperature and pressure, implying energy saving and better quality products. Accomplishing the hydrolysis in a MBR, further benefits are added to the process: the two phases remain separated during the reaction and there is no emulsion formed and no separation needed, since the products are separated by the membrane.

The enzyme suitable for the hydrolysis is called lipase. It can be immobilized onto the membrane. During the reaction, the pure triacylglycerol substrate (no solvent is present) and water are flown separately into the two sides of the membrane. Both hydrophilic and hydrophobic membranes can be applied [19]. In case of hydrophilic membrane, the substrate should be circulated on the enzyme side of the membrane, while water is on the other side. Thus fatty acids formed are remaining in the oily phase (and accumulated there), while glycerol is passing through the hydrophilic membrane into the water phase. Applying hydrophobic membrane the arrangement is the other way around: the substrate is flown on the nonenzyme side of the membrane. In our experiments lipase from *Candida rugosa* (Sigma) and hydrophilic cellulose acetate membrane (cut off 3000) was used [19] successfully for the hydrolysis of various oils and fats [20, 21].

In hydrolytic degradation of polymers (polysaccharides and proteins), the smallsized products (e.g., glucose) often have inhibitory effect, and thus their removal is beneficial for the reaction. MBRs provide a simple solution, since the membrane applied serves for rejection of the long polymer chains as well as the biocatalysts (enzymes or cells), while the product can easily pass through the membrane.

Hydrolysis of various proteins—e.g., from milk, whey, plasma—is an important step in industrial processes, and it is realized by protease enzymes. The MBRs provide significant advantages for the process [18] including retention and reuse of the biocatalysts, avoiding product inhibition—a possibility for continuous operation.

Regarding enzymatic hydrolysis of polysaccharides, starch, cellulose, and pectin are considered. Hydrolytic products of starch are utilized widely in food industry (e.g., maltodextrin, dextrose, and high fructose syrups). The reaction is catalyzed by amylase enzymes. When the hydrolysis is carried out in MBRs, higher effectivity can be achieved [22, 23]. In our work hollow fiber cellulose acetate (UF) membrane module (jacketed) was used, and the experimental results confirmed that not only purified amylase preparations but a crude fermentation filtrate, an enzyme complex solution (mainly glucoamylase) produced by *Aspergillus awamori*, was capable for degradation of starch effectively and continuously.

Cellulose is a long-chain polysaccharide containing glucose (monomer) similarly to starch. Enzymatic hydrolysis of cellulose, however, is more difficult [24]. The reaction can be carried out in MBRs to overcome some of the problems. A special tubular membrane module was used to carry out the reaction, where a fine, hairy surface membrane was built in [24]. The hydrolysis was catalyzed by a Celluclast preparation (Novozymes), and it was found that the special MBR had a positive effect on the process and enhanced conversion and productivity of the enzyme reaction was accomplished.

Pectin is a polysaccharide occurring in the cell walls of certain plants, mainly in the fruits like apple, orange, some berried, etc. It is important to use pectolytic enzymes in processing of fruits (production of fruit juices) [25, 26] to enhance yields and improve liquefaction and clarification. As a result of the hydrolytic degradation of pectin, galacturonic acid (monomer) is formed. It was assumed that the hydrolysis was inhibited by the monomer; therefore MBRs seemed a promising reactor type to realize the process effectively. Regenerated cellulose UF membrane (30 kD) and polygalacturonase enzyme preparation (Sigma) from *Aspergillus niger* were used in the experiments, and it was found that the MBR worked reliably with

excellent stability for long and higher volumetric productivity was achieved than the batch system. To enhance the effectiveness of the process, vacuum was applied in the secondary side of the membrane [27].

The reaction between acids and alcohol results in esters and water. It can be catalyzed by lipase enzymes. The reaction is reversible and product removal enhances the yield. Both products can be separated from the reaction mixture by membranes. If the substrates are low molecular weight acids and alcohols, flavor esters are formed, which are volatile compounds. Thus pervaporation (PV) can be applied for their recovery that is quite easy to connect online to the reaction, forming an integrated MBR system. Ethyl acetate [32] and other flavor esters [33] were manufactured effectively by a lipase preparation (*Candida antarctica* immobilized onto a resin, from Novozymes) and separated online by PV using hydrophobic membranes, and the permeate was obtained by using vacuum traps cooled by dry ice.

PV is not suitable for the separation of higher molecular weight ester products, but it is possible to apply it for removal of water formed during the esterification. Ester type biolubricant from oleic acid and alcohols [34] and ester of 2-choloropropionic acid and 1-butanol [35] were manufactured by using integrated PV for water removal.

Electrodialysis also can be combined to enzymatic processes, where charged compounds are formed and they can be separated online. This kind of MBR was applied for continuous recovery of galacturonic acid obtained from the hydrolysis of pectin by a pectinase preparation [28]. Both monopolar and bipolar membranes were successfully used [29, 30].

Summarizing the application possibilities of MBRs in enzymatic processes, it can be concluded that they provided real advantages for the bioprocesses listed above and—beyond process intensification [31, 36]—these systems can be operated in continuous mode of operation: constant uptake of substrate (and biocatalyst, if necessary) and release of product can be achieved.

#### **3.3 Microbial MBRs**

In microbial MBRs a single microorganisms can work, but sterile conditions should be provided. One of the examples given here is the biohydrogen production, where gas separation is connected and the other one is manufacture of organic acids, where electrodialysis is used for in-situ separation of the products.

Biohydrogen can be produced by fermentation using various microbes. Some of them need light for the operation (photofermentation), while others do not (dark fermentation). *Escherichia coli* belong to the dark fermentative group and is able to form biogas containing mainly hydrogen and CO2 in the headspace of the bioreactor. When the bioreactor is integrated by a membrane gas separation unit (MBR), higher productivity was possible to achieve, as proven in Veszprem [37–42]. Polyimide hollow fiber membranes and *E. coli* XL-1 Blue strain were used for the experiments, which resulted in a feasible concept for the integrated production and purification of the promising energy carrier, hydrogen gas [43–46]. Additionally, hydrogen fermenters could be attached to microbial electrochemical technologies, thus giving opportunity for adequate treatment of the effluents containing residual organic matter [47–50].

Transformation of fumaric acid into L-malic acid—which is the second most popular food acid—is an equilibrium reaction; thus, product removal could increase the yield. The reaction is catalyzed by immobilized cells of *Leuconostoc* and *Brevibacterium* species containing fumarase enzyme, and the acid (in salt form) can be separated by electrodialysis [51] integrated to the reaction.

**111**

*Integration of Membranes and Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.84513*

**4. Conclusions**

**Acknowledgements**

**Abbreviations**

ED Electrodialysis GS Gas separation

MF Microfiltration NF Nanofiltration PV Pervaporation RO Reverse osmosis UF Ultrafiltration

MBRs Membrane bioreactors

monopolar or bipolar electrodialysis systems.

*coelicolor*, in a vertically oriented capillary MGR.

Malic acid is not the only acid that can be produced in integrated (MBR) system, other organic acids—manufactured by fermentation—can be online separated by

A relatively novel area of MBR applications is the immobilization and cultivating of microbes; the system is called membrane gradostat reactor (MGR) [15]. The biofilm growing (and attached) on the surface has a significant effect on the permeability. The behavior of these systems was studied in case of, e.g., *Streptomyces* 

Bioprocesses integrated with membrane separations (MBRs) are able to provide more effective, successful bioconversions and bioreactions. The MBRs have been used in large scale worldwide in wastewater treatments, on one hand, while there are other, special applications in smaller scale (and sometimes only in a developing stage), on the other hand. Examples for both areas were given in this chapter.

The research work was partly supported by the project EFOP-3.6.1-16-2016- 00015. Peter Bakonyi acknowledges the support received from National Research, Development and Innovation Office (Hungary) under grant number PD 115640.

*Integration of Membranes and Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.84513*

Malic acid is not the only acid that can be produced in integrated (MBR) system, other organic acids—manufactured by fermentation—can be online separated by monopolar or bipolar electrodialysis systems.

A relatively novel area of MBR applications is the immobilization and cultivating of microbes; the system is called membrane gradostat reactor (MGR) [15]. The biofilm growing (and attached) on the surface has a significant effect on the permeability. The behavior of these systems was studied in case of, e.g., *Streptomyces coelicolor*, in a vertically oriented capillary MGR.

#### **4. Conclusions**

*Biotechnology and Bioengineering*

cooled by dry ice.

PV for water removal.

**3.3 Microbial MBRs**

organic matter [47–50].

were successfully used [29, 30].

necessary) and release of product can be achieved.

in the secondary side of the membrane [27].

excellent stability for long and higher volumetric productivity was achieved than the batch system. To enhance the effectiveness of the process, vacuum was applied

The reaction between acids and alcohol results in esters and water. It can be catalyzed by lipase enzymes. The reaction is reversible and product removal enhances the yield. Both products can be separated from the reaction mixture by membranes. If the substrates are low molecular weight acids and alcohols, flavor esters are formed, which are volatile compounds. Thus pervaporation (PV) can be applied for their recovery that is quite easy to connect online to the reaction, forming an integrated MBR system. Ethyl acetate [32] and other flavor esters [33] were manufactured effectively by a lipase preparation (*Candida antarctica* immobilized onto a resin, from Novozymes) and separated online by PV using hydrophobic membranes, and the permeate was obtained by using vacuum traps

PV is not suitable for the separation of higher molecular weight ester products, but it is possible to apply it for removal of water formed during the esterification. Ester type biolubricant from oleic acid and alcohols [34] and ester of 2-choloropropionic acid and 1-butanol [35] were manufactured by using integrated

Electrodialysis also can be combined to enzymatic processes, where charged compounds are formed and they can be separated online. This kind of MBR was applied for continuous recovery of galacturonic acid obtained from the hydrolysis of pectin by a pectinase preparation [28]. Both monopolar and bipolar membranes

Summarizing the application possibilities of MBRs in enzymatic processes, it can be concluded that they provided real advantages for the bioprocesses listed above and—beyond process intensification [31, 36]—these systems can be operated in continuous mode of operation: constant uptake of substrate (and biocatalyst, if

In microbial MBRs a single microorganisms can work, but sterile conditions should be provided. One of the examples given here is the biohydrogen production, where gas separation is connected and the other one is manufacture of organic acids, where electrodialysis is used for in-situ separation of the products.

Biohydrogen can be produced by fermentation using various microbes. Some of them need light for the operation (photofermentation), while others do not (dark fermentation). *Escherichia coli* belong to the dark fermentative group and is able to form biogas containing mainly hydrogen and CO2 in the headspace of the bioreactor. When the bioreactor is integrated by a membrane gas separation unit (MBR), higher productivity was possible to achieve, as proven in Veszprem [37–42]. Polyimide hollow fiber membranes and *E. coli* XL-1 Blue strain were used for the experiments, which resulted in a feasible concept for the integrated production and purification of the promising energy carrier, hydrogen gas [43–46]. Additionally, hydrogen fermenters could be attached to microbial electrochemical technologies, thus giving opportunity for adequate treatment of the effluents containing residual

Transformation of fumaric acid into L-malic acid—which is the second most

increase the yield. The reaction is catalyzed by immobilized cells of *Leuconostoc* and *Brevibacterium* species containing fumarase enzyme, and the acid (in salt form) can

popular food acid—is an equilibrium reaction; thus, product removal could

be separated by electrodialysis [51] integrated to the reaction.

**110**

Bioprocesses integrated with membrane separations (MBRs) are able to provide more effective, successful bioconversions and bioreactions. The MBRs have been used in large scale worldwide in wastewater treatments, on one hand, while there are other, special applications in smaller scale (and sometimes only in a developing stage), on the other hand. Examples for both areas were given in this chapter.

#### **Acknowledgements**

The research work was partly supported by the project EFOP-3.6.1-16-2016- 00015. Peter Bakonyi acknowledges the support received from National Research, Development and Innovation Office (Hungary) under grant number PD 115640.

#### **Abbreviations**


*Biotechnology and Bioengineering*

#### **Author details**

Katalin Belafi-Bako\* and Peter Bakonyi University of Pannonia, Veszprem, Hungary

\*Address all correspondence to: bako@almos.uni-pannon.hu

© 2019 The Author(s). Licensee IntechOpen. 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.

**113**

*Integration of Membranes and Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.84513*

[1] Mandenius CF, editor. Bioreactors: Design, Operations and Novel

Applications. 1st ed. Oxford, UK: Verlag-Wiley; 2016. DOI: 10.1002/9783527683369 bioreactors – A mini review with emphasis on industrial

limitations and perspectives. Desalination and Water Treatment.

2016;**57**:19062-19076. DOI: 10.1080/19443994.2015.1100879

wastewater treatment: Applications,

[11] Szentgyörgyi E, Bélafi-Bakó K. Anaerobic membrane bioreactors. Hungarian Journal of Industrial Chemistry. 2010;**38**:181-185

[12] Bakonyi P, Kumar G, Bélafi-Bakó K, Sang-Hyoun Kim SH, Koter S, Kujawski W, et al. A review of the innovative gas separation membrane bioreactor with mechanisms for integrated production and purification of biohydrogen. Bioresource Technology. 2018;**270**:643- 655. DOI: 10.1016/j.biortech.2018.09.020

[13] Cheng XY, Tong YW, Loh KC.

membrane bioreactor for enhanced biotransformation of indene to cis-indandiol using pseudomonas putida. Biochemical Engineering Journal. 2014;**87**:1-7. DOI: 10.1016/j.

[14] Godongwana B, De Jager D, Sheldon MS, Edwards W. The effect of Streptomyces ceolicolor development on the hydrodynamics of a vertically oriented capillary membrane gradostat reactor. Journal of Membrane Science.

2009;**333**:79-87. DOI: 10.1016/j.

[15] Szentgyörgyi E, Nemestóthy N, Bélafi-Bakó K. Application of membranes in biogas production.

Treatment;**210**(14):112-115. DOI:

[16] Bélafi-Bakó K, Gubicza L. Biocatalysts and membranes. In: Bélafi-Bakó K, Gubicza L, Mulder M, editors. Integration of Membrane

memsci.2009.01.051

Desalination and Water

10.5004/dwt.2010.2582

An immersed hollow fiber

bej.2014.03.011

[2] Drioli E, Giorno L. Comprehensive Membrane Science and Engineering (I-IV). 1st ed. Amsterdam, NL: Elsevier;

[3] Baker RW. Membrane Technology and Applications. Wiley; 2012

[4] Purkait MK, Singh R. Membrane Technology in Separation Science. Boca

[5] Yoon SH. Membrane Bioreactor Processes. Boca Raton: CRC Press; 2016

[6] Cicek N. A review on membrane bioreactors and their potential application

in the treatment of agricultural wastewater. Canadian Biosystems Engineering. 2003;**45**:637-649

[7] Huang L, Lee DJ. Membrane bioreactor: A mini review on recent R+D works. Bioresource Technology. 2015;**194**:383-388. DOI: 10.1016/j.

[8] Krzeminski P, Leverette L, Malamis S, Katsou E. Membrane bioreactors – A review on recent developments in energy reduction, fouling control, novel configurations, LCA and market prospects. Journal of Membrane Science. 2017;**527**:207-227. DOI: 10.1016/j.memsci.2016.12.010

[9] Shin C, McCarty PL, Kim J, Bae J. Pilot scale temperature-climate treatment of domestic wastewater with a staged anaerobic fluidized membrane bioreactor. Bioresource Technology. 2014:95-103. DOI: 10.1016/j.

[10] Dvorak L, Gomez M, Dolina J, Cernin A. Anaerobic membrane

biortech.2015.07.013

biortech.2014.02.060

Raton: CRC Press; 2017

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*Integration of Membranes and Bioreactors DOI: http://dx.doi.org/10.5772/intechopen.84513*

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*Biotechnology and Bioengineering*

**112**

**Author details**

provided the original work is properly cited.

Katalin Belafi-Bako\* and Peter Bakonyi University of Pannonia, Veszprem, Hungary

\*Address all correspondence to: bako@almos.uni-pannon.hu

© 2019 The Author(s). Licensee IntechOpen. 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,

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[13] Cheng XY, Tong YW, Loh KC. An immersed hollow fiber membrane bioreactor for enhanced biotransformation of indene to cis-indandiol using pseudomonas putida. Biochemical Engineering Journal. 2014;**87**:1-7. DOI: 10.1016/j. bej.2014.03.011

[14] Godongwana B, De Jager D, Sheldon MS, Edwards W. The effect of Streptomyces ceolicolor development on the hydrodynamics of a vertically oriented capillary membrane gradostat reactor. Journal of Membrane Science. 2009;**333**:79-87. DOI: 10.1016/j. memsci.2009.01.051

[15] Szentgyörgyi E, Nemestóthy N, Bélafi-Bakó K. Application of membranes in biogas production. Desalination and Water Treatment;**210**(14):112-115. DOI: 10.5004/dwt.2010.2582

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[17] Bélafi-Bakó K. Simultaneous application of enzymes and membranes in food processing. In: Jerrod MC, editor. Food Engineering Research Trends. New York: Nova Science Publishers; 2008. pp. 263-279

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bioreactor. Enzyme and Microbial Technology. 2006;**38**:155-161. DOI: 10.1016/j.enzmictec.2005.05.012

[25] Bélafi-Bakó K, Eszterle M, Kiss K, Nemestóthy N, Kovács S, Gubicza L. Utilisation of a membrane bioreactor for pectin hydrolysis by Aspergillus Niger polygalacturonase. Desalination. 2006;**200**:507-508. DOI: 10.1016/j.

[26] Bélafi-Bakó K, Eszterle M, Kiss K, Nemestóthy N, Gubicza L. Hydrolysis

of pectin by Aspergillus Niger polygalacturonase in a membrane bioreactor. Journal of Food Engineering.

2007;**78**:438-442. DOI: 10.1016/j.

[27] Kiss K, Nemestóthy N, Gubicza L, Bélafi-Bakó K. Vacuum assisted membrane bioreactor for enzymatic hydrolysis of pectin from various sources. Desalination. 2009;**241**:29-33. DOI: 10.1016/j.desal.2008.01.055

[28] Molnár E, Eszterle M, Kiss K, Nemestóthy N, Fekete J, Bélafi-Bakó K. Utilisation of electrodialysis for galacturonic acid recovery. Desalination. 2009;**241**:81-85. DOI:

[29] Molnár E, Nemestóthy N, Bélafi-Bakó K. Galacturonic acid recovery from pectin rich agro-wastes by

Hungarian Journal of Industrial Chemistry. 2008;**36**:95-99. DOI:

10.1515/186

DOI: 10.1515/343

electrodialysis with bipolar membranes.

[30] Belafi-Bako K, Molnar E, Csanadi Z, Nemestothy N. Comparative study on electrodialysis systems for galacturonic acid recovery. Hungarian Journal of Industrial Chemistry. 2012;**40**:65-67.

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10.1016/j.desal.2008.01.059

desal.2006.03.414

jfoodeng.2005.10.012

[17] Bélafi-Bakó K. Simultaneous application of enzymes and membranes in food processing. In: Jerrod MC, editor. Food Engineering Research Trends. New York: Nova Science Publishers; 2008. pp. 263-279

[18] Rios GM, Belleville MP, Paolucci D, Sanchez J. Progress in enzymatic membrane reactors—Review. Journal of Membrane Science. 2004;**242**:189-196. DOI: 10.1016/j.memsci.2003.06.004

[19] Bélafi-Bakó K, Dombi Á, Szabó L, Nagy E. Triacylglycerol hydrolysis

bioreactor. Biotechnology Techniques.

[20] Csányi E, Bélafi-Bakó K. Semicontinuous fatty acid production by lipase. Hungarian Journal of Industrial

[21] Keöves E, Csányi E, Bélafi-Bakó K, Gubicza L. Membrane reactors for biochemical reactions. Hungarian Journal of Industry and Chemistry.

[22] Koutinas A, Bélafi-Bakó K, Kabiri-Badr A, Tóth A, Gubicza L, Webb C. Enzymatic hydrolysis of polysaccharides, hydrolysis of starch by an enzyme complex from fermentation by *Aspergillus awamori*. Food and Bioproducts Processing. 2001;**79**:41-45. DOI: 10.1205/09603080151123353

[23] Bélafi-Bakó K, Nemestóthy N, Milisic V, Gubicza L. Membrane bioreactor for utilisation of carbohydrates in waste streams. Desalination. 2002;**149**:329-330. DOI: 10.1016/S0011-9164(02)00803-2

[24] Bélafi-Bakó K, Koutinas A, Nemestóthy N, Gubicza L, Webb C. Continuous enzymatic cellulose hydrolysis in a tubular membrane

by lipase in a flat membrane

Chemistry. 1999;**27**:293-295

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[49] Rózsenberszki T, Koók L, Bakonyi P, Nemestóthy N, Logroño W, Pérez M, et al. Municipal waste liquor treatment via bioelectrochemical and fermentation (H2 + CH4) processes: Assessment of various technological sequences. Chemosphere. 2017;**171**:692-701. DOI: 10.1016/j.chemosphere.2016.12.114

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**117**

**Chapter 9**

Applied

*Memory Tekere*

**Abstract**

**1. Introduction**

membrane and fluidized bed reactors [4–8].

Microbial Bioremediation and

Different Bioreactors Designs

Microbial remediation of pollutants involves the use of microorganisms to degrade pollutants either completely to water and carbon dioxide (for organic pollutants) or into less toxic forms. In the case of nonbiodegradable inorganic compounds, bioremediation takes the form of bioaccumulation or conversion of one toxic species to a less toxic form for example Cr(VI) is converted to less toxic (III). Bioremediation is considered an environmentally friendly way for pollution cleanup. Microbial clean up can be applied *in situ* (in place of contamination) or *ex situ* (off the site of contamination). *In situ* remediation in the natural environment is deemed slow and often times difficult to control and optimize the different parameters affecting the bioremediation. To this end, use of engineered bioreactors is preferred. Engineered bioreactors providing for optimum conditions for microbial growth and biodegradation have been developed for use in bioremediation processes to achieve the different desired remediation goals. Bioreactors in use range in mode of operation from batch, continuous, and fed batch bioreactors and are designed to optimize microbial processes in relationship to contaminated media and nature of pollutant. Designed bioreactors for bioremediation range from packed, stirred tanks, airlift, slurry phase, and partitioning phase reactors amongst others.

**Keywords:** bioremediation, bioreactors, pollution, microorganisms, degradation

Bioremediation is a natural process that relies on microorganisms and plants and/or their derivatives (enzymes or spent biomass) to degrade or alter environmental contaminants as these organisms carry out their normal life functions [1, 2]. Bioremediation is considered an economical, versatile, efficient and eco-friendly way of dealing with environmental pollutants as compared to the physico-chemical methods [1–3]. The use of well-designed microbial bioreactors is acknowledged as an efficient way to ensure that microbial growth and processes occur in a controlled environment that provides the necessary optimum conditions [3–5]. This chapter focuses on microbial remediation in bioreactors so phytoremediation as facilitated by plants is not discussed. Several studies describe microbial remediation in designed bioreactors ranging from batch, continuous, and fed-batch operated mode which can be in different designs such as suspended carrier, slurry and fixed bed,

#### **Chapter 9**

*Biotechnology and Bioengineering*

production and purification in a double-membrane bioreactor system. International Journal of Hydrogen Energy. 2015;**40**:1690-1697. DOI: 10.1016/j.ijhydene.2014.12.002

[47] Rivera I, Buitrón G, Bakonyi P, Nemestóthy N, Bélafi-Bakó K. Hydrogen production in a microbial electrolysis cell fed with a dark fermentation effluent. Journal of Applied

Electrochemistry. 2015;**45**:1223-1229. DOI: 10.1007/s10800-015-0864-6

[48] Kumar G, Bakonyi P, Kobayashi T, Xu KQ , Sivagurunathan P, Kim SH, et al. Enhancement of biofuel

production via microbial augmentation: The case of dark fermentative hydrogen. Renewable and Sustainable Energy Reviews. 2016;**57**:879-891. DOI: 10.1016/j.rser.2015.12.107

[49] Rózsenberszki T, Koók L, Bakonyi P, Nemestóthy N, Logroño W, Pérez M, et al. Municipal waste liquor treatment via bioelectrochemical and fermentation (H2 + CH4) processes: Assessment of various technological sequences. Chemosphere. 2017;**171**:692-701. DOI: 10.1016/j.chemosphere.2016.12.114

[50] Bakonyi P, Kumar G, Koók L, Tóth G, Rózsenberszki T, Bélafi-Bakó K, et al. Microbial electrohydrogenesis linked to dark fermentation as integrated application for enhanced biohydrogen production: A review on process characteristics, experiences and lessons. Bioresource Technology. 2018;**251**:381-389. DOI: 10.1016/j.

[51] Bélafi-Bakó K, Nemestóthy N, Gubicza L. Study on application of membrane techniques in bioconversion

of fumaric acid to L-malic acid. Desalination. 2004;**162**:301-306. DOI: 10.1016/S0011-9164(04)00063-3

biortech.2017.12.064

**116**
