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An Overview on Polymer Gels Applied to Enzyme and Cell Immobilization

  • Gustavo Pagotto Borin
  • Ricardo Rodrigues de Melo
  • Elaine Crespim
  • Helia Harumi Sato
  • Fabiano Jares Contesini
Chapter
Part of the Gels Horizons: From Science to Smart Materials book series (GHFSSM)

Abstract

Immobilization of enzymes and cells is crucial in several industrial areas. This is mainly due to the possibility to improve enzyme properties including thermal stability, substrate selectivity, and biocatalyst reuse. These modifications allow for a considerable decrease in the cost of many commercial applications. The use of polymer gels for cell and enzyme immobilization presents numerous advantages over other immobilization supports, since they allow the protein or cell entrapment to be performed in a more efficient and simpler way. The polymers used here include polysaccharides and synthetic polymers in which several industrially relevant enzymes were immobilized with positive results. In addition to the immobilization of enzymes, there are many studies reporting the immobilization of microbial cells in polymers for enzyme production. Enzyme and cell immobilization in polymer gels show potential to deliver useful and efficient strategies to make use of microbial enzymes from an industrial point of view. However, further efforts must be made to better understand and apply immobilization of biocatalysts and to develop new technologies. This chapter focuses on general aspects of polymer gels, particularly regarding the immobilization of enzymes and microbial cells in different industrial fields.

Keywords

Polymer gel Immobilization Microbial enzymes Whole-cells 

1 Introduction

Polymer gels comprise a great variety of different polymeric compounds that present innumerable industrial applications. Polymers can be naturally produced, in which case the most representative group is polysaccharides (Thakur and Thakur 2014a, b). This includes alginate, chitosan, and agar-agar. Some polysaccharides form gels through ionic gelation in the presence of ions like calcium (Ca2+), such as alginate or pectin, while other gels solidify in high temperatures. In addition to polysaccharides, lignin based hydrogels have recently been studied and reviewed (Thakur and Thakur 2015). On the other hand, a wide variety of synthetic polymers capable of forming gels presents different industrial applications, such as polyacrylamide and polyvinyl alcohol.

An extremely important use of polymer gels is for biocatalyst immobilization, fixing in place microbial enzymes or cells (Lahiri 2015; Wilkowska et al. 2015). In the latter case, the cells can be entrapped and used for biotransformation or they can be used for the production of enzymes with industrial interest (Kamble and Banoth 2013; Hemachander et al. 2001). Immobilization of biocatalysts takes place via the fixation of the protein or cells to a support medium through any of various means including adsorption, covalent bonding, or entrapment inside a polymeric matrix. The last technique is the main focus of this review, as several polymer gels can be used for enzyme or cell entrapment and is a method in which activity loss is low when compared to other techniques.

Enzyme or cell immobilization allows the reuse of the biocatalyst and reduces cost if the catalytic activity is maintained. Furthermore, the enzyme immobilization process is able to allow for an improvement in the stability of the enzyme (Milessi et al. 2015). Lastly, both the advantages and disadvantages of immobilizing enzymes as well as comparing this to microbial cells will be discussed.

In this chapter, various aspects of polymer gels applied to enzyme and cell immobilization will be discussed. Initially, polymer gels used for biocatalyst immobilization will be introduced, followed by a more detailed discussion on enzyme immobilization. Afterwards, the results of cell entrapment in gels will be reported.

2 Supports for Entrapment Techniques

Determining the appropriate immobilization support is an important step in the development of feasible and efficient processes. The choice of polymeric matrix determines the rigidity, absorption capacity, mechanical strength, and porosity characteristics of the beads used in immobilization. Physical entrapment of cells and enzymes within a polymeric matrix is recognized as being one of the most used immobilization techniques. The entrapment method utilizes a simple and single-step procedure for both cell and enzyme immobilization within a polymeric network. Typically, entrapment techniques utilize any of several natural and synthetic supports for efficient immobilization. The use of natural polymers such as chitosan, chitin, alginate, and many others have been applied as important matrices for immobilization (Table 1) (Hsuanyu 2004; Taylor et al. 2010; Zajkoska et al. 2013). Natural polymeric materials used in entrapment processes demonstrate important characteristics, such as their ability to be gathered from many sources, ease of modification, and lack of pollutants. Furthermore, they have a range of functional groups and good biocompatible properties such as being non-toxic and inert to organisms that are often necessary for applications in the pharmaceutical and food industries (Zhang et al. 2013).
Table 1

Commonly used materials for entrapment techniques

 

Compounds carriers

Formation principle

Gel geometry

Natural polymers

Alginate

Ionotropic gelation

Gel cylinder/beads

Cellulose

Ionotropic gelation

Beads

Chitosan

Ionotropic gelation

Beads

Chitin

Ionotropic gelation

Beads

Pectin

Ionotropic gelation

Beads

Pectate

Ionotropic gelation

Beads

Carrageenan

Ionotropic/thermal gelation

Gel cylinder

Gelatin

Thermal gelation

Gel cylinder

Agar

Thermal gelation

Gel cylinder/beads

Agarose

Thermal gelation

Gel cylinder

Synthetic polymers

Polyvinyl alcohol—PVA

Thermal gelation

Lentikats/gel

Polyacrylamide

Chemical gelation

Gel cylinders/beads

Polyacrylonitrile

Thermal gelation

Beads

In other science sectors, synthetic polymeric matrices are also efficiently applied for the immobilization of cells and enzymes. Synthetic polymers present several advantages: good mechanical rigidity, high specific surface area, easy alteration of their surface characteristics, and their potential for providing specific functional groups that match the individual needs of each bioprocess. Other advantages offered by synthetic polymers are that they can be synthesized in larger amounts more easily than most natural polymers, and do not suffer batch-to-batch variations like natural polymers (Zhang et al. 2013; De Vos et al. 2014). In general, polyvinyl alcohol, polyacrylamide, and polyacrylonitrile are often the most suitable synthetic matrices for gel immobilization (Table 1).

2.1 Natural Polymers

2.1.1 Immobilization in Ionic Hydrogels

A wide variety of ionic hydrogels are used as supports for cell and enzyme entrapment. Normally, ionic hydrogels used in immobilization are alginate, cellulose, chitosan, chitin, pectin, and pectate. Ionic hydrogels contain ionizable groups in their structures such as amino groups, carboxylates, sulfates, and hydroxyls that possess varying degrees of affinity with water molecules. In addition, several anionic polymers are also utilized with their own benefits and drawbacks.

Alginate is a natural anionic biomaterial formed by β-d-mannuronic acid and α-l-glucuronic acid chains. The l-glucuronic acid content present in the alginate’s structure has great importance on its mechanical rigidity and can influence the stability of the gels in the presence of anti-gelling ions and calcium ion sequesters. Structurally, alginates rich in glucuronic acid exhibit high porosity and low shrinkage capacity during gel formation and do not swell when dried. In contrast, alginates showing higher mannuronic acid levels make the gels softer and more elastic (Thu et al. 1996). Alginate gels can be constructed by an ionic network in the presence of cations such as Ca2+ or other multivalent counterions (Orive et al. 2006).

Cellulose is a natural, semi-crystalline polysaccharide that is very abundant on earth, typically composed of 1,4-linked β-d-glucopyranosyl chains. Cellulose is a very useful matrix for enzyme immobilization due to its low cost and commercial availability both in fibrous and granular forms. However, this support is accompanied by some drawbacks, such as susceptibility to hydrolysis by microbial enzymes (cellulases) and low particle size, which impairs their use in rapid high-pressure applications (Hsuanyu 2004; Agbor et al. 2011).

Chitosan is a polymer obtained from deacetylated chitin chains, and is the second most abundant support compound that can obtained from nature after cellulose. It is a natural product, non-toxic, biocompatible, biodegradable, and inexpensive, making it very important, both economically and environmentally. Chitosan is a polymer that has a molecular structure similar to cellulose, only differing in a few specific functional groups. The main difference between these biopolymers is the presence of amino groups (−NH2) in the chitosan structure. Chitosan is recognized for its solubility in dilute acid media, forming a polymer with cationic characteristics due to the protonation of the amino group, generating an ammonium (−NH3+) ion, which gives special properties to this biomaterial (Berger et al. 2004; Mendes et al. 2011).

Carrageenans are described as a family of linear, sulfated polysaccharides and are isolated from certain species of red seaweed. These polysaccharides are inexpensive and possess distinct, flexible helical structures that give them the capacity to form a variety of distinct gels. The formation of carrageenan gels can be carried out either by cooling or by interacting with an aqueous solution containing cations (i.e. K+, Cu2+, Fe3+, Mg2+, NH4+, Ca2+, and Mn2+), amine-containing compounds, and\or water-miscible organic solvents (Van De Velde et al. 2002; Zajkoska et al. 2013).

Pectins are one of the main water-soluble structural polysaccharides present in plant cell walls. Structurally, pectins are formed by biopolymers of 1,4-α-d-galacturonic acid partially linked by methyl esterification. The process of pectin gelation is carried out by connection between the divalent cations and polygalacturonate structure. The gelling reaction is depicted using the egg-box model where the divalent cations form non-covalent interpolymer associations with clusters of two or sometimes four adjacent polygalacturonate structures (Braccini and Pérez 2001; Müller-Maatsch et al. 2016).

2.1.2 Immobilization in Thermogels

Gelatin consists of proteins and peptides derived from the partial hydrolysis of collagen extracted from skin and bones. The proteins found in gelatin are applicable in various sectors in the food industry due to their physical properties, such as a melting point similar to physiological temperatures. The gelatin-based immobilization process has already been developed for the immobilization of different cells and enzymes. The gelation process of gelatin can occur reversibly with temperature; however, when solutions are cooled to 30–35 °C, more efficient immobilization occurs. However, some techniques have been described as achieving an irreversible gelation process, for example, using cross-linking compounds (Tanriseven and Doǧan 2002; Yang and Ou 2005; Górecka and Jastrzębska 2011).

Agar and agarose are polysaccharides used as entrapment supports for cells and enzymes. Agar is an inert polymer composed of two principal components: agarose and agaropectin. Agarose is a heteropolysaccharide with neutral properties that has a strong ability for gelation. Agar and agarose are both advantageous in their price, availability, acid tolerance, and low reactivity with other biomolecules (Duckworth and Yaphe 1971). However, their use is limited by their low melting points (Zajkoska et al. 2013).

2.2 Synthetic Polymers

Among synthetic gels, polyacrylamide was the first matrix used for cell immobilization and is currently the most used matrix for enzyme entrapment. The polyacrylamide matrix has the advantage of being nonionic and the properties of immobilized enzymes are only minimally altered when in its gel matrix. However, the initiator of the polymerization process, dimethylaminopropionitrile, is highly toxic and requires great care (Hsuanyu 2004).

An alternative is polyvinyl alcohol (PVA), a non-toxic thermoplastic polymer, and is biodegradable, water-soluble, and biocompatible. Polyvinyl alcohol is commercially obtained from polyvinyl acetate, in which the vinyl acetate is hydrolyzed to form vinyl alcohol groups. PVA is completely insoluble in organic solvents with the exception of ethanol, in which it exhibits low solubility. As a thermoplastic polymer, it can be converted to different structures by freeze-thawing processes and as such, the gelation of PVA is performed through repeated freeze-thawing cycles (Qi et al. 2004; Vrana et al. 2009).

Polyacrylonitrile is a synthetic polymer that has attracted considerable attention due to some important features, such as chemical resistance, abrasion resistance, and thermal stability. However, a major obstacle to the long-term use of a polyacrylonitrile matrix is its biodegradability and low hydrophilicity (Stoilova et al. 2010; Potvorova et al. 2012; Feng et al. 2013).

3 Enzyme Immobilization in Polymer Gels

Polymer gels, whether natural or synthetic, can be used for entrapment by utilizing the porous matrix of the gel. In this context, biocatalyst immobilization is rising as a useful and robust technology to facilitate and improve many bioconversion processes based on whole cells or enzymes. Among the many advantages of this technology are the following: (i) increase of the robustness of the biocatalyst, (ii) possibility of reuse, (iii) improvement of the product yield, and (iv) reduction or even elimination of hazardous and toxic substances from the process (Zajkoska et al. 2013; Kras et al. 2016).

According to IUPAC (1997), biocatalyst immobilization is the fixation of a cell or its derivatives (e.g. organelles and proteins) into or onto a support to increase their stability and prolong their use. In other words, the immobilized structure may be inside or alongside a support depending on the desired purpose and type of immobilization method applied. Likewise, the cells and their derivatives may be retained by a membrane (Kras et al. 2016). In choosing the best method of immobilization, four criteria must be considered: (1) the substrates that will be used for biotransformation or production and the type of biocatalyst used (enzyme or whole-cell); (2) the equipment to be used for the biocatalysis reaction; (3) the downstream-process technology necessary for the purification of the product(s); and (4) how to avoid the release of compounds toxic to humans or the environment (Bianchini et al. 2015).

In this section, different studies on enzyme immobilization using various polymer gels are discussed.

3.1 Enzyme Immobilization Using Alginate

For a more efficient and feasible process, the immobilization conditions using calcium alginate must be optimized. Alginate concentration is a very important parameter, since higher concentrations of sodium alginate may result in a decrease of pore size which can limit the ability of compounds to reach the enzymes (Kumar et al. 2009). Another important parameter is CaCl2 concentration, since high concentrations of this compound can modify the pH of the system and affect enzyme activity (Rehman et al. 2014).

It is possible that some of an enzyme’s characteristics can suffer alterations after immobilization in gels, such as an increase of the optimum temperature of its reaction (likely due to physical limitations of enzymes within the interior of the gel). This results in a higher activation energy needed for the substrate to easily diffuse into the beads and bind to the biocatalyst (Kara et al. 2006). In addition, thermal stability of a confined protein can be altered compared to the free form, often likely due to the restriction in conformational changes allowed, directly caused by the support matrix (Shah et al. 2008).

Abdulla and Ravindra (2013) described the immobilization of a microbial lipase using glutaraldehyde and gel confinement into a hybrid matrix of equal amounts of κ-carrageenan and alginate. The immobilization of the lipase improved certain properties of the enzyme, including its thermal stability and hydrolysis performance. The immobilized enzyme showed an activity yield of 89.26%, and maintained 84.02% of its activity after being stored at 4 °C for 14 days. And subsequently, after being run for 10 cycles, it was still possible to observe 75.54% of enzymatic activity. As for the evaluation of the influence of immobilization on the enzyme properties, both enzyme preparations were characterized, showing that Vmax values remained approximately similar, while Km values changed considerably between the free and immobilized lipase.

Awad et al. (2015) studied the immobilization of phytase from Penicillium purpurogenum GE1 on grafted alginate/carrageenan beads. The authors observed maximum loading capacity after 20 h at the enzyme: acetate buffer dilution ratio of 1:2. Some properties of the confined enzyme were different from those observed in the free form. The optimal temperature and pH of the immobilized preparation were greater than the soluble one. The immobilized biocatalyst was more stable in higher temperatures (50 °C for 1 h) and acidic pH conditions (pH 4 for 45 min). In addition, the immobilization allowed 100% activity retention when the enzyme was incubated at 4 °C for 3 months, while the free form preparation completely lost activity after 4 weeks when incubated at the same temperature. Regarding the reusability of the immobilized enzyme, the gel beads containing the enzyme maintained 100% activity for more than 12 repeated batch reutilizations.

The immobilization of a pectinase from Bacillus licheniformis KIBGE-IB21 into calcium alginate has been studied by Rehman et al. (2014). These enzymes are particularly important since they catalyze the hydrolysis of pectin and are industrially applied such as in the clarification of fruit juices. The authors optimized the concentration of calcium chloride and sodium alginate, 0.2 M and 3.0%, respectively. The immobilized enzyme showed increased optimal reaction time for the degradation of pectin, showing an increased optimal activity. In addition, the entrapped enzyme showed increased thermal stability.

In another experiment, a commercial α-amylase (Diastase) was entrapped in calcium alginate beads. The best conditions for immobilization were 4% (wt/v) sodium alginate, 1 M calcium chloride, and 2 h of curing time and as a result, 85% of immobilization yield was obtained. Immobilized enzymes showed a higher Km than the free enzyme, indicating that the substrate affinity of α-amylase was decreased after immobilization (Lahiri 2015).

3.2 Enzyme Immobilization Using Agar

An important gel for the immobilization of enzymes is agar, since it is biocompatible, non-toxic, and has strong solidifying properties. As in the case of other gels, the concentration of agar is very important for protein immobilization, since the porosity of the agar matrix is dependent on the concentration of agar used. And once again, the optimal pH and temperature of the enzyme can be altered after immobilization in agar (Rehman et al. 2014).

Xylooligosaccharides are important, small oligosaccharides (2–10 units) that present interesting functional properties. These compounds are produced through hydrolysis of xylan, catalyzed by endoxylanases. As such, the use of immobilization for the entrapment of xylanase can be of great interest, since it can reduce the cost of the industrial process. In the study by Milessi et al. (2015), a recombinant endoxylanase from Bacillus subtilis was immobilized in different gel-based supports, with immobilization yields above 80%. A remarkable increase was observed in the stability of the proteins immobilized in chitosan or agarose activated by glyoxal groups. The authors explained that the thermal stability observed for the agarose-glyoxal derivative was probably due to lysine residues present in unstable sites.

Bibi et al. (2015) studied an endoxylanase that hydrolyzes xylan, presenting several applications, including in the pulp and paper industry. The authors immobilized this xylanase from Geobacillus stearothermophilus KIBGE-IB29 in agar–agar gel beads and the best results were found using 2.5% of the polymer. The optimum temperature increased by 10 °C (from 50 to 60 °C) for the immobilized biocatalyst. In addition, the immobilization led to an increase in thermal stability, retaining 79.0% of activity at 80 °C while the free enzyme completely lost activity at this temperature. The immobilized enzyme was successfully reused for up to six reaction cycles.

3.3 Enzyme Immobilization Using Chitosan

Chitosan is a well-studied support for the immobilization of enzymes because of the availability of various chitosan products with different N-deacetylation grades (Betigeri and Neau 2002). It is frequently studied for the immobilization of enzymes in combination with other gels. This is based on the intense electrostatic interactions of the carboxyl groups of supports like alginate with the amine groups of chitosan, resulting in chitosan–alginate hybrid gels. Frequently, these hybrid gels are stronger than pure chitosan and is what makes them have a longer activity span under intense conditions of temperature and mechanical agitation (Mi et al. 2002). Finally, chitosan has low cost and a lack of toxicity and biological reactivity.

In one experiment, the purified α-amylase from Exiguobacterium sp. DAU5 was immobilized in chitosan beads, utilizing glutaraldehyde (Fang et al. 2016). The immobilized enzyme displayed higher organic-solvent tolerance than free enzymes. The specific activity of the enzyme immobilized in chitosan beads and chitosan-carbon beads was 2240 and 2320 U/g, respectively. The optimal conditions for the immobilized protein were 50 °C and pH 8.5.

In a study by Xie and Wang (2011), the commercial lipase from Candida rugosa was immobilized in magnetic chitosan microspheres using glutaraldehyde. The esterification of soybean oil had a maximum yield of 87% using the ratio of methanol:oil of 4:1, at 35 °C, after a 30 h reaction time. Furthermore, the immobilized protein was used 4 times without a significant decrease in performance.

In a different experiment, a C. rugosa lipase was covalently immobilized in magnetic Fe3O4–chitosan nanoparticles. The best results obtained were 20 U/g of Fe3O4–chitosan. The immobilized preparation was utilized in twenty repeated uses, retaining more than 83% of its initial activity. In addition, lipases after immobilization remained stable over wide ranges of pH and temperature (Kuo et al. 2012).

3.4 Enzyme Immobilization in Synthetic Polymers

Non-natural polymers have also been studied for enzyme immobilization. These matrices allow the obtention of particles with good mechanical and chemical stability and appear to be promising alternatives to natural polymers to overcome the disadvantages of some polysaccharides (Lozinsky et al. 2003; Schlieker and Vorlop 2006).

A commercial invertase was immobilized in a system composed of polyacrylamide and gelatin (Emregul 2006). The authors observed that the Km values were 166 and 86 mM for immobilized and free enzymes, respectively. All immobilized preparations were successfully utilized twenty times within 60 days approximately, still showing good activity.

Fernandes et al. (2009) studied the immobilization of an inulinase with invertase activity in particles composed of PVA. After immobilization, the enzyme showed a broadened optimal pH towards lower values, as well as good mechanical stability of the particles when the temperature was above 55 °C. The authors observed a 1.8-fold increase in Km, probably due to diffusion limitations. In addition, a 10% decrease in the reaction yield was observed at a reaction temperature of 50 °C, after 20 repeated, consecutive batches.

4 Immobilization of Whole Cells Using Polymer Gels Applied to Biocatalysis and Biotransformation

Immobilized cells in gelatinous and porous matrices have been extensively employed in the last decades, with reports dating back to before the 1960s. In 1960, Hattori and Furusaka published a work evaluating cells of the bacterium Escherichia coli in relation to the changes in its chemical activity after immobilization in a resin. They observed a decrease in substrate oxidation activity that persisted even after the detachment of the adsorbed cells. In 1974, a strategy was announced for the use of immobilized cells for the continuous fermentation of l-aspartic acid (Chibata et al. 1974a, b; Ramakrishna and Prakasham 1999; Martins et al. 2013).

The ability to immobilize microorganisms like bacteria, yeasts, and fungi (Meunier et al. 2010) combined with their capacity to display the functional properties of their proteins on the cell surface or at the intracellular microenvironment has made it possible for many species to be used for cell immobilization. In addition, this has been accomplished in diverse types of natural (agar, agarose, and pectin), synthetic (PVA and polyacrylamide), and hybrid supports (derived from the incorporation of organic and inorganic materials) (Ramakrishna and Prakasham 1999; Fukuda et al. 2008; Desimone et al. 2009; Léonard et al. 2011). For immobilization, filamentous fungi and yeasts have often been chosen as some of the most resistant cell biocatalyst systems for industrial purposes, such as the filamentous fungi Rhizopus niveous, Rhizopus chinensis, Mucor circinelloides, Rhizomucor miehei, Trichoderma reesei, Aspergillus niger, and the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica (Kumakura et al. 1989; Ramakrishna and Prakasham 1999; Fukuda et al. 2008; Robles-Medina et al. 2009; Andrade 2012).

In this section, special attention will be given to whole-cell immobilization. Basically, an immobilization process consists of the attachment of cells to a solid phase (matrix or membrane support), enabling the exchange of nutrients and gases between the cells and the broth medium (Ramakrishna and Prakasham 1999).

Cell immobilization techniques have been described for many plant and mammalian cells (Seifert and Phillips 1997; Uludag et al. 2000; Murthy et al. 2014), as well as bacterial and fungal strains, with positive results for both natural and genetically modified types (Shriver-Lake et al. 2002; Prasad et al. 2005; Mrudula and Shyam 2012; Bisht et al. 2013). As previously mentioned, the main methods used are adsorption, covalent bonding/cross-linking, entrapment, and encapsulation (Fig. 1). Cell-matrix interaction, biocompatibility of the carrier, mass transfer, and aeration of the cells are some of the factors that affect whole-cell immobilization (Nedovic and Willaert 2004; Liu and Wang 2010) and thus influence the parameters for an experiment. Each method has its pros and cons, making one more suited for a specific application than another (Desimone et al. 2009; Pajic-lijakovic et al. 2015), but entrapment is the most used cell immobilization method (Trelles and Rivero 2013).
Fig. 1

Different strategies for whole-cell immobilization

Some studies have been carried out to understand the physiological and morphological changes that cells may undergo when they are immobilized (Omar et al. 1992; Niu et al. 2013). Due to various physical characteristics of supports, the choice of polymer may also influence cell productivity, seemingly even differing across microorganisms (Bisht et al. 2013; Chandorkar et al. 2014). Further building upon that data, attempts to attain the optimal conditions for a particular product’s production or bioconversion (Chen et al. 2012) as well as studies focusing on the development of new polymer gels with different characteristics (Niu et al. 2013) were also carried out.

4.1 Whole-Cell Immobilization Versus Enzyme Immobilization

In contrast with individual enzymes, whole-cell immobilization is preferred for having all the needed catalysts and cofactor regeneration for a specific reaction (Bianchini et al. 2015). This technology is cheaper than enzyme immobilization and has excellent operational stability, providing a valid alternative to improve industrial processes (Fukuda et al. 2008). Likewise, immobilization is exceptionally useful for certain microorganisms that exhibit different morphologies throughout their life cycles, as is the case of fungi (Prasad et al. 2005). These cells can be immobilized in a morphological state that allows their highest catalytic activity, therefore assuring the maintenance of their highest productivity form for a longer period (Covizzi et al. 2007). In addition, immobilized cells can work in a higher concentration than free cells, which increases fermentation speed and throughput, guarantees the synthesis of the metabolites, and protects the living cells from environmental stress factors that might arise from the process such as pH changes, high concentrations of the end-products, or phenols and other toxic compounds (Bisht et al. 2013; Martins et al. 2013; Vilela et al. 2013; Lin et al. 2015).

Still, the major advantage of cell immobilization, often cited in literature, is that they are easily recovered and can be reused for many cycles, remaining nearly just as effective as in the first use for a long period (Pradella 2001). Additionally, continuous systems can be operated above the usual µmax (maximum specific growth rate) observed for free cells. In this sense, Chandorkar et al. (2014) observed that lipase production by A. niger entrapped in sodium alginate remained almost the same after 4 cycles, whereas Bisht et al. (2013) described the maintenance of lipase activity by Pseudomonas aeruginosa for 7 cycles in a bioreactor. In another study (Duarte et al. 2013), S. cerevisiae cells were evaluated for ethanol production through the fermentation of glucose and sucrose after immobilization using two different substrates, calcium alginate and calcium alginate covered with chitosan. In both cases, the immobilized cells could withstand 8 fermentation cycles of 10 h each, with no observed contamination.

In contrast with the easier whole-cell recovery, enzyme immobilization is an expensive technology due to the enzyme recovery and purification steps that are required to obtain the desired products from the fermentation broth. Furthermore, there may be losses during the enzyme purification process, as well as catalytic activity reduction (Sührer et al. 2015). Conversely, some disadvantages of whole-cell immobilization must be considered, namely: (1) it can generate undesired byproducts and/or toxic metabolites which might damage the cell biocatalyst; (2) possibility of cell leakage from the carrier; and (3) alteration in the physiology and growth kinetics of the cells (Hattori and Furusaka 1959; Mattiasson and Hahn-Hägerdal 1982; Robles-Medina et al. 2009; Martins et al. 2013; Sührer et al. 2015). Mattiasson and Hahn-Hägerdal (1982) discussed that many studies had demonstrated that immobilized cells have alterations in metabolism in comparison to the same free cells. According to these authors, while in some cases growth rates might be reduced, specific metabolites can become highly produced, of which industrial processes take advantage. They propose that the microenvironment of entrapped cells might be responsible for such changes, more specifically due to a decreased water activity and/or oxygen deficiency. Vilela et al. (2013) also discuss that the interior of the polymer beads, having a limited access to the substrate, does not promote cellular growth. The authors point to other studies that suggest it is the microenvironment inside the gelatinous bead—and not the polymer itself—that causes changes in the entrapped microbial cell physiology.

The advances in the understanding of the metabolism and physiology of microorganisms and cells and the development of new matrices allowed the use of cell immobilization for various applications, predominantly the production of ethanol, biodiesel and alcohols, organic acids, antibiotics and enzymes; aroma formation; the bioremediation of waste residues; and biosensors. Some of these practical applications are described below, concretely showing the vast potential of this technology.

4.2 Applications of Whole-Cell Immobilization

4.2.1 Biodiesel Production

Biodiesel fuel (BDF) is composed of fatty acid alkyl esters or methyl esters (MEs) derived from vegetable oil or animal fats and may be produced from any of three different reactions: (i) pyrolysis (or cracking), (ii) microemulsion, or (iii) transesterification. The first two options are considered too expensive for industrialization and yield low-quality biodiesel. The transesterification (i.e. acidolysis, alcoholysis, or interesterification, depending on the acyl group acceptor) is the most common reaction used to produce biodiesel (Robles-Medina et al. 2009). It consists of a reaction between triglycerides (TAGs) from an oil or fat along with an alcyl-acceptor, such as alcohols like methanol and ethanol. As products of this reaction, methyl esters (MEs or biodiesel) and glycerol (via alcoholysis) can be formed, as well as another triacylglycerol (via interesterification) (Fig. 2) (Huang et al. 2012).
Fig. 2

Example of transesterification reactions (alcoholysis) and types of catalysts used for biodiesel production

Transesterification can be catalyzed by acids such as sulfuric acid (H2SO4) or phosphoric acid (H3PO4), bases such as potassium hydroxide (KOH) or sodium hydroxide (NaOH), enzymatically, or through lipases attached to the surface of immobilized cells or overexpressed in the intracellular environment (Fig. 2) (Fukuda et al. 2008; Robles-Medina et al. 2009). An alkaline biocatalyst is the most used process worldwide for transesterification catalysis, contributing to almost 100% of the biodiesel production process as of 2008 (Demirbas 2008; Robles-Medina et al. 2009).

Facing the need to discover another low-cost and more environmentally friendly way to produce biodiesel than by an alkaline biocatalyst or enzymatic process, cell immobilization recently appeared as an attractive substitute due its aforementioned advantages. The first project with this type of technology aimed at BDF production was described by Ban et al. (2001). In this work, the 1,3-specific fungal lipase (ROL) of Rhizopus oryzae cells was immobilized within biomass support particles (BSPs). They were then used for the catalysis of the transesterification reactions and ME production. Interestingly, ME content reached 90% yield, the same percentage obtained by extracellular lipases (Ban et al. 2001), and these results supported further research regarding cell immobilization as a good alternative to produce biodiesel.

Since then, many other studies have been conducted to discover the most appropriate culture conditions and microorganisms for whole-cell immobilization systems and to attain the same—or higher—levels of biodiesel production obtained through the enzymatic process. Table 2 shows a small bibliographic survey with some promising studies of biodiesel production.
Table 2

Whole-cell immobilization of microorganisms, culture conditions, and biodiesel yield (ME %)

Microorganism

Support

Carbon source

Oil

Alcohol

ME** (%)

References

R. oryzae

BSP*

Olive oil and glucose

Soybean oil

Methanol

90

Ban et al. (2001, 2002), Hama et al. (2007), Hama et al. (2015)

R. oryzae

BSP*

Fatty acids

Soybean oil

Methanol

80

Hama et al. (2004)

R. oryzae

BSP*

Olive oil or glucose

Jatropha oil

Different alcohols

89

Tamalampudi et al. (2008)

R. oryzae

BSP*

Soybean oil

Soybean oil

Methanol

70

Li et al. (2007a)

R. oryzae

BSP*

Soybean oil

Rapeseed oil

Methanol

72

Li et al. (2007b)

R. oryzae

BSP*

Soybean oil

Oleic acid

Methanol

90

Li et al. (2008)

R. oryzae

BSP*

Olive oil

Soybean oil

Methanol

73

Chen and Lin (2010)

R. oryzae

Sodium alginate

Olive oil

Jatropha oil

Methanol

80.5

Ganesan et al. (2011)

A. niger

BSP*

Olive oil

Palm oil

Methanol

87

Xiao et al. (2010)

S. cerevisiae (intracellular ROL)

Glucose

Soybean oil

Methanol

71

Matsumoto et al. (2001)

S. cerevisiae

Glucose

Soybean oil

Methanol

78

Matsumoto et al. (2002)

(cell surface ROL)

Hama et al. (2007) studied the methanolysis of soybean oils for the production of biodiesel using R. oryzae biomass immobilized in polyurethane foam. The repeated methanolysis reaction was carried out in a 20-L air-lift bioreactor (also named packed-bed reactor (PBR) system) for 20 batch cycles and a ME content of 65–80% was reached and maintained during the entire process. In addition, when methanol (four molar equivalents to oil) was added in the first cycle, 90% of the ME conversion was achieved. The disadvantages observed in the PBR system, however, included cellular exfoliation when the reaction flow rate was high (near 55 L/h) and a decrease in the conversion rate at a flow of 5 L/h because of the inefficiency of the mixture inside the reactor (Hama et al. 2007). One of the reasonable explanations for exfoliation is the shear stress at high flow rates that damages the cells and causes loss of lipase activity (Hama et al. 2007; Andrade 2012).

Ban et al. (2001; 2002) investigated the immobilization of R. oryzae, the effects of pretreatments on the cell biomass, the effect of water on the transesterification reaction, and also studied vegetable oils used for biodiesel production. The researchers found that in the presence of 15% water, methanolysis increased up to 90%, a conversion rate similar to that reached with extracellular lipases. They used a discontinuous mode process and after six cycles, the biocatalyst cells retained a yield of 70-80% of ME.

Comparing the enzymatic transesterification of fungal lipases with cell immobilization, the latter is still disadvantageous because it does not attain the high ME mixture content and has a lower reaction rate than the former. However, the results observed so far point to a hopeful future, where cell immobilization can be as efficient as free enzymes, overcoming the current major bottlenecks and increasing economic viability.

4.2.2 Enzyme Production

Immobilized cells are showing great potential for the production of enzymes, particularly those of industrial interest. There are already a myriad of examples in published literature. Siddiqui et al. (2016) used A. niger cells immobilized by entrapment in calcium-alginate beads for the production of native cellulolytic enzymes, achieving up to 0.37 IU/mL of enzymatic activity on glucose after 48 h of incubation. In comparison, the same A. niger free cells took twice the time to obtain enzymatic activity close to this value. Bacterial cells immobilized in calcium alginate beads were also successfully employed by Darah et al. (2015) for polygalacturonase production. Entrapped Enterobacter aerogenes NBO2 cells produced 23.48 U/mL of the enzyme, while 18.54 U/mL were obtained using the free cells. In these two studies, there is a clear observation that, given the appropriate optimization, cell immobilization can significantly enhance enzyme production.

4.2.3 Aroma Formation

Another industrial application for cell immobilization is aroma formation. The flavor industry has a huge market around the world, having dealt with about US$20.3 billion in 2009 (Markets andMarkets 2015). The compounds produced by fungi, yeasts, and bacteria that confer aromas are metabolites resulting from the catabolism of sugars and nitrogenous and sulfur compounds, as well as from the synthesis of essential molecules for the growth of microorganisms like nucleic acids, amino acids, and lipids (Nedovi et al. 2015). These metabolites include alcohols (ethanol), esters (phenylethyl acetate, ethyl hexanoate), carbonyls (acetaldehyde, diketones), organic acids, and sulfur compounds (hydrogen sulfide, sulfur dioxide) and their use in the industrial field has high added value, mainly because flavor is an important ally for the acceptance of many available food products (Lalou et al. 2013).

Aroma compounds have been studied since the 1960s and 1970s with jasmonates and lenthionin production by the fungi ascomycete Lasiodiplodia theobromae (synonym Botryodiplodia theobromae) and basidiomycete Lentinus edodes, respectively (Yasumoto et al. 1974; Krings and Berger 1998). However, only a few years ago cell immobilization started to be employed for aroma production as an advantageous biocatalyst. Some works have showed positive results with this technology for beer and wine making (Tataridis et al. 2005; Willaert and Nedovic 2006; Vilela et al. 2013).

Wilkowska et al. (2015) observed abundant production of volatiles when using apple/cranberry and apple/chokeberry pomaces with the yeast Kluyveromyces marxianus cells immobilized in foamed alginate. It was possible to identify 11 aroma compounds, esters and alcohols, with fruity and floral aromas (e.g. ethyl acetate, isoamyl alcohol, and ethyl butyrate). Thus, depending on the desired flavor compounds, the fermentation of fruit pomaces using immobilized cells can be an effective method of producing commercially valuable volatiles (Wilkowska et al. 2015).

4.2.4 Bioremediation of Wastewater and Organophosphates, and Biosensors

Whole-cell immobilization has also been used in wastewater treatment, as one of the most widely used uses for immobilized cells (Pradella 2001). Usually, wastewater has to undergo two separate processes to avoid the proliferation of nitrogen compound-consuming cyanobacteria in the water: nitrification and denitrification. Nitrification is the oxidation of ammonia to nitrites and subsequently nitrites to nitrates by the action of bacteria, such as Nitrosomonas and Nitrobacter, respectively. Denitrification must then be carried out under anoxic conditions and can be performed by other bacterial genera, i.e. Micrococcus and Pseudomonas. These two reactions occur in treatment plants and require a high flow rate to degrade the nitrogen contaminants. In this system, the active sludge formed by free bacteria is responsible for both reactions. However, the sludge needs additional organic compounds, such as acetic acid, glucose, or methanol, because the organic molecules existing in the wastewater are not sufficient for bacterial growth and proliferation, the addition of which increases wastewater treatment costs. Hence, there are some disadvantages in the conventional process that can be overcome by bacterial cell immobilization (Kras et al. 2016).

One example of this is the LentiKats® technology developed, patented, and improved in recent years by a group from the Czech Republic. The last update of this technology initiative consisted of two tanks: the first one is filled with immobilized bacteria of nitrifiers N. europaea and N. winogradskyi, while the second has immobilized denitrifiers P. denitrificans and P. fluorescens. These strains are immobilized on a porous hydrogel matrix made of polyvinyl alcohol (PVA), being non-toxic and non-biodegradable. The manufacturer states that this technology can treat waste residues containing 800 mg/L of N–NH4+ or 1000 mg/L of N–NO3− with an efficiency of 98% (Cechovská et al. 2009; Bousková 2010). Several different waste sources have been treated with LentiKats®, from industrial wastewater to groundwater from uranium mining sites (Kras et al. 2016). LentiKats® still remains an expensive investment for wastewater treatment, but it can be used for other applications, like ethanol production, bioconversion of malic to lactic acid present in apple juices, and even could potentially be used for xylitol production from sugarcane bagasse (lignocellulosic residue, abundant in Brazil). Thus, despite being costly, this technology may be a good long-term alternative for various applications.

Another application for whole-cell immobilization is the bioremediation of organophosphates (OPs). These neurotoxic compounds are found in pesticides and insecticides and their removal or decomposition is currently done by chemical treatment, incineration, or deposition in landfills. It is of paramount importance that these OPs be detoxified and converted in new, non-harmful compounds in order to not damage the environment and human health (Kim et al. 2014). Organophosphorus hydrolases (OPHs) are enzymes produced by soil microorganisms and are able to degrade the OPs. Many attempts have been made to improve OP degradation efficiency by immobilizing these metalloenzymes in different supports (Kim et al. 2014); however whole-cell immobilization has also attracted attention because of its many advantages already cited, including higher enzyme stability and lower cost.

Bacteria and yeasts have been used as OP degradation biocatalysts, such as E. coli, Moraxella sp., Pseudomonas putida, Stenotrophomonas sp., and Y. lipolytica. Their OPHs were attached on the cell surface through fusion systems (like lipoproteinand outer membrane protein A, Lpp-OmpA system; protein InaV from Pseudomonas syringae; GPI-based; or Flo1p-based) or were located inside the periplasm. Different enzymatic activities were found against each class of OP (paraoxon, parathion, diazinon, fenitrothion) and the whole-cell immobilization stability lasted up to weeks (Kim et al. 2014).

Besides the ability to detoxify OPs, whole-cell immobilization also can be applied to construct biosensors to detect different compounds such as heavy metals (Shing et al. 2013), pesticides (Anu Prathap et al. 2012), sugar (Kitova et al. 2010), urea (Jha et al. 2009), lactate (Smutok et al. 2007), and even carry out quorum sensing related to N-acylhomoserine lactones (Struss et al. 2010). Notably, a cell-based system was developed by Grosh et al. (2008) to measure the levels of cytokines in the sera of cancer patients. For this, a cellulose triacetate (CTA) membrane of an ion-selective electrode was used to immobilize human umbilical vein endothelial cells (HUVEC). The electrode was exposed to the serum of healthy and cancer patients and after one hour, it was possible to note differences between cytokine (β-FGF, HGF, TNF-α, etc.) levels of the two patient groups and to correctly correlate the response of the biosensor with the stage of cancer (Ghosh et al. 2008). Despite promising results, the hype created from the use of biosensors for medical applications remains in the numerous research reports instead of transitioning to the commercialization of practical biosensors. In addition to the initial studies, it would be more than reasonable to expect the development of new biosensor technologies (D’Orazio 2011).

5 Conclusions and Future Perspectives

As described in this chapter, polymer gels show tremendous potential for the immobilization of industrially relevant enzymes, such as lipases, amylases, and xylanases. In general, it can be expected that enzyme immobilization results in increased enzyme stability, reaction efficiency, and reusability. All of these factors are involved in the cost reduction of enzyme-related industrial processes. To achieve the best results, the enzyme immobilization must be individually optimized, using simple and low-cost supports and processes.

In addition, depending on the conditions and goal, whole-cell immobilization appears to be an effective and less expensive strategy, while remaining just as efficient as other methods. Different types of cells can be immobilized in several matrices for biocatalytic reactions. Their applicability ranges from environmental issues, like heavy metal and pesticide contamination of the soil, to more industrial and clinical uses, like complex chemical production and biosensors for medical tests.

Despite many efforts to better understand and optimize whole-cell immobilization, including better understanding cell physiology and metabolism, selecting the type of matrix, dealing with inhibitory compounds released during biocatalysis steps, and optimizing cell growth conditions, a lot has yet to be done to best benefit from this technology and promote more efficient applications in all fields. Genetic improvement of microorganisms, development of new materials for support, and perfecting growth conditions are just some of the details that deserve more attention to translate the preliminary results into industrial reality.

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Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Gustavo Pagotto Borin
    • 1
    • 2
  • Ricardo Rodrigues de Melo
    • 1
    • 3
  • Elaine Crespim
    • 1
  • Helia Harumi Sato
    • 3
  • Fabiano Jares Contesini
    • 1
    • 2
  1. 1.Laboratório Nacional de Ciência E Tecnologia Do Bioetanol (CTBE)Centro Nacional de Pesquisa Em Energia E Materiais (CNPEM)CampinasBrazil
  2. 2.Institute of BiologyUniversity of CampinasUnicampBrazil
  3. 3.College of Food EngineeringUniversity of CampinasCampinasBrazil

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