Recent Advances in Microbial Biopolymer Production

1

IntroductionDue to extensive research over the past decades micro-bial biopolymers offer a wide variety of new applica-tions and have the potential to replace common, less favorable, materials. In particular, the substitution of non-degradable plastics is of considerable interest as it allows for the environmentally and economically ben-eficial disposal of major waste streams (Luckachan and Pillai, 2011). Furthermore, a replacement with polymers derived from plants or algae often allows for improved physical properties of the polymeric materials (Freitas et al., 2011).

Among the most commercially important and prom-ising microbial polymers (Table 1), xanthan gum was the first to be produced at the industrial scale and still is one of the most significant biopolymer currently on the market. Its worldwide annual production amounts to approximately 100,000 metric tons with a market price of 3 to 5 US$ per kg (Freitas et al., 2011; Rehm, 2010). Compared to an estimated production of 260 million

tons of petrochemical polymers in 2007 (Hopewell et al., 2009) it can be seen that the market share of biopolymers and particularly bioplastics is relatively small. In 2006 approximately 350,000 metric tons of bioplastics, like polyhydroxyalkanoates (PHA), were produced, which accounted for a market share of 0.2% of the worldwide plastics production. However, with annual growth rates of 25–30% a market share of up to 5% and production capacities of roughly 3 million metric tons per year are estimated for 2020 (Beucker et al., 2007).

The current low market share is mainly due to higher production costs and technical requirements for microbial production, as opposed to chemical synthesis from non-renewable resources or extraction from plant, animal or algal biomass. The production costs of microbial polyhydroxybutyrate (PHB) – a potential bioplastic – for instance are still 5 to 10 times higher than costs for the synthesis of common petrochemical polymers (Rehm, 2010). In Figure 1 an overall scheme of biopolymer production and purification process

REVIEW ARTICLE

Recent Advances in Microbial Biopolymer Production and Purification

Dirk Kreyenschulte1,2, Rainer Krull1, and Argyrios Margaritis2

1Institute of Biochemical Engineering, Technical University Braunschweig, Braunschweig, Germany and 2Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario, N6A 5B9, Canada

AbstractOver the past decades a large amount of biopolymers originating from various types of microorganisms have been reported. With ongoing research the number of possible applications has increased rapidly, ranging from use as food additives and biomedical agents to biodegradable plastics from renewable resources. In spite of the plethora of applications, the large-scale introduction of biopolymers into the market has often been forestalled by high production costs mainly due to complex or inefficient downstream processing. In this article, state-of-the-art methods and recent advances in the separation and purification of microbial polymers are reviewed, with special focus on the biopolymers, γ-polyglutamic acid and xanthan gum. Furthermore, a study of the general factors affecting production and purification is presented, including biopolymer rheology, enzymatic degradation and production of biopolymer mixtures.Keywords: Biopolymer applications, biopolymer market, biotechnological polymer production, broth viscosity, downstream processing, γ-polyglutamic acid, polyamides, polyesters, polysaccharides, xanthan gum

Address for Correspondence: Dr. Argyrios Margaritis, Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario, N6A 5B9, Canada. Tel: 519-661-2146. E-mail: [email protected]

(Received 25 May 2012; revised 18 September 2012; accepted 20 September 2012)

Critical Reviews in Biotechnology, 2012; Early Online: 1–16© 2012 Informa Healthcare USA, Inc.ISSN 0738-8551 print/ISSN 1549-7801 onlineDOI: 10.3109/07388551.2012.743501

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Microbial Biopolymer Production and Purification

D. Kreyenschulte et al.

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2 D. Kreyenschulte et al.


Table 1. List of commercially important microbial biopolymers.Polymer Class Source Substrates Applications ReferencesPolyamidesCyanophycin Cyanobacteria,

Acinetobacter spp., Bordetella spp., Desulfitobacterium hafniense, Nitrosomonas europaea

• Arginine, (NH4)

2SO

4

• Protein hydrolysate• ProtamylasseTM

• Water softener• Metal ion-exchange

system• Hydrogels• Synthesis of chemicals• Nutrition

Elbahloul et al., 2005Mooibroek et al., 2007Solaiman et al., 2011

γ-Polyglutamic acid

Bacillus spp., Staphylococcus epidermis, Natrialba aegyptiaca, Natronococcus occultus, Fusobacterium nucleatum

• Glycerol,L-glutamic-acid, citric acid

• Biodegradable plastics• Fertilizer• Food thickener• Hydrogels• Medical adhesives• Nanoparticle drug/gene

delivery• Skin care• Tissue scaffolds• Wastewater treatment

Buescher and Margaritis, 2007Rehm, 2010Bajaj and Singhal, 2011

Poly-ε-lysine Streptomyces albulus ssp. lysinopolymerus

• Glucose, (NH4)

2SO

4• Coating material• Dietary agent• Drug/Gene delivery• Emulsifying agent• Endotoxin removal• Food preservative• Hydrogels• Interferon inducer

Hamano, 2011Shih et al., 2006

PolyanhydridesPolyphosphate Eukaryotic and prokaryotic

cells• Sodium acetate, KH

2PO

4

and NH4Cl e.g. from

wastewater

• Antibacterial agent• ATP substitute• Food additive• Insulating fiber

Achberge-rová and Nahalká, 2011Kishida et al., 2006Kornberg et al., 1999

PolyestersPolyhydroxy-alkanoates

Prokaryotes • Carbohydrates• Starch• Alcohols• Industrial waste

products

• Biodegradable plastics• Drug delivery• Tissue engineering

Chanpra-teep, 2010Koller et al., 2010Zinn et al., 2001

PolysaccharidesAlginate Pseudomonas and

Azotobacter spp. (mostly A. vinelandii)

• Sucrose • Cell immobilization• Drug delivery• Food additive• Textile/paper industry• Wound dressing• Water treatment

Remming-horst and Rehm, 2006Sabra et al., 2001Rehm and Valla, 1997

Bacterial cellulose Gluconacetobacter, Agrobacterium, Aerobacter, Achromobacter, Azotobacter, Escherichia, Rhizobium, Sarcina, and Salmonella spp.

• Glucose• Sucrose• Other carbohydrates

• Food additive• Membrane material• Oil recovery• Paper industry• Wound dressing

Chawla et al., 2009Shoda and Sugano, 2005

Curdlan Agrobacterium, Rhizobium and Cellulomonas spp.

• Glucose• Sucrose• Other carbohydrates

• Food additive• Concrete additive• Drug delivery• Immune stimulator• Heavy metal removal

McIntosh et al., 2005Salah et al., 2011b

Dextran Leuconostoc, Streptococcus and Lactobacillus spp. (mostly L. mesenteroi-des), Gluconobacter sp., Pediococcus pentosaceus

• Sucrose• Maltodextrins

• Blood-plasma substitute• Molecular sieves

(Sephadex)• Heavy metal removal• Cosmetics• Emulsifying and thickening

agent

Naessens et al., 2005Patel et al., 2010

(Continued)

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Microbial Biopolymer Production and Purification 3


is depicted, showing the general stages of up- and downstream processing that are required in order to achieve the final polymer product. It is important to note that the costs for downstream processing usually account for 40 to 60% of the total production costs (Reif and Scheper, 2006), further optimizations in the separation and purification process are of paramount importance to the economics of biopolymer production.

This review discusses microbial polymer production with special emphasis on recent advances in separa-tion and purification. Using the examples of xanthan gum and γ-polyglutamic acid (γ-PGA), a biopolymer of emerging commercial interest, widely established and newly developed techniques for biopolymer production and purification are outlined. Furthermore, we present a critical review of the general factors affecting microbial production and downstream processing of biopolymers.

General factors affecting the production and purification of biopolymersNext to the control of common cultivation conditions, such as temperature, medium composition, aeration and

agitation, necessary for almost every bioprocess there are some specific factors that have to be considered when dealing with biopolymers. A short overview of some of the key factors is presented.

Rheology of biopolymersThe rheology of the cultivation broth has a major impact on mass, oxygen and heat transfer characteristics and consequently on the outcome and success of a bio-process. Due to the secretion of polymers, the viscos-ity of the culture fluid can increase dramatically in the course of a cultivation and hence differ from the com-mon Newtonian behavior of aqueous solutions. Thus, a basic knowledge of fundamental rheological properties is essential to understanding the requirements of the production, as well as the separation and purification of microbial polymers.

In general, the relationship between shear stress τ and velocity gradient or shear rate γ is given by the Herschel–Bulkley equation (1), which utilizes the consistency index K, the yield stress τ

0 and the flow index n.

τ τ γ= + ⋅0 K n( )

(1)

Table 1. (Continued).Polymer Class Source Substrates Applications References

Gellan Pseudomonas elodea, Sphingomonas spp. (mostly S. paucimobilis ATCC 31461)

• Carbohydrates• Industrial waste products

• Agar substitute• Cell immobilization• Food additive• Gel electrophoresis• Tissue engineering

Bajaj et al., 2007Fialho et al., 2008

Hyaluronic acid Streptococcus zooepidemicus, S. equi, Pasteurella multocida

• Glucose, amino acids, nucleo-tides, salts, trace ele-ments and vitamins

• Cosmetics• Drug/gene delivery• Viscosupple-mentation• Wound dressing

Chong et al., 2005Kogan et al., 2007

Levan Zymomonas mobilis, Bacillus spp., Streptococcus spp., Alcaligenes viscosus and other prokaryotes

• Sucrose• Lactose

• Blood-plasma substitute• Cosmetics• Emulsifying agent• Food additive

de Oliveira et al., 2007Senthil-kumar and Gunase-karan, 2005Shih et al., 2005

Pullulan Aureobasidium pullulans, Tremella mesenterica, Cytaria sp., Cryphonectria parasitica, Rhodototula bacarum

• Carbohydrates• Industrial waste products

• Blood-plasma substitute• cosmetics• Enzyme immobilization• flocculating agent• Food additive• Pharmaceutical coating

Leathers. 2003Singh et al., 2008

Scleroglucan Sclerotium rolfsii and S. glucanicum, Schizophyllum commune, Botrytis cinerea, Epicoccum nigrum

• Glucose• Sucrose

• Cosmetics• Drug delivery• Immune stimulator• Oil recovery• Pharmaceutical coating

Schmid et al., 2011Survase et al., 2007

Succinoglycan Sinorhizobium meliloti, Agrobacterium sp., Alcaligenes faecalis var. myxogenes, Pseudomonas sp.

• Sucrose and other carbo-hydrates

• Food additive• Oil recovery

Freitas et al., 2011Moosavi-Nasab et al., 2010Simsek et al., 2009Stredansky et al., 1998

Xanthan gum Xanthomonas campestris • Glucose• Sucrose• Other Carbohydrates

• Agricultural products• Coatings• Cosmetics• Food additive• Oil recovery• Paper industry• Thickener

Palaniraj and Jayaraman, 2011

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Depending on the values for K, τ0 and n, fluids can

be classified as Newtonian and non-Newtonian fluids (dilatant, pseudoplastic, Bingham plastic or Casson flu-ids). For Newtonian fluids τ

0 = 0 and n = 1, so there is a

linear relationship between shear stress τ and shear rate γ . In this case, the slope in a plot of τ against γ is defined

as the viscosity μ. For non-Newtonian dilatants (τ0 = 0,

n > 1) and pseudoplastic fluids (τ0 = 0, n < 1) equation

(1) changes into the Ostwald-de-Waele or into the more common power-law model. The relationship between τ and γ differs so that the viscosity can depend on the shear rate. It is then defined as the apparent viscosity μ

a

(shown in equation 2).

µ γanK= ⋅ −( ) 1

(2)

Dilatant fluids increase in viscosity with increas-ing shear rate, whereas pseudoplastic fluids exhibit a decrease in viscosity with increasing shear rate. Furthermore, for Bingham plastic (n = 1, τ

0 ≠ 0, τ > τ

0)

and Casson fluids (n = 0.5, τ0 ≠ 0, τ > τ

0) a yield stress

τ0 has to be overcome before flowing occurs (Margaritis

and Pace, 1985). Figure 2, shows a plot of shear stress τ versus shear rate γ indicating the flow characteristics of different fluids.

The most frequent behavior of biopolymer solutions is pseudoplastic (Seviour et al., 2011), also called “shear-thinning”. An example of a biopolymer with pseudoplas-tic behavior is an aqueous solution of the biopolymer γ-PGA. Richard and Margaritis (2003) studied the oxygen transfer, as well as the rheological characteristics of the

broth during microbial PGA production. The flow index n decreased from 1 (Newtonian behavior) to 0.885 dem-onstrating the pseudoplasticity of the γ-PGA biopolymer solution. In this case the viscosity did not seem to limit the oxygen transfer as the oxygen demand for growth and PGA production was highest in the early exponential growth phase. As shown in Figure 3, γ-PGA concentration and broth viscosity reached their maximum values in the late exponential and early stationary phase (Richard and Margaritis, 2003).

Besides the γ-PGA concentration, broth viscosity is also dependent on the molecular weight of the secreted biopolymer. A linear correlation between the intrinsic viscosity μ and the molecular weight M of biopolymer was found to correspond to the Mark–Houwink constant a = 1. The empirical Mark–Houwink equation describing this relationship is given by equation (3) with the propor-tionality coefficient k (Richard and Margaritis, 2001).

µ = ⋅K M a

(3)

As shown in Figure 4, xanthan solutions at low concen-trations exhibit high viscosities. Regarding the necessity

Figure 1. Schematic diagram of overall biopolymer production and purification process.

Figure 2. Shear stress versus shear rate relationship for different types of fluids.

Figure 3. Oxygen uptake rate Qo2X (■) and broth viscosity (▲)

during batch cultivation of B. subtilis IFO 3335 (adapted from Richard and Margaritis, 2003).

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of a high product concentration for a cost-effective down-stream processing, it is important to have efficient mix-ing and oxygen transfer in the bioreactor. This is further emphasized by the fact that oxygen depletion leads to reduced productivity and decreased molecular weight of the biopolymer. High molecular weight is desired for most applications, which implies that both quality and quantity of the biopolymer product are affected (Herbst et al., 1992; Suh et al., 1990).

While, increasing the mixing power input is one way of increasing mass transfer, this may also result in cell disruption. Another possibility is to utilize alternative impeller types that are designed for highly viscous media. Previous work, by Amanullah et al., 1998, has shown that the Rushton turbine impeller improves both mixing and mass transfer.

Enzymatic degradation of secreted biopolymersThe production and secretion of biopolymers by micro-organisms serves the purpose of protection against unfavorable environmental conditions or the establish-ment of extracellular energy reserves. In order to be able to metabolize these energy reserves efficiently, the secreted biopolymer has to be broken down into smaller fragments, which implies the presence of extracellular enzymes capable of degrading the biopolymer mole-cules. If high molecular weights are desired, the presence of these enzymes in the cultivation broth can affect the quality of the product. Therefore, it is important to know the characteristics and conditions of microbial biopoly-mer degradation.

For γ-PGA it was found that degradation occurs in the culture fluid even after removal of the cells. It was sug-gested that Bacillus subtilis IFO3335 secreted a γ-PGA degrading enzyme in the course of the cultivation (Goto and Kunioka, 1992). This enzyme was later identified as endo-γ-glutamyl hydrolase in various Bacillus species (King et al., 2000; Kimura et al., 2004). The breakdown of PGA occurs due to a hydrolysis of γ-glutamyl bonds

thereby cleaving PGA into the oligomers of glutamic acid. In the late stationary phase of PGA production, this leads to a decrease in molecular weight, concentration and viscosity of the PGA solution possibly triggered by a lack of nutrients (Goto and Kunioka, 1992). To produce high molecular weight PGA with Bacillus species the duration of the cultivation has to be chosen carefully and under the consideration of various aspects such as substrate, prod-uct and biomass concentration, viscosity, and molecular weight of the biopolymer. A long cultivation time may have the advantage of a lower biomass concentration due to autolysis and low substrate concentration, which simplifies product recovery, yet product concentration and molecular weight decrease as well. If, on the other hand, the time of cultivation is too short, product con-centration and molecular weight may not have reached their maximum or there is still a considerable amount of unconverted substrate making product recovery less cost-efficient.

Depending on the requirements of the biopolymer application, enzymatic degradation can also be advanta-geous as shown by Richard and Margaritis (2006). The authors studied the in situ depolymerization of PGA in cell free broth after stopping the cultivation during the stationary phase at maximum product concentration. In this case, the molecular weight of the microbial PGA could be reduced to meet the requirements of drug deliv-ery applications (Richard and Margaritis, 2006). This example clearly indicates the influence of the final appli-cation on the biopolymer production process.

Production of biopolymer mixturesIn general, the production of by-products is undesired because it has significant impact on downstream pro-cessing and reduces the overall product yield due to inef-ficient nutrient conversion. For example, during pullulan production with Aureobasidium pullulans the occur-rence of dark melanin-like pigments requires additional purification steps, including adsorption on activated charcoal (Singh et al., 2008).

A factor that often is not accounted for is the synthesis of biopolymer mixtures. In the case of pullulan, a linear polysaccharide composed of maltotriose units, β-glucans can be produced that are structurally different from the α-glucan pullulan (Leathers, 2003; Shingel, 2004). As these other polysaccharides are usually not separated in the purification process the final pullulan product can be impure. Several researchers have examined the effect of culture conditions on polysaccharide synthesis by A. pullulans. It was found that the nature and chemi-cal composition of the secreted polysaccharides strongly depend on the nitrogen source and concentration, bio-reactor design, and morphology of the fungus (Campbell et al., 2004; Orr et al., 2009; Seviour et al., 2011; Simon et al., 1993). Therefore, to avoid the production of unde-sired biopolymer mixtures close attention has to be paid to establish and maintain adequate cultivation condi-tions. Other examples for biopolymer mixtures are the

Figure 4. Flow behaviour of aqueous xanthan solutions of varying concentrations (Wyatt and Liberatore, 2009).

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simultaneous production of alginate and polyhydroxy-butyrate (PHB) by Azotobacter vinelandii (Clementi, 1997; Da Silva and Garcia-Cruz, 2010) or γ-PGA and levan by Bacillus subtilis ssp. natto. For the latter, separate biopolymer production could be achieved by varying the carbon source as levan synthesis requires the addition of sucrose (Shih and Yu, 2005).

Commercially important microbial biopolymersWith considerable research being conducted in this highly interesting and promising field the amount and variety of microbial polymers, and their applications are increasing rapidly. As can be seen in Table 1, there are numerous economically relevant biopolymers that can-not all be discussed in detail here. Thus, this review will focus on γ-PGA and xanthan gum, a new polymer with potential industrial applications with strong future pros-pects and a well-established biopolymer, respectively. Although the exact purification procedures strongly depend on the particular product characteristics, general principles and new downstream processing approaches can be illustrated using these two examples and applied to other biopolymers in a modified form. As separation and purification steps are always embedded into the whole production process different cultivation strategies, as well as desired applications, are discussed.

γ-Polyglutamic acidStructure and propertiesPGA is an anionic extracellular polyamide consisting of glutamic acid repeat units linked between the α-amino and γ-carboxylic acid functional groups, as depicted in Figure 5. Therefore, it can be classified as pseudo-poly(amino acid) (Richard and Margaritis, 2001). PGA was first identified in the capsule of Bacillus anthracis, where it is an important virulence factor protecting the pathogen against the immune response of the infected host (Ivanovics and Bruckner, 1937). Among other pro-karyotes and even some eukaryotic cells, Bacillus subtilis and B. licheniformis are the most important strains for PGA production (Bajaj and Singhal, 2011; Buescher and Margaritis, 2007). The PGA produced by these organisms contains d- and l-glutamic acid repeated units, whereas PGA from B. anthracis consists solely of d-glutamic acid

(Makino et al., 1988). Besides its immunoevasive func-tion in capsular form, free extracellular PGA can serve as a nitrogen or carbon source or supports biofilm forma-tion (Stanley and Lazazzera, 2005; Rehm, 2010).

Applications of γ-PGA biopolymerAs shown in Table 1, γ-PGA has a wide variety of pos-sible applications ranging from drug delivery systems to wastewater treatment. Owing to the large number of reported applications, which have recently been reviewed (Buescher and Margaritis, 2007; Bajaj and Singhal, 2011; Sung et al., 2005), no attempt is made to discuss all of them in detail. The key properties of PGA are its anionic nature, biodegradability and sustain-ability, as it can be produced from renewable resources. Especially when applying for medical purposes, it is of paramount importance that PGA causes no toxic effect or severe immune response in the human body. This could be proved upon examining the immunoevasive effect of PGA as a virulence factor of human pathogens and is further confirmed by the fact that PGA can naturally be found in mammalian cells, such as murine neurons (Edde et al., 1990; Kocianova et al., 2005).

An important application, that is currently undergo-ing extensive research, is the use of biopolymers as drug delivery systems, in particular their application in cancer treatment. Due to its highly anionic nature, Manocha and Margaritis (2010) investigated the use of γ-PGA as a car-rier for the cationic anticancer drug doxorubicin (DOX). In this study, stable polymer-drug complexes could be formed that showed a slow pH-dependent release of the drug in vitro (Manocha and Margaritis, 2010). More studies are needed to improve the production, as well as the characterization of γ-PGA-nanoparticles. However, this field of research has great potential, since the suc-cessful use of α-PGA, which has similar properties, has already been demonstrated (Li et al., 1999; Shih et al., 2004; Singer et al., 2005; Whelan, 2002). Other biomedi-cal applications include its use for vaccination, where high molecular weight PGA can stimulate the immune response against bound antigens (Kubler-Kielb, 2006), tissue engineering, where PGA/chitosan-hydrogels serve as tissue scaffolds (Hsieh et al., 2005), and medical adhe-sives, as PGA was reported to be an alternative material for the formation of glass ionomer cements (Ledezma-Pérez, 2005).

In the food industry, the addition of PGA serves to improve the appearance and the texture of products (Bajaj and Singhal, 2011). It can moreover be used to promote the absorption of minerals, like Ca2+, which is beneficial for the prevention of osteoporosis (Ashiuchi and Misono, 2002). The most common example of PGA in food is the traditional Japanese Nattō, in which PGA occurs due to the cultivation of soybeans with Bacillus subtilis. Furthermore, successful applications of PGA in a fertilizer, in skin care and in wastewater treatment were reported (Buescher and Margaritis, 2007; Hoppensack et al., 2003; Poetter et al., 2001; Sung et al., 2008). In the latter, it was shown to bind Figure 5. Chemical structure of γ-PGA.

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heavy metal ions or dyes and flocculate various organic and inorganic compounds (Inbaraj and Chen, 2011; Inbaraj et al., 2009; Shih et al., 2001).

Production of γ-PGAA great deal of research has been conducted on the opti-mization of microbial PGA production. Special emphasis was given to the effect of medium composition, which has a major impact on quantity and quality of the PGA prod-uct. As the optimal medium depends on the microbial strain used for cultivation a great number of publications are available. The two most frequently utilized species, are Bacillus subtilis and Bacillus licheniformis, which proved to be best fitted for large-scale production. These efforts have been reviewed and summarized elsewhere (Bajaj and Singhal, 2011; Richard and Margaritis, 2001).

In general, bacteria capable of producing PGA can be divided into those that require additional glutamic acid and those that do not. An example of a glutamic acid dependent strain is Bacillus licheniformis ATCC 9945A, which has been used by many research groups. It was found to exhibit optimum growth and PGA production in Medium E, proposed by Leonard and colleagues (Buescher and Margaritis, 2007; Leonard et al., 1958). In conformance with the dependency on glutamic acid, Medium E contains 20 g/L of l-glutamic acid. In addi-tion, 80 g/L of glycerol was added as a carbon source, as well as 12 g/L of citric acid and other components.

Cultivation of various Bacillus species was performed in either batch or fed-batch mode with the former being the predominant approach. In batch cultivation with 250 mL-flasks, Jung et al. reported a peak PGA concentra-tion of 83.2 g/L; extensively exceeding the average con-centrations previously reported. However, cultivation in a 7 L bioreactor utilizing the optimized medium did not yield comparable amounts of PGA (28.4 g/L) (Jung et al., 2005). Huang et al. (2005). examined the effect of glucose-feeding in a 10 L bioreactor. With a working vol-ume of 4 L a concentration of just above 80 g/L at the end of the cultivation and a momentary maximum concen-tration of 101.1 g/L PGA could be achieved. The process was successfully scaled up in a 100 L bioreactor yielding a concentration of 83.8 g/L PGA after 46 h. This clearly demonstrates that the fed-batch approach is a promising operation mode for a more efficient and cost-effective production of PGA at the industrial scale.

Separation and purification of γ-PGAIn general, there are three different strategies for the separation and purification of PGA from a cultivation broth: precipitation of the polymer by adding certain amounts of alcohol, precipitation by complex forma-tion with metal ions and filtration methods utilizing the higher molecular weight of PGA, as compared to other components.

However, all strategies require the removal of biomass and cell debris prior to biopolymer separation. The cell

removal can be achieved by centrifugation or filtration of the culture fluid. The usually high viscosity of the broth caused by extracellular accumulation of PGA can be a problem as it slows the settling velocity in the case of centrifugation and reduces the transmembrane flow in the case of filtration methods. Do et al. (2001) studied the recovery of PGA from a highly viscous cultivation broth and found that an acidification of the broth prior to centrifugation could significantly improve the separation process. Lowering the pH of the broth to 3 resulted in a reduced viscosity and zeta potential of the cells, which in turn led to increased aggregation. During aggregation of the cells and lower viscosity, the energy demand of the subsequent centrifugation step proved to be less than 25% of the previously reported values. As centrifugation is usually one of the downstream processing steps with the highest energy consumption, this report contributes to considerably reducing the costs of large-scale PGA purification. However, the cost of reducing the pH at the industrial scale should also be considered as part of the total separation and purification step.

The most common method for PGA recovery is the alcohol induced precipitation developed by Goto and Kunioka (1992). After centrifugation the cell free cultiva-tion broth is poured into three or four volumes of cold methanol, ethanol, 2-propanol or n-propanol:ether (1:1 v/v) (Birrer et al., 1994; Do et al., 2001; Goto and Kunioka, 1992; Kubota et al., 1993). The addition of alco-hol reduces the water activity leading to a precipitation of the polymer molecules, which can then be separated from the supernatant and further purified by dialysis. The major disadvantage of this approach is the co-precipita-tion of other substances, like extracellular proteins due to a lack of specificity. The presence of these impurities in the precipitated crude product requires additional purification steps, such as washing, dialysis or enzymatic degradation, thereby increasing the cost of downstream processing. Furthermore, the addition of alcohol cannot guarantee a complete precipitation of the produced PGA leading to losses of up to 15% of the original PGA content in the precipitation step (Manocha and Margaritis, 2010).

Another drawback of this strategy is the amount of alcohol needed for precipitation. As mentioned above, usually three or four volumes of alcohol have to be added to the culture supernatant equaling 75–80% (v/v). Thus, further costs arise from the acquisition, regeneration and disposal of these toxic chemicals questioning the applica-bility in large-scale downstream processing. An attempt has been made to reduce the amount of alcohol needed by Do and coworkers (2001). Prior to precipitation, the cell free culture supernatant was concentrated from 20 to 60 g/L PGA by ultrafiltration under optimized conditions. As the necessary amount of alcohol generally decreases at higher PGA concentrations, the required volume of ethanol, methanol or 2-propanol could be reduced by 50% (v/v) (Do et al., 2001). At the same time, this study demonstrates that the aforementioned strategies can

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successfully be combined with each other in order to achieve an efficient separation and purification process.

Prescott and Stock (2005) patented a method for producing medical and commercial grade PGA of high molecular weight. The method involved combinations of different filtration steps, which made use of the molecu-lar weight difference between PGA and other undesired substances. After an initial optional diafiltration of the culture broth for buffer exchange (MWCO 30–100 kDa) the cells are separated either by centrifugation or filtra-tion through a 0.22 µm filter. The neutralized filtrate containing PGA is then buffer exchanged and further concentrated by diafiltration. Depending on the required purity and application of the product various purification steps like sterile filtration, alcohol precipitation or freeze drying can follow up.

An advantage of this strategy is that the addition of precipitating agents or other substances that have to be separated again later on is avoided. Still, the use of diafil-tration membranes or filtration in general is associated with high membrane costs and fouling due to high broth viscosity. Dilution of the culture fluid can decrease the viscosity, but is economically disadvantageous because of a prolonged operation time.

Since PGA is an anionic polymer it is capable of bind-ing certain metal cations through electrostatic interac-tions. As a component of bacterial capsules this binding or trapping phenomenon serves to provide essential metal cations for enzymatic reactions or structural com-ponents of the cell (Beveridge and Murray, 1976). Hence there have been several studies examining the metal-binding characteristics of PGA (Beveridge and Murray 1976 and 1980; McLean et al., 1990). It was found that the addition of specific metal ions, such as Cu2+, Al3+, Cr3+ or Fe3+, induced flocculation of dissolved PGA (McLean et al., 1990). Manocha and Margaritis (2010) investigated whether this metal-induced precipitation could be used for the separation of PGA from Bacillus licheniformis cultivation broth. Of all metal cations tested divalent copper ions exhibited the best properties for selective precipitation and subsequent resolubilization of PGA. Thus, copper sulfate solution was added to the cell free culture broth in concentrations up to 500 mM, where it quickly induced the formation of insoluble PGA pre-cipitates that could be separated from the broth. After resolubilization of the precipitate in EDTA-solution, diafiltration and freeze-drying the purified product was obtained.

In order to assess the efficiency of this new separation strategy, it was compared to the conventional ethanol induced precipitation, regarding the co-precipitation of other proteins and the amount of PGA recovered. In Figure 6, a flow diagram of both separation processes is depicted. It was shown that only 3% of the proteins pres-ent in the supernatant were precipitated by Cu2+, whereas 48% were precipitated using ethanol. Of the total PGA content a fraction of 85% could be recovered by copper sulfate and 82% by ethanol induced precipitation. A

further characterization of the purified product revealed no detectable amounts of residual copper ions (Manocha and Margaritis, 2010).

Thus, a new strategy for the separation and purifica-tion of PGA could be developed that is not only more selective, but also more efficient than other existing approaches as a product of better quality and at less expense. With copper induced precipitation, the utili-zation of expensive and environmentally problematic chemicals, as well as the number of filtration steps, can be reduced to a minimum making it a promising strategy for PGA production at the industrial scale. Still, a scale-up and a further optimization of this method are needed to prove its applicability and cost-effectiveness with respect to commercial applications.

Xanthan gumStructure and propertiesAs depicted in Figure 7, xanthan gum consists of a cellulose-like backbone of β-d-glucose units that is linked at the positions 1 and 4. Every other glucose unit is connected to a trisaccharide side chain, composed of glucuronic acid and two mannose residues (Rosalam and England, 2006). Approximately 50% of the terminal mannose residues carry a pyruvate substituent group, whereas the mannose unit connected to the backbone usually carries an acetyl group at C-6 (Palaniraj and Jayaraman, 2011; Garcia-Ochoa et al., 2000). Due to the different types of monosaccharides involved, xanthan belongs to the heteropolysaccharides. After its discovery as an exo-polysaccharide of Xanthomonas campestris in the 1950s, xanthan gum was the first biopolymer to be produced at the industrial scale, starting in 1964. The main properties causing the commercial exploitation are its high viscosity at comparably low concentrations and its non-toxic character, which led to its approval for the use in foods by the FDA in 1969 (Faria et al., 2011; Garcia-Ochoa et al., 2000).

Applications of xanthan gumWith a market capitalization of 235–270 million US$ and an annual production of approximately 90,000 to 100,000 tons, xanthan gum is one of the most significant micro-bial polymers on the market (Faria et al., 2011; Freitas et al., 2011). Until 2015, a further increase of market value to more than 400 million US$ is expected (Pradella, 2006). Therefore, a great variety of well-established appli-cations can be found, particularly in the food and oil industries. The commercial value of xanthan gum arises from its unique rheological properties combined with its biodegradable and non-toxic characteristics. Even at low concentrations xanthan induces high viscosities and pseudoplastic flow behavior. In addition, it exhibits better stability against high temperatures, salinities or extreme pH values than most other biopolymers (Rosalam and England, 2006).

Since its FDA approval for use in foods in 1969, xanthan gum has been used as a suspending and thickening agent

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to improve the viscosity, appearance and texture of vari-ous products, like salad dressings, beverages and dairy products (Faria et al., 2011; Palaniraj and Jayaraman, 2011). This stabilizing and thickening effect is also

utilized for cosmetics and personal care applications, for example to adjust the viscosity of shampoo, and in the pharmaceutical industry for stabilizing suspensions and emulsions. Furthermore, it can delay drug release when used as a coating for tablets (Palaniraj and Jayaraman, 2011).

Another important field considering the applications of xanthan gum is the oil industry. Here the biopolymer can be utilized for tertiary or enhanced oil recovery (EOR). To increase the amount of crude oil obtained from an oil field a viscous mixture of water and xan-than can be injected into the reservoir thereby increas-ing the pressure. Ideally residual oil is displaced by the viscous solution and then extracted from the well. The increased viscosity of the solution improves oil recovery, as the mobility of the water is reduced and “fingering” of the water can be avoided (Nasr et al., 2007; Wang and Dong, 2009).

Figure 6. Bioprocess flow diagram showing the recovery and purification of γ-polyglutamic acid from B. licheniformis cell-free cultivation broth using (A) copper sulfate induced precipitation; (B) ethanol induced precipitation (adapted from Manocha and Margaritis, 2010).

Figure 7. Chemical structure of xanthan gum (adapted fromRehm, 2010).

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Production of xanthan gumThe microorganism commonly used for the production of xanthan is the bacterium Xanthomonas campestris NRRL B-1459 (Garcia-Ochoa et al., 2000). As the medium composition not only influences the biopolymer yield, but also the product quality, with regards to the molecu-lar weight or the presence of specific functional groups, considerable research has been conducted on the nutri-ent requirements of microbial growth and product for-mation. During extensive studies on xanthan production, the highest yield was achieved with 2–5% glucose as the main carbon source. Besides glucose, other carbohy-drates such as sucrose, maltose and soluble starch were also shown to be efficient substrates (Leela and Sharma, 2000; Psomas et al., 2007; Souw and Demain, 1979). Even industrial or agricultural waste products, like sugar cane broth, sugar-beet molasses and cheese whey, are poten-tial low-cost substrates for industrial xanthan produc-tion (Faria et al., 2011; Moosavi and Karbassi, 2010; Silva et al., 2009).

For the biosynthesis of xanthan a high ratio of carbon to nitrogen (C/N) is beneficial, whereas fast microbial growth requires both high nitrogen and carbon source concentrations (Becker et al., 1998; De Vuyst et al., 1987). Based on these findings Lo et al., (1997a) suggested a two step cultivation process, including a shift from an ini-tially low C/N ratio allowing cell growth to a comparably higher level promoting xanthan production. The shift in the C/N ratio was achieved through a single addition of glucose at the early stationary phase and led to a maxi-mum xanthan concentration of approximately 38 g/L (Lo et al., 1997a). A comparison of different fed-batch strategies showed that xanthan production could be sig-nificantly enhanced by multiple pulse and continuous glucose feeding. Here the addition of glucose was initi-ated at low nitrogen concentrations in the broth, thereby considerably increasing the C/N ratio. As substrate inhi-bition had to be prevented the glucose concentration was maintained at about 30 g/L. With this approach xanthan concentrations of up to 62 g/L could be obtained, pro-vided that sufficient oxygen and substrate supply were ensured. Hence a fed-batch strategy demonstrates a sig-nificantly improved performance in comparison to com-mon batch processes (Amanullah et al., 1998b). Since the productivity did not seem to decrease at the end of these fed-batch experiments, continuous cultivation seems to be a promising approach. By maintaining relatively constant reaction conditions a constant product qual-ity and high productivity could be achieved. Evans and coworkers demonstrated the feasibility of continuous processes for xanthan production with a safe operation for more than 2,000 h (Evans et al., 1979). Still, the major drawbacks of continuous cultivations, i.e. the high risk of contamination with other microorganisms and the risk of strain mutation, prevent its application at the indus-trial scale. Thus, attempts were made to further develop and optimize the fed-batch xanthan production. Here an interesting new strategy is the water-in-oil cultivation,

where the aqueous phase containing medium and cells is dispersed in an organic phase, like n-hexadecane, per-fluorocarbon or vegetable oil (Ju and Zhao, 1993; Kuttuva et al., 2004). By using low-cost vegetable oil, a maximum xanthan concentration of 120 g/L was reported. The main advantage of this strategy is the reduced viscosity of the culture fluid, as high viscosities caused by high xanthan concentrations are confined within the aqueous droplets. A disadvantage is the difficult phase separa-tion complicating the product recovery in downstream processing (Kuttuva et al., 2004). In spite of these encour-aging results, the application of water-in-oil cultivation for xanthan production needs to be further researched and optimized. An important aspect is the prevention of phase inversion, which occurs due to the addition of aqueous solutions for pH control or substrate feeding, and dramatically increases broth viscosity.

A new approach to bioreactor design was presented by Yang et al. (1996) who developed a centrifugal packed-bed reactor (CPBR) for xanthan cultivation (Yang et al., 1996). After an initial growth phase the cells were immo-bilized on cotton fibers in a rotating matrix by natural adsorption. The immobilized bacteria provided good xanthan production in repeated batch operation for more than three weeks and a high product yield of 85% xanthan from glucose. Still, the immobilization caused oxygen transfer limitations, which led to reduced cell viability. This had to be compensated for by increasing medium recirculation or packed-bed rotation, for example by increasing the overall energy input. However, it could be shown that the volumetric mass transfer coefficient k

La

reached similar values for CPBR and water-in-oil culti-vation, respectively. Among the examined systems the conventional stirred tank reactor exhibited the lowest oxygen mass transfer (Lo et al., 2001).

Separation and purification of xanthan gumThe recovery of xanthan gum from the viscous cultivation broth is a difficult process that can account for up to 70% of the total production cost (Torrestiana-Sanchez et al., 2007). In general, downstream processing can be divided into the deactivation and/or removal of the bacterial cells, the precipitation, dewatering, drying and milling of the biopolymer product. The exact sequence of separa-tion steps strongly depends on the intended use of the purified xanthan, since requirements differ between the various applications (Garcia-Ochoa et al., 2000). In con-trast to many other biopolymer separation processes, cell separation is optional meaning that industrial xanthan can contain residual biomass, again depending on the intended application. An example flow sheet of an indus-trial xanthan production process is given in Figure 8.

The deactivation of the cells can be achieved by chem-ical, enzymatic, mechanical or thermal treatment of the cultivation broth. Chemical and enzymatic treatment can alter the structure and composition of the product and involves the addition of agents that need to be removed in the following purification steps, cell deactivation is

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usually achieved by a heat treatment (pasteurization) (Garcia-Ochoa et al., 2000). Thermal degradation of the biopolymer can be avoided under the appropriate con-ditions, due to the high temperature stability of xanthan (Becker et al, 1998). Besides deactivation, heating of the cultivation broth also has the advantage of an improved xanthan recovery from the cells (Galindo and Albiter, 1996) and a reduced broth viscosity. As mentioned before, high viscosities considerably complicate cell separation, so that thermal treatment or dilution of the culture fluid represent necessary steps prior to centrifu-gation or filtration.

As is the case for many biopolymers, precipitation is the most common strategy for xanthan separation and purification. Agents used for the precipitation of xanthan are water-miscible non-solvents, like isopropyl alcohol, ethanol or acetone (Garcia-Ochoa et al., 2000; Psomas et al., 2007; Salah et al., 2011b; Zhang and Chen, 2010), or polyvalent cations, like calcium, aluminum or quaternary ammonium salts that form complexes with the polyanionic xanthan (Pace, 1980; Palaniraj and Jayaraman, 2011). An advantage of using alcohols is that impurities, like certain salts and cell debris, are washed out simultaneously. The addition of divalent salts can furthermore lead to a less soluble xanthan product, meaning alcohol precipitation utilizing isopropyl

alcohol is generally favored in industrial applications (Becker et al., 1998; Garcia-Ochoa et al., 1993). Upon considering the process cost of the precipitation step, the required volume of alcohol is a decisive parameter that is influenced by the salt and biopolymer concentra-tions. It was found that the addition of electrolytes, like potassium or sodium chloride, to the cultivation broth could reduce the amount of isopropyl alcohol needed from 3 to approximately 1.4 volumes of alcohol per volume of the broth (Galindo and Albiter, 1996; Garcia-Ochoa et al., 2000). Lo et al. evaluated the application of an additional ultrafiltration step in xanthan downstream processing. After heat treatment the cultivation broth containing xanthan gum was concentrated via ultrafil-tration, which reduced the necessary volume of alcohol, as well as the energy costs by 80%. Up to 45% of the over-all downstream processing cost could be saved, which would represent a considerable competitive advantage in industrial xanthan production (Lo et al., 1997b). Although, no significant fouling of the ultrafiltration membranes could be observed it is important to notice that the transmembrane flux decreases with increasing xanthan concentration, due to high viscosities. Thus, the use of ultrafiltration is naturally associated with a prolonged operation time, which can be problematic for large-scale productions.

Figure 8. Bioprocess flow sheet of xanthan gum production in a conventional stirred tank bioreactor (adapted from Rosalam and England, 2006).

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While usually the precipitation succeeds the ultra-filtration step, it was attempted to combine these two operations into one process referred to as membrane-assisted precipitation by Torrestiana-Sanchez et al. (2007). During ultrafiltration the concentration polar-ization generally induces the formation of a biopolymer fouling layer with a high specific flux resistance. Here the effect of potassium chloride and isopropyl alcohol on the structure and resistance of this layer was exam-ined. It was found that a separate addition of these compounds further increased resistance, leading to a decrease in transmembrane flux. Still, a combination of at least 1% (w/v) KCl and more than 30% (v/v) isopropyl alcohol resulted in a highly porous fouling layer with a significantly reduced flux resistance. Thus, by combin-ing ultrafiltration and precipitation the consumption of precipitating agents can be considerably reduced, compared to the conventional recovery process, which utilizes up to 75% (v/v) alcohol. Another advantage is the substantial increase in transmembrane flux in spite of high broth viscosities, since one of the major draw-backs of ultrafiltration is the long operation time. With membrane-assisted precipitation a new strategy for the separation and concentration of xanthan gum was established that shows great potential for future applica-tions. Further research has to be conducted on scale-up and incorporation of this process into industrial scale downstream processing.

Another approach to increasing transmembrane flux in biopolymer filtration was reported by Hofmann and Posten who examined the application of pres-sure electrofiltration for the concentration of xanthan solutions (Hofmann and Posten, 2003). As depicted in Figure 9, a common dead-end filtration is supported by an electrical field, which leads to the migration of anionic xanthan polymers in the direction of the positively charged anode. Thus, the thickness of the fouling layer on the cathode side membrane can be considerably reduced, resulting in an enhanced filtrate flux. Compared to conventional pressure filtration, the filtration time decreased from days to a few hours (Goezke and Posten, 2010).

Based on these results a pilot-scale pressure electro-filtration system was developed and further optimized. It was shown that aqueous xanthan solution could be successfully concentrated from 5 to 220 g/L with an average filtrate flux of 82.7 L/(m2 h) and a retention of almost 100% of the xanthan present. Again, filtration time could be reduced by more than 90% compared to common press filtration (Hofmann et al., 2006). As the electrofiltration has to be ended when the anode-side filter cake reaches the cathode-side membrane, it can only be operated batch-wise and has to be disassem-bled in order to obtain the product. This can generally be disadvantageous for industrial applications because of higher idle times and personnel costs. Thus, cross-flow filtration is more common for the separation of biopolymers than a dead-end approach. However, new

pressure electrofiltration systems provide a consider-ably increased filtrate flux and reduced shear forces that can lead to biopolymer degradation in the case of cross-flow filtration. In addition, electrofiltration can be used to concentrate polymer solutions to a far greater extent, as the highly viscous fluid in the filtra-tion chamber does not have to be pumped (Goezke and Posten, 2010).

In spite of these promising results it has to be noted that the electrofiltration experiments were carried out with aqueous xanthan solutions that exhibit low ionic strength and a well-defined composition. No attempts were made to concentrate preheated xanthan cultivation broths, which can exhibit considerably different proper-ties. The ionic strength for instance has a major impact on the electrophoretic velocity of xanthan in the electric field, as cations originating e.g. from medium salts mask the negative charges and thus decrease migration veloc-ity (Hofmann and Posten, 2003). Thus, a desalination step may be necessary prior to electrofiltration increasing the costs and complexity of downstream processing. Further studies have to investigate the applicability of pressure electrofiltration for the separation of xanthan from the actual cultivation broth. Nonetheless, this strategy has the potential of improving large-scale recovery and puri-fication of xanthan gum.

Future developments in biopolymer applicationsIn spite of a rapidly increasing number of applica-tions and considerable progress in production and

Figure 9. Press electro filtration using an electrofilterplate,FR:hydro dynamic force and Fel: electric force (adapted from Hofmann et al., 2006).

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purification processes, the worldwide market for poly-mers is still dominated by materials derived from non-renewable resources or animal and plant biomass. The reason for this is the high production costs associated with microbial cultivation, which originate to a major extent from downstream processing. Thus, currently a lot of research is being conducted to facilitate separation and purification of polymeric biomolecules, in order to render large scale production more cost-effective and competitive.

Despite the production costs, various advantages and applications of microbial polymers already justify a replacement of the common dominating materials. Especially polymers derived from petrochemicals, which have the major disadvantage of being non-biodegradable and creating a dependency on limited non-renewable resources. Due to these environmental and cost con-cerns of hydrocarbon-based chemical polymers, an increased emphasis on the use of microbial biopolymers within the market is expected in the future. It has been forecast that the global market for biological polymers could potentially reach $3.5 billion over the next decade, a significant increase from the current $600 million value (Singh, 2011).

Declaration of interestThis work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada through Discovery Grant No. 4388 awarded to Dr. A. Margaritis. The authors report no declarations of interest.

Nomenclature and unitsa Mark-Houwink constant (Dimensionless, Equation 3)k Proportionality coefficient (Dimensionless, Equation 3)K Consistency index (Pa•sn, Equations 1 and 2)M Molecular weight (g/mol, Equation 3)n Flow behavior index (Dimensionless, Equations 1

and 2)μ Intrinsic viscosity (L•g-1, Equation 3)μa Apparent viscosity (Pa•s, Equation 2)τ Shear stress (Pa, Equation 1)τ0 Yield stress (Pa, Equation 1)γ Shear rate (s-1, Equations 1 and 2)

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