Critical Reviews in Biotechnology, 2012; Early Online: 116 2012 Informa Healthcare USA, Inc.

. ISSN 0738-8551 print/ISSN 1549-7801 online DOI: 10.3109/07388551.2012.743501

REVIEW ARTICLE

 ecent Advances in Microbial Biopolymer Production R and Purification

Dirk Kreyenschulte1,2, Rainer Krull1, and Argyrios Margaritis2
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.
1 2

Institute of Biochemical Engineering, Technical University Braunschweig, Braunschweig, Germany and Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario, N6A 5B9, Canada
Abstract Over 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, -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, -polyglutamic acid, polyamides, polyesters, polysaccharides, xanthan gum

Introduction
Due to extensive research over the past decades microbial biopolymers offer a wide variety of new applications 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 beneficial 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 promising 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 2530% 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

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: amarg@uwo.ca (Received 25 May 2012; revised 18 September 2012; accepted 20 September 2012)

2 D. Kreyenschulte et al.
Table 1. List of commercially important microbial biopolymers. Polymer Class Source Substrates Polyamides Cyanophycin Cyanobacteria, Arginine, (NH4)2SO4 Acinetobacter spp., Protein hydrolysate Bordetella spp., ProtamylasseTM Desulfitobacterium hafniense, Nitrosomonas europaea -Polyglutamic acid Bacillus spp., Staphylococcus epidermis, Natrialba aegyptiaca, Natronococcus occultus, Fusobacterium nucleatum

Applications Water softener Metal ion-exchange  system Hydrogels Synthesis of chemicals Nutrition

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

 lycerol,L-glutamic-acid, G citric acid

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

Poly--lysine

Streptomyces albulus ssp. lysinopolymerus

Glucose, (NH4)2SO4

Biodegradable plastics Fertilizer Food thickener Hydrogels Medical adhesives  Nanoparticle drug/gene delivery Skin care Tissue scaffolds Wastewater treatment Coating material Dietary agent Drug/Gene delivery Emulsifying agent Endotoxin removal Food preservative Hydrogels Interferon inducer

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

Hamano, 2011 Shih et al., 2006

Polyanhydrides Polyphosphate

Eukaryotic and prokaryotic cells

Sodium acetate, KH2PO4  and NH4Cl e.g. from wastewater

Antibacterial agent ATP substitute Food additive Insulating fiber

Achberge-rov and Nahalk, 2011 Kishida et al., 2006 Kornberg et al., 1999 Chanpra-teep, 2010 Koller et al., 2010 Zinn et al., 2001

Polyesters Polyhydroxyalkanoates

Prokaryotes

Carbohydrates Starch Alcohols  Industrial waste products

Biodegradable plastics Drug delivery Tissue engineering

Polysaccharides Alginate

Pseudomonas and Azotobacter spp. (mostly A. vinelandii)

Sucrose

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

Glucose Sucrose Other carbohydrates

Cell immobilization Drug delivery Food additive Textile/paper industry Wound dressing Water treatment Food additive Membrane material Oil recovery Paper industry Wound dressing

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

Chawla et al., 2009 Shoda and Sugano, 2005

Glucose Sucrose Other carbohydrates

Dextran

Leuconostoc, Streptococcus and Lactobacillus spp. (mostly L. mesenteroides), Gluconobacter sp., Pediococcus pentosaceus

Sucrose Maltodextrins

Food additive Concrete additive Drug delivery Immune stimulator Heavy metal removal Blood-plasma substitute  Molecular sieves (Sephadex) Heavy metal removal Cosmetics  Emulsifying and thickening agent

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

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

(Continued)

Critical Reviews in Biotechnology

Microbial Biopolymer Production and Purification 3

Table 1. (Continued). Polymer Class Source Gellan Pseudomonas elodea, Sphingomonas spp. (mostly S. paucimobilis ATCC 31461) Streptococcus zooepidemicus, S. equi, Pasteurella multocida Zymomonas mobilis, Bacillus spp., Streptococcus spp., Alcaligenes viscosus and other prokaryotes Aureobasidium pullulans, Tremella mesenterica, Cytaria sp., Cryphonectria parasitica, Rhodototula bacarum Sclerotium rolfsii and S. glucanicum, Schizophyllum commune, Botrytis cinerea, Epicoccum nigrum Sinorhizobium meliloti, Agrobacterium sp., Alcaligenes faecalis var. myxogenes, Pseudomonas sp. Xanthomonas campestris Substrates Carbohydrates Industrial waste products

Applications

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

Hyaluronic acid

Levan

Pullulan
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

Scleroglucan

Succinoglycan

Agar substitute Cell immobilization Food additive Gel electrophoresis Tissue engineering  Glucose, amino acids, Cosmetics nucleo-tides, salts, trace ele- Drug/gene delivery ments and vitamins Viscosupple-mentation Wound dressing Sucrose Blood-plasma substitute Lactose Cosmetics Emulsifying agent Food additive Carbohydrates Blood-plasma substitute Industrial waste products cosmetics Enzyme immobilization flocculating agent Food additive Pharmaceutical coating Glucose Cosmetics Sucrose Drug delivery Immune stimulator Oil recovery Pharmaceutical coating  Sucrose and other Food additive carbo-hydrates Oil recovery

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

de Oliveira et al., 2007 Senthil-kumar and Gunase-karan, 2005 Shih et al., 2005 Leathers. 2003 Singh et al., 2008

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

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

Xanthan gum

Glucose Sucrose Other Carbohydrates

Agricultural products Coatings Cosmetics Food additive Oil recovery Paper industry Thickener

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 separation and purification. Using the examples of xanthan gum and -polyglutamic acid (-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.

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 biopolymers
The 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 bioprocess. Due to the secretion of polymers, the viscosity of the culture fluid can increase dramatically in the course of a cultivation and hence differ from the common 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 and is given by the Herschel velocity gradient or shear rate Bulkley equation (1), which utilizes the consistency index K, the yield stress 0 and the flow index n.  )n = 0 + K ( (1)

General factors affecting the production and purification of biopolymers

Next to the control of common cultivation conditions, such as temperature, medium composition, aeration and

2012 Informa Healthcare USA, Inc.

4 D. Kreyenschulte et al. Depending on the values for K, 0 and n, fluids can be classified as Newtonian and non-Newtonian fluids (dilatant, pseudoplastic, Bingham plastic or Casson fluids). For Newtonian fluids 0 = 0 and n = 1, so there is a linear relationship between shear stress and shear rate . In this case, the slope in a plot of against is defined as the viscosity . For non-Newtonian dilatants (0 = 0, n > 1) and pseudoplastic fluids (0 = 0, n < 1) equation (1) changes into the Ostwald-de-Waele or into the more common power-law model. The relationship between differs so that the viscosity can depend on the and shear rate. It is then defined as the apparent viscosity a (shown in equation 2). )n 1 a = K ( (2)  Dilatant fluids increase in viscosity with increasing shear rate, whereas pseudoplastic fluids exhibit a decrease in viscosity with increasing shear rate. Furthermore, for Bingham plastic (n = 1, 0 0, > 0) and Casson fluids (n = 0.5, 0 0, > 0) a yield stress 0 has to be overcome before flowing occurs (Margaritis and Pace, 1985). Figure 2, shows a plot of shear stress indicating the flow characteristics of versus shear rate different fluids. The most frequent behavior of biopolymer solutions is pseudoplastic (Seviour et al., 2011), also called shearthinning . An example of a biopolymer with pseudoplastic behavior is an aqueous solution of the biopolymer -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 demonstrating the pseudoplasticity of the -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, -PGA concentration and broth viscosity reached their maximum values in the late exponential and early stationary phase (Richard and Margaritis, 2003). Besides the -PGA concentration, broth viscosity is also dependent on the molecular weight of the secreted biopolymer. A linear correlation between the intrinsic viscosity and the molecular weight M of biopolymer was found to correspond to the MarkHouwink constant a = 1. The empirical MarkHouwink equation describing this relationship is given by equation (3) with the proportionality coefficient k (Richard and Margaritis, 2001).

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

= K M a (3)  As shown in Figure 4, xanthan solutions at low concentrations exhibit high viscosities. Regarding the necessity

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

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



Figure 3. Oxygen uptake rate Qo2X () and broth viscosity () during batch cultivation of B. subtilis IFO 3335 (adapted from Richard and Margaritis, 2003).
Critical Reviews in Biotechnology

Microbial Biopolymer Production and Purification 5 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, product 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 concentration 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 advantageous 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 delivery applications (Richard and Margaritis, 2006). This example clearly indicates the influence of the final application on the biopolymer production process.

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

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

of a high product concentration for a cost-effective downstream processing, it is important to have efficient mixing 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.

Production of biopolymer mixtures

In general, the production of by-products is undesired because it has significant impact on downstream processing and reduces the overall product yield due to inefficient nutrient conversion. For example, during pullulan production with Aureobasidium pullulans the occurrence 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, -glucans can be produced that are structurally different from the -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 chemical composition of the secreted polysaccharides strongly depend on the nitrogen source and concentration, bioreactor 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 undesired biopolymer mixtures close attention has to be paid to establish and maintain adequate cultivation conditions. Other examples for biopolymer mixtures are the

Enzymatic degradation of secreted biopolymers

The production and secretion of biopolymers by microorganisms serves the purpose of protection against unfavorable environmental conditions or the establishment 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 molecules. 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 biopolymer degradation. For -PGA it was found that degradation occurs in the culture fluid even after removal of the cells. It was suggested that Bacillus subtilis IFO3335 secreted a -PGA degrading enzyme in the course of the cultivation (Goto and Kunioka, 1992). This enzyme was later identified as endo--glutamyl hydrolase in various Bacillus species (King et al., 2000; Kimura et al., 2004). The breakdown of PGA occurs due to a hydrolysis of -glutamyl bonds

2012 Informa Healthcare USA, Inc.

6 D. Kreyenschulte et al. simultaneous production of alginate and polyhydroxybutyrate (PHB) by Azotobacter vinelandii (Clementi, 1997; Da Silva and Garcia-Cruz, 2010) or -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). (Makino et al., 1988). Besides its immunoevasive function in capsular form, free extracellular PGA can serve as a nitrogen or carbon source or supports biofilm formation (Stanley and Lazazzera, 2005; Rehm, 2010). Applications of -PGA biopolymer As shown in Table 1, -PGA has a wide variety of possible 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 sustainability, 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 undergoing 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 -PGA as a carrier 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 -PGA-nanoparticles. However, this field of research has great potential, since the successful use of -PGA, which has similar properties, has already been demonstrated (Li et al., 1999; Shih et al., 2004; Singer et al., 2005; Whelan, 2002). Other biomedical 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 adhesives, as PGA was reported to be an alternative material for the formation of glass ionomer cements (LedezmaPrez, 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 Natt, 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
Critical Reviews in Biotechnology

Commercially important microbial biopolymers

With 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 cannot all be discussed in detail here. Thus, this review will focus on -PGA and xanthan gum, a new polymer with potential industrial applications with strong future prospects 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.

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

-Polyglutamic acid
Structure and properties PGA is an anionic extracellular polyamide consisting of glutamic acid repeat units linked between the -amino and -carboxylic acid functional groups, as depicted in Figure 5. Therefore, it can be classified as pseudopoly(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 prokaryotes 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

Figure 5. Chemical structure of -PGA.



Microbial Biopolymer Production and Purification 7 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 -PGA A great deal of research has been conducted on the optimization 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 product. 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 addition, 80g/L of glycerol was added as a carbon source, as well as 12g/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 concentration of 83.2g/L; extensively exceeding the average concentrations 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 10L bioreactor. With a working volume of 4L a concentration of just above 80g/L at the end of the cultivation and a momentary maximum concentration of 101.1g/L PGA could be achieved. The process was successfully scaled up in a 100L 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 -PGA In 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 formation 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 cultivation 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 alcohol 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-precipitation 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 7580% (v/v). Thus, further costs arise from the acquisition, regeneration and disposal of these toxic chemicals questioning the applicability 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 60g/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

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

2012 Informa Healthcare USA, Inc.

8 D. Kreyenschulte et al. 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 molecular weight difference between PGA and other undesired substances. After an initial optional diafiltration of the culture broth for buffer exchange (MWCO 30100 kDa) the cells are separated either by centrifugation or filtration through a 0.22 m 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 diafiltration 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 binding certain metal cations through electrostatic interactions. As a component of bacterial capsules this binding or trapping phenomenon serves to provide essential metal cations for enzymatic reactions or structural components of the cell (Beveridge and Murray, 1976). Hence there have been several studies examining the metalbinding 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 precipitates 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 present 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 purification 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 utilization 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.

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

Xanthan gum
Structure and properties As depicted in Figure 7, xanthan gum consists of a cellulose-like backbone of -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; GarciaOchoa et al., 2000). Applications of xanthan gum With a market capitalization of 235270 million US$ and an annual production of approximately 90,000 to 100,000 tons, xanthan gum is one of the most significant microbial 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 applications 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
Critical Reviews in Biotechnology

Microbial Biopolymer Production and Purification 9

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

Figure 6. Bioprocess flow diagram showing the recovery and purification of -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).

to improve the viscosity, appearance and texture of various 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 xanthan can be injected into the reservoir thereby increasing 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).

2012 Informa Healthcare USA, Inc.

10 D. Kreyenschulte et al. Production of xanthan gum The 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 molecular weight or the presence of specific functional groups, considerable research has been conducted on the nutrient requirements of microbial growth and product formation. During extensive studies on xanthan production, the highest yield was achieved with 25% glucose as the main carbon source. Besides glucose, other carbohydrates 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 potential low-cost substrates for industrial xanthan production (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 initially 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 maximum xanthan concentration of approximately 38 g/L (Lo et al., 1997a). A comparison of different fed-batch strategies showed that xanthan production could be significantly enhanced by multiple pulse and continuous glucose feeding. Here the addition of glucose was initiated at low nitrogen concentrations in the broth, thereby considerably increasing the C/N ratio. As substrate inhibition had to be prevented the glucose concentration was maintained at about 30g/L. With this approach xanthan concentrations of up to 62 g/L could be obtained, provided that sufficient oxygen and substrate supply were ensured. Hence a fed-batch strategy demonstrates a significantly improved performance in comparison to common 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 quality 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 2,000h (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 industrial 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,


Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

where the aqueous phase containing medium and cells is dispersed in an organic phase, like n-hexadecane, perfluorocarbon 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 separation complicating the product recovery in downstream processing (Kuttuva et al., 2004). In spite of these encouraging 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 packedbed reactor (CPBR) for xanthan cultivation (Yang et al., 1996). After an initial growth phase the cells were immobilized 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 kLa reached similar values for CPBR and water-in-oil cultivation, 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 gum The 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 separation steps strongly depends on the intended use of the purified xanthan, since requirements differ between the various applications (Garcia-Ochoa et al., 2000). In contrast 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 industrial xanthan production process is given in Figure 8. The deactivation of the cells can be achieved by chemical, 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
Critical Reviews in Biotechnology

Microbial Biopolymer Production and Purification 11 usually achieved by a heat treatment (pasteurization) (Garcia-Ochoa et al., 2000). Thermal degradation of the biopolymer can be avoided under the appropriate conditions, 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 centrifugation 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 concentrations. 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; GarciaOchoa 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 ultrafiltration, which reduced the necessary volume of alcohol, as well as the energy costs by 80%. Up to 45% of the overall 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.

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

Figure 8. Bioprocess flow sheet of xanthan gum production in a conventional stirred tank bioreactor (adapted from Rosalam and England, 2006).
2012 Informa Healthcare USA, Inc.

12 D. Kreyenschulte et al. While usually the precipitation succeeds the ultrafiltration step, it was attempted to combine these two operations into one process referred to as membraneassisted precipitation by Torrestiana-Sanchez et al. (2007). During ultrafiltration the concentration polarization 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 examined. 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 combining 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 drawbacks 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 applications. 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 pressure 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 electrofiltration 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 82.7L/(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 disassembled in order to obtain the product. This can generally be disadvantageous for industrial applications because of higher idle times and personnel costs. Thus, crossflow filtration is more common for the separation of biopolymers than a dead-end approach. However, new


Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

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

pressure electrofiltration systems provide a considerably 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 filtration 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 properties. 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 velocity (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 purification of xanthan gum.

Future developments in biopolymer applications

In spite of a rapidly increasing number of applications and considerable progress in production and
Critical Reviews in Biotechnology

Microbial Biopolymer Production and Purification 13 purification processes, the worldwide market for polymers is still dominated by materials derived from nonrenewable 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 concerns 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).
Bajaj IB, Survase SA, Saudagar PS, Singhal RS. 2007. Gellan gum: Fermentative production, downstream processing and applications. Food Technology and Biotechnology 45: 341354. Bajaj I, Singhal R. 2011. Poly (glutamic acid)an emerging biopolymer of commercial interest. Bioresour Technol 102: 55515561. Becker A, Katzen F, Phler A, Ielpi L. 1998. Xanthan gum biosynthesis and application: a biochemical/genetic perspective. Appl Microbiol Biotechnol 50: 145152. Beucker S, Marscheider-Weidemann F, Carus M. (2007). Zukunftsmarkt Biokunststoffe. Umweltbundesamt online, available at: http:// www.umweltdaten.de/publikationen/fpdf-l/3451.pdf Beveridge TJ, Murray RG. 1976. Uptake and retention of metals by cell walls of Bacillus subtilis. J Bacteriol 127: 15021518. Beveridge TJ, Murray RG. 1980. Sites of metal deposition in the cell wall of Bacillus subtilis. J Bacteriol 141: 876887. Birrer GA, Cromwick AM, Gross RA. 1994. Gamma-poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies. Int J Biol Macromol 16: 265275. Buescher JM, Margaritis A. 2007. Microbial biosynthesis of polyglutamic acid biopolymer and applications in the biopharmaceutical, biomedical and food industries. Crit Rev Biotechnol 27: 119. Campbell BS, Siddique AB, McDougall BM, Seviour RJ. 2004. Which morphological forms of the fungus Aureobasidium pullulans are responsible for pullulan production? FEMS Microbiol Lett 232: 225228. Chanprateep S. 2010. Current trends in biodegradable polyhydroxyalkanoates. J Biosci Bioeng 110: 621632. Chawla PR, Bajaj IB, Survase SA, Singhal RS. 2009. Microbial cellulose: Fermentative production and applications. Food Technology and Biotechnology 47: 107124. Chong BF, Blank LM, Mclaughlin R, Nielsen LK. 2005. Microbial hyaluronic acid production. Appl Microbiol Biotechnol 66: 341351. Clementi F. 1997. Alginate production by Azotobacter vinelandii. Crit Rev Biotechnol 17: 327361. Da Silva AN, Garcia-Cruz CH. 2010. The response surface methodology as a tool for assessing the production of alginate and polyhydroxybutirate by Azotobacter vinelandii. Acta Scientiarum Technology, 32: 105112. De Oliveira MR, da Silva RSSF, Buzato JB, Celligoi MAPC. 2007. Study of levan production by Zymomonas mobilis using regional lowcost carbohydrate sources. Biochemical Engineering Journal, 37: 177183. De Vuyst L, Van Loo J, Vandamme EJ. 1987. Two-step fermentation process for improved xanthan production by Xanthomonas Campestris NRRL-B-1459. Journal of Chemical Technology and Biotechnology 39: 263273. Do JH, Chang HN, Lee SY. 2001. Efficient recovery of gamma-poly (glutamic acid) from highly viscous culture broth. Biotechnol Bioeng 76: 219223. Edd B, Rossier J, Le Caer JP, Desbruyres E, Gros F, Denoulet P. 1990. Posttranslational glutamylation of alpha-tubulin. Science 247: 8385. Elbahloul Y, Krehenbrink M, Reichelt R, Steinbchel A. 2005. Physiological conditions conducive to high cyanophycin content in biomass of Acinetobacter calcoaceticus strain ADP1. Appl Environ Microbiol 71: 858866. Evans CGT, Yeo RG, Ellwood DC. 1979. Continuous culture studies on the production of extracellular polysaccharides by Xanthomonas juglandis. Microbial Polysaccharides and Polysaccharases 5168. Faria S, De Oliveira Petkowicz CL, De Morais SAL, Terrones MGH, De Resende MM, De Frana FP, Cardoso VL. 2011. Characterization of xanthan gum produced from sugar cane broth. Carbohydrate Polymers 86: 469476. Fialho AM, Moreira LM, Granja AT, Popescu AO, Hoffmann K, S-Correia I. 2008. Occurrence, production, and applications of gellan: current state and perspectives. Appl Microbiol Biotechnol 79: 889900.

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

Declaration of interest
This 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 units

a Mark-Houwink constant (Dimensionless, Equation 3) k Proportionality coefficient (Dimensionless, Equation 3) K Consistency index (Pasn, Equations 1 and 2) M Molecular weight (g/mol, Equation 3) n Flow behavior index (Dimensionless, Equations 1 and 2) Intrinsic viscosity (Lg-1, Equation 3) a Apparent viscosity (Pas, Equation 2) Shear stress (Pa, Equation 1) 0 Yield stress (Pa, Equation 1) Shear rate (s-1, Equations 1 and 2)

References
Achbergerov L, Nahlka J. 2011. Polyphosphatean ancient energy source and active metabolic regulator. Microb Cell Fact 10: 63. Amanullah A, Serrano-Carreon L, Castro B, Galindo E, Nienow AW. 1998. The influence of impeller type in pilot scale xanthan fermentations. Biotechnol Bioeng 57: 95108. Amanullah A, Satti S, Nienow AW. 1998. Enhancing xanthan fermentations by different modes of glucose feeding. Biotechnol Prog 14: 265269. Ashiuchi M, Misono H. 2002. Biochemistry and molecular genetics of poly-gamma-glutamate synthesis. Appl Microbiol Biotechnol 59: 914.

2012 Informa Healthcare USA, Inc.

14 D. Kreyenschulte et al.
Freitas F, Alves VD, Reis MA. 2011. Advances in bacterial exopolysaccharides: from production to biotechnological applications. Trends Biotechnol 29: 388398. Galindo E, Albiter V. 1996. High-yield recovery of xanthan by precipitation with isopropyl alcohol in a stirred tank. Biotechnology Progress, 12: 540547. Garcia-Ochoa F, Casas JA, Mohedano AF. 1993. Xanthan precipitation from solutions and fermentation broths. Separation Science and Technology 28: 13031313. Garca-Ochoa F, Santos VE, Casas JA, Gmez E. 2000. Xanthan gum: production, recovery, and properties. Biotechnol Adv 18: 549579. Goto A, Kunioka M. 1992. Biosynthesis and hydrolysis of poly(glutamic acid) from Bacillus subtilis IFO3335. Biosci Biotechnol Biochem 56: 10311035. Goezke G, Posten C. 2010. Electrofiltration of biopolymers. Food Engineering Reviews 2: 131146. Hamano Y. 2011. Occurrence, biosynthesis, biodegradation, and industrial and medical applications of a naturally occurring e-poly-L-lysine. Biosci Biotechnol Biochem 75: 12261233. Herbst H, Schumpe A, Deckwer WD. 1992. Xanthan production in stirred tank fermenters: oxygen transfer and scale-up. Chemical Engineering and Technology 15: 425434. Hofmann R, Kppler T, Posten C. 2006. Pilot-scale press electrofiltration of biopolymers. Separation and Purification Technology 51: 303309. Hofmann R, Posten C. 2003. Improvement of dead-end filtration of biopolymers with pressure electrofiltration. Chemical Engineering Science 58: 38473858. Hopewell J, Dvorak R, Kosior E. 2009. Plastics recycling: challenges and opportunities. Philos Trans R Soc Lond, B, Biol Sci 364: 21152126. Hoppensack A, Oppermann-Sanio FB, Steinbchel A. 2003. Conversion of the nitrogen content in liquid manure into biomass and polyglutamic acid by a newly isolated strain of Bacillus licheniformis. FEMS Microbiol Lett 218: 3945. Hsieh CY, Tsai SP, Wang DM, Chang YN, Hsieh HJ. 2005. Preparation of gamma-PGA/chitosan composite tissue engineering matrices. Biomaterials 26: 56175623. Huang J, Du Y, Xu G, Zhang H, Zhu F, Huang L, Xu Z. 2011. High yield and cost-effective production of poly(-glutamic acid) with Bacillus subtilis. Engineering in Life Sciences 11: 291297. Inbaraj BS, Wang JS, Lu JF, Siao FY, Chen BH. 2009. Adsorption of toxic mercury(II) by an extracellular biopolymer poly(gamma-glutamic acid). Bioresour Technol 100: 200207. Inbaraj BS, Chen BH. 2011. Dye adsorption characteristics of magnetite nanoparticles coated with a biopolymer poly(?-glutamic acid). Bioresour Technol 102: 88688876. Ivanovics G, Bruckner V. 1937. ber die chemische Natur der immunspezifischen Kapselsubstanz der Milzbrandbazillen. Die Naturwissenschaften 25: 250. Ju L-K, Zhao S. 1993. Xanthan fermentations in water/oil dispersions. Biotechnology Techniques 7: 463468. Jung D-Y, Jung S, Yun J-S, Kim J-N, Wee Y-J, Jang H-G, Ryu H-W. 2005. Influences of cultural medium component on the production of poly(-glutamic acid) by Bacillus sp. RKY 3. Biotechnology and Bioprocess Engineering 10: 289295. Kimura K, Tran LS, Uchida I, Itoh Y. 2004. Characterization of Bacillus subtilis gamma-glutamyltransferase and its involvement in the degradation of capsule poly-gamma-glutamate. Microbiology (Reading, Engl) 150: 41154123. King EC, Blacker AJ, Bugg TD. 2000. Enzymatic breakdown of polygamma-D-glutamic acid in Bacillus licheniformis: identification of a polyglutamyl gamma-hydrolase enzyme. Biomacromolecules 1: 7583. Kishida N, Kim J, Tsuneda S, Sudo R. 2006. Anaerobic/oxic/anoxic granular sludge process as an effective nutrient removal process utilizing denitrifying polyphosphate-accumulating organisms. Water Res 40: 23032310. Kocianova S, Vuong C, Yao Y, Voyich JM, Fischer ER, DeLeo FR, Otto M. 2005. Key role of poly-gamma-DL-glutamic acid in immune  evasion and virulence of Staphylococcus epidermidis. J Clin Invest 115: 688694. Kogan G, Solts L, Stern R, Gemeiner P. 2007. Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol Lett 29: 1725. Koller M, Salerno A, Dias M, Reiterer A, Braunegg G. 2010. Modern biotechnological polymer synthesis: A review. Food Technology and Biotechnology 48: 255269. Kornberg A. 1999. Inorganic polyphosphate: a molecule of many functions. Prog Mol Subcell Biol 23: 118. Kubler-Kielb J, Liu TY, Mocca C, Majadly F, Robbins JB, Schneerson R. 2006. Additional conjugation methods and immunogenicity of Bacillus anthracis poly-gamma-D-glutamic acid-protein conjugates. Infect Immun 74: 47444749. Kubota H, Matsunobu T, Uotani K, Takebe H, Satoh A, Tanaka T, Taniguchi M. 1993. Production of Poly(-glutamic acid) by Bacillus subtilis F-2-01. Biosci Biotech Biochem 57: 12121213. Kuttuva SG, Restrepo AS, Ju LK. 2004. Evaluation of different organic phases for water-in-oil xanthan fermentation. Appl Microbiol Biotechnol 64: 340345. Leathers TD. 2003. Biotechnological production and applications of pullulan. Appl Microbiol Biotechnol 62: 468473. Ledezma-Prez AS, Romero-Garca J, Vargas-Gutirrez G, Arias-Marn E. 2005. Cement formation by microbial poly(-glutamic acid) and fluoroalumino-silicate glass. Materials Letters, 59: 31883191. Leela JK, Sharma G. 2000. Studies on xanthan production from Xanthomonas campestris. Bioprocess Engineering 23: 687689. Leonard CG, Housewright RD, Thorne CB. 1958. Effects of some metallic ions on glutamyl polypeptide synthesis by Bacillus subtilis. J Bacteriol 76: 499503. Li C, Price JE, Milas L, Hunter NR, Ke S, Yu DF, Charnsangavej C, Wallace S. 1999. Antitumor activity of poly(L-glutamic acid)-paclitaxel on syngeneic and xenografted tumors. Clin Cancer Res 5: 891897. Lo Y-M, Yang S-T, Min DB. (1997a). Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris. Applied Microbiology and Biotechnology 47: 689694. Lo Y-M, Yang S-T, Min DB. 1997b. Ultrafiltration of xanthan gum fermentation broth: Process and economic analyses. Journal of Food Engineering, 31: 219236. Lo YM, Hsu CH, Yang ST, Min DB. 2001. Oxygen transfer characteristics of a centrifugal, packed-bed reactor during viscous xanthan fermentation. Bioprocess and Biosystems Engineering, 24: 187193. Luckachan GE, Pillai CKS. 2011. Biodegradable Polymers - A Review on Recent Trends and Emerging Perspectives. Journal of Polymers and the Environment, 19: 637676. Makino S, Sasakawa C, Uchida I, Terakado N, Yoshikawa M. 1988. Cloning and CO2-dependent expression of the genetic region for encapsulation from Bacillus anthracis. Mol Microbiol 2: 371376. Manocha B, Margaritis A. 2010. A novel Method for the selective recovery and purification of gamma-polyglutamic acid from Bacillus licheniformis fermentation broth. Biotechnol Prog 26: 734742. Manocha B, Margaritis A. 2010. Controlled release of doxorubicin from doxorubicin/-polyglutamic acid ionic complex. Journal of Nanomaterials, doi 780171. Margaritis A, Pace GW. 1985. Microbial Polysaccharides. In: MooYoung M, ed. Comprehensive Biotechnology Volume 3. Pergamon Press Ltd., 10051044. McIntosh M, Stone BA, Stanisich VA. 2005. Curdlan and other bacterial (1>3)-beta-D-glucans. Appl Microbiol Biotechnol 68: 163173. McLean RJ, Beauchemin D, Clapham L, Beveridge TJ. 1990. MetalBinding Characteristics of the Gamma-Glutamyl Capsular Polymer of Bacillus licheniformis ATCC 9945. Appl Environ Microbiol 56: 36713677. Mooibroek H, Oosterhuis N, Giuseppin M, Toonen M, Franssen H, Scott E, Sanders J, Steinbchel A. 2007. Assessment of technological options and economical feasibility for cyanophycin biopolymer Critical Reviews in Biotechnology

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

Microbial Biopolymer Production and Purification 15

and high-value amino acid production. Appl Microbiol Biotechnol 77: 257267. Moosavi A, Karbassi A. 2010. Bioconversion of sugar-beet molasses into xanthan gum. Journal of Food Processing and Preservation, 34: 316322. Moosavi-Nasab M, Taherian AR, Bakhtiyari M, Farahnaky A, Askari H. 2010. Structural and Rheological Properties of Succinoglycan Biogums Made from Low-Quality Date Syrup or Sucrose Using Agrobacterium radiobacter Inoculation. Food and Bioprocess Technology, 110. Naessens M, Cerdobbel A, Soetaert W, Vandamme EJ. 2005. Leuconostoc dextransucrase and dextran: Production, properties and applications. Journal of Chemical Technology and Biotechnology, 80: 845860. Nasr S, Soudi MR, Haghighi M. 2007. Xanthan production by a native strain of X. campestris and evaluation of application in EOR. Pak J Biol Sci 10: 30103013. Orr D, Zheng W, Campbell BS, McDougall BM, Seviour RJ. 2009. Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans. J Appl Microbiol 107: 691698. Pace GW, Righelato RC. 1980. Production of extracellular microbial polysaccharides. Advances in Biochemical Engineering, 15, 4170. Palaniraj A, Jayaraman V. 2011. Production, recovery and applications of xanthan gum by Xanthomonas campestris. Journal of Food Engineering, 106: 112. Patel S, Kasoju N, Bora U, Goyal A. 2010. Structural analysis and biomedical applications of dextran produced by a new isolate Pediococcus pentosaceus screened from biodiversity hot spot Assam. Bioresour Technol 101: 68526855. Ptter M, Oppermann-Sanio FB, Steinbchel A. 2001. Cultivation of bacteria producing polyamino acids with liquid manure as carbon and nitrogen source. Appl Environ Microbiol 67: 617622. Pradella JGDC. 2006. Biopolmeros e Intermedirios Qumicos. Centro de Gesto e Estudos Estratgicos, available at: http://www.anbio. org.br/pdf/2/tr06_biopolimeros.pdf Prescott AG, Stock LR. 2005. Method for producing medical and commercial grade poly-gamma-glutamic acid of high molecular weight. United States Patent 7,364,875 B2. Psomas SK, Liakopoulou-Kyriakides M, Kyriakidis DA. 2007. Optimization study of xanthan gum production using response surface methodology. Biochemical Engineering Journal, 35: 273280. Rehm BH. 2010. Bacterial polymers: biosynthesis, modifications and applications. Nat Rev Microbiol 8: 578592. Rehm BH, Valla S. 1997. Bacterial alginates: biosynthesis and applications. Appl Microbiol Biotechnol 48: 281288. Reif O-W, Scheper T. 2006. Aufreinigung. In: Anthranikian G. Angewandte Mikrobiologie. Springer Verlag, Berlin, 427443. Remminghorst U, Rehm BH. 2006. Bacterial alginates: from biosynthesis to applications. Biotechnol Lett 28: 17011712. Richard A, Margaritis A. 2001. Poly(glutamic acid) for biomedical applications. Crit Rev Biotechnol 21: 219232. Richard A, Margaritis A. 2003. Rheology, oxygen transfer, and molecular weight characteristics of poly(glutamic acid) fermentation by Bacillus subtilis. Biotechnol Bioeng 82: 299305. Richard A, Margaritis A. 2006. Kinetics of molecular weight reduction of poly(glutamic acid) by in situ depolymerization in cell-free broth of Bacillus subtilis. Biochemical Engineering Journal, 30: 303307. Rosalam S, England R. 2006. Review of xanthan gum production from unmodified starches by Xanthomonas comprestris sp. Enzyme and Microbial Technology, 39: 197207. Sabra W, Zeng AP, Deckwer WD. 2001. Bacterial alginate: physiology, product quality and process aspects. Appl Microbiol Biotechnol 56: 315325. Salah RB, Chaari K, Besbes S, Blecker C, Attia H. 2011b. Production of xanthan gum from Xanthomonas campestris NRRL B-1459 by fermentation of date juice palm by-products (Phoenix dactylifera L.). Journal of Food Process Engineering, 34: 457474. Salah R, Jaouadi B, Bouaziz A, Chaari K, Blecker C, Derrouane C, Attia H, Besbes S. 2011a. Fermentation of date palm juice by curdlan gum production from Rhizobium radiobacter ATCC 6466: Purification, rheological and physico-chemical characterization. LWT - Food Science and Technology, 44: 10261034. Schmid J, Meyer V, Sieber V. 2011. Scleroglucan: biosynthesis, production and application of a versatile hydrocolloid. Appl Microbiol Biotechnol 91: 937947. Senthilkumar V, Gunasekaran P. (2005). Influence of fermentation conditions on levan production by Zymomonas mobilis CT2. Indian Journal of Biotechnology 4: 491496. Seviour RJ, McNeil B, Fazenda ML, Harvey LM. 2011. Operating bioreactors for microbial exopolysaccharide production. Crit Rev Biotechnol 31: 170185. Shih IL, Van YT, Yeh LC, Lin HG, Chang YN. 2001. Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties. Bioresour Technol 78: 267272. Shih IL, Van YT, Shen MH. 2004. Biomedical applications of chemically and microbiologically synthesized poly(glutamic acid) and poly(lysine). Mini Rev Med Chem 4: 179188. Shih IL, Yu YT, Shieh CJ, Hsieh CY. 2005. selective production and characterization of levan by Bacillus subtilis (Natto) Takahashi. J Agric Food Chem 53: 82118215. Shih IL, Shen MH, Van YT. 2006. Microbial synthesis of poly(epsilonlysine) and its various applications. Bioresour Technol 97: 11481159. Shih IL, Yu YT. 2005. Simultaneous and selective production of levan and poly(gamma-glutamic acid) by Bacillus subtilis. Biotechnol Lett 27: 103106. Shingel KI. 2004. Current knowledge on biosynthesis, biological activity, and chemical modification of the exopolysaccharide, pullulan. Carbohydr Res 339: 447460. Shoda M, Sugano Y. 2005. Recent advances in bacterial cellulose production. Biotechnology and Bioprocess Engineering 10: 18. Silva MF, Fornari RCG, Mazutti MA, de Oliveira D, Padilha FF, Cichoski AJ, Cansian RL, Di Luccio M, Treichel H. 2009. Production and characterization of xantham gum by Xanthomonas campestris using cheese whey as sole carbon source. Journal of Food Engineering 90: 119123. Simon L, Caye-Vaugien C, Bouchonneau M. 1993. Relation between pullulan production, morphological state and growth conditions in Aureobasidium pullulans: New observations. Journal of General Microbiology 139: 979985. Simsek S, Mert B, Campanella OH, Reuhs B. 2009. Chemical and rheological properties of bacterial succinoglycan with distinct structural characteristics. Carbohydrate Polymers, 76: 320324. Singer JW, Shaffer S, Baker B, Bernareggi A, Stromatt S, Nienstedt D, Besman M. 2005. Paclitaxel poliglumex (XYOTAX; CT-2103): an intracellularly targeted taxane. Anticancer Drugs 16: 243254. Singh RS, Saini GK, Kennedy JF. 2008. Pullulan: Microbial sources, production and applications. Carbohydrate Polymers, 73: 515531. Singh R. 2011. Facts, growth, and opportunities in industrial biotechnology. Organic Process Research & Development, 15: 175179. Solaiman DK, Garcia RA, Ashby RD, Piazza GJ, Steinbchel A. 2011. Rendered-protein hydrolysates for microbial synthesis of cyanophycin biopolymer. N Biotechnol 28: 552558. Souw P, Demain AL. 1979. Nutritional Studies on Xanthan Production by Xanthomonas campestris NRRL B1459. Appl Environ Microbiol 37: 11861192. Stanley NR, Lazazzera BA. 2005. Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-gamma-dl-glutamic acid production and biofilm formation. Mol Microbiol 57: 11431158. Stredansky M, Conti E, Bertocchi C, Matulova M, Zanetti F. 1998. Succinoglycan production by Agrobacterium tumefaciens. Journal of Fermentation and Bioengineering 85: 398403. Suh I-S, Herbst H, Schumpe A, Deckwer W-D. 1990. The molecular weight of xanthan polysaccharide produced under oxygen limitation. Biotechnology Letters 12: 201206.

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

2012 Informa Healthcare USA, Inc.

16 D. Kreyenschulte et al.
Sung MH, Park C, Kim CJ, Poo H, Soda K, Ashiuchi M. 2005. Natural and edible biopolymer poly-gamma-glutamic acid: synthesis, production, and applications. Chem Rec 5: 352366. Sung M-H, Park C, Kim C-J, Park GS, Uyama H, Poo HR, Song JJ. 2008. Poly-gamma-glutamic acid-vitamin complex and use thereof. United States Patent 0132440 A1. Survase SA, Saudagar PS, Bajaj IB, Singhal RS. 2007. Scleroglucan: Fermentative production, downstream processing and applications. Food Technology and Biotechnology 45: 107118. Torrestiana-Sanchez B, Balderas-Luna L, Brito-De la Fuente E, Lencki RW. 2007. The use of membrane-assisted precipitation for the concentration of xanthan gum. Journal of Membrane Science, 294: 8492. Wang J, Dong M. 2009. Optimum effective viscosity of polymer solution for improving heavy oil recovery. Journal of Petroleum Science and Engineering, 67: 155158. Whelan J. 2002. Targeted taxane therapy for cancer. Drug Discov Today 7: 9092. Wyatt NB, Liberatore MW. 2009. Rheology and viscosity scaling of the polyelectrolyte xanthan gum. Journal of Applied Polymer Science 114: 40764084. Yang S-T, Lo Y-M, Min DB. 1996. Xanthan gum fermentation by Xanthomonas campestris immobilized in a novel centrifugal fibrous-bed bioreactor. Biotechnology Progress 12, 630637. Zhang Z, Chen H. 2010. Fermentation performance and structure characteristics of xanthan produced by Xanthomonas campestris with a glucose/xylose mixture. Appl Biochem Biotechnol 160: 16531663. Zinn M, Witholt B, Egli T. 2001. Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 53: 521.

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by HINARI on 12/09/12 For personal use only.

Critical Reviews in Biotechnology