Applied Microbiology and Biotechnology (2018) 102:1587–1598 https://doi.org/10.1007/s00253-018-8745-7

MINI-REVIEW

Towards a better production of bacterial exopolysaccharides by controlling genetic as well as physico-chemical parameters

Dipanjan Sengupta1 & Sriparna Datta1 & Dipa Biswas1

Received: 9 September 2017 /Revised: 22 December 2017 /Accepted: 27 December 2017/Published online: 17 January 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Bacterial extracellular polymeric substances, which are basically bacterial metabolites, have currently become a subject of great concern of modern day microbiologists and biotechnologists. Among these metabolites, bacterial exopolysaccharides or EPS, in particular, have gained a significant importance. EPS are formed by the bacteria in their late exponential or stationary phase of growth under special situations for specific purposes. They take part in the formation of bacterial biofilms. There is a great diversity in the types of EPS. Strikingly enough, a same species of bacterium can produce different types of EPS under different situations. The importance of EPS is largely because of their different applications in various industries. Now that the bacterial EPS has got the potentiality to become an upcoming tool in various futuristic applications of human benefit, the focus currently develops towards how better they can be produced in the laboratory by promoting the favorable factors for their production. While studying with different EPS forming bacteria, both the intrinsic factors like genetic configuration of the bacteria and the extrinsic factors like culture conditions under the influence of different physico-chemical parameters in order to maximize the EPS production have been taken into consideration. Both the factors have proved their worth. Hence, towards a better outcome for EPS production, it is indicated that a genetic manipulation of the bacteria should be synchronized with a proper selection of its culture condition by controlling different physico-chemical parameters.

Keywords Exopolysaccharides (EPS) . Genetic manipulation . Culture conditions . Physico-chemical parameters . Quorum sensing

Introduction 2015) because of their potential in commercial applications for human benefit (Freitas et al. 2011). In the last couple of decades, a good deal of enthusiasm has Unlike bacterial intracellular metabolites, which are not been noted in carrying out intensive studies in the field of only scarce but also difficult to isolate for detailed study, the applied microbiology and industrial biotechnology regarding extracellular metabolites, particularly the exopolysaccharide the discovery, isolation, characterization, and optimization of or EPS component of bacterial biofilms, have great potentials different bacterial extracellular polymeric substances, particu- in industrial applications for human welfare (Madhuri and larly the bacterial exopolysaccharides or EPS (Ruas-Madiedo Prabhakar 2014). Hence, different types of bacterial EPS have and de los Reyes-Gavilán 2005; Bramhachari and Dubey been isolated, characterized, and utilized in different domains 2006; Paulo et al. 2012; Bales et al. 2013; Torres-Rodríguez of human interest like food and beverage industry, preserva- et al. 2014; Madhuri and Prabhakar 2014; Liang and Wang tion of dairy products, cosmetics, textiles, agriculture, waste management, bioremediation, pharmaceuticals, medical ap- plication in wound healing, and so on (Ates 2015). Bacterial exopolysaccharides, as the name implies, are ex- tracellular metabolites secreted by the organism especially in * Sriparna Datta [email protected] the late log (or exponential) phase or at the onset of stationary phase of growth when there is either a scarcity of further

1 nutritional and favorable factors or the organisms are unable Department of Chemical Technology, University of Calcutta, 92, to utilize them. EPS are high molecular weight biopolymers Acharya Prafulla Chandra Road, Kolkata 700009, India 1588 Appl Microbiol Biotechnol (2018) 102:1587–1598 and, though structurally diverse, they are essentially made up eventually resulting in a matrix-like network. This very much of a carbohydrate moiety and a non-carbohydrate moiety like mimics the cell adhesion molecule (CAM) in the eukaryotic pyruvate, acetate, succinate, and phosphate (Nwodo and Okoh cells. 2013). It is interesting to note that in biofilms, the microor- In reality, the isolation and characterization of bacterial ganism accounts for only about 10% of the dry mass EPS have not been very smooth because the yield of EPS from while the matrix accounts for the remaining 90% the bacteria is often very inadequate and is dependent on the (Flemming and Wingender 2010). Such extracellular nature of the culture media and on the concerned bacterial matrices impart several functions like microbial cell to species. Further, it is interesting to note that a particular bac- cell adhesion; microbial adhesion to a surface; signaling terial species can produce different types of EPS having dif- molecule for other cells to interact; inhibiting cellular ferent potentialities when the substrate of the culture media or desiccation; tolerating excessive changes in temperature, the culture condition changes (Dertli et al. 2013; Matsuyama pH, salinity, or radiation; fighting against antibiotics or et al. 2003). On the other hand, different species of bacteria toxic molecules; and even acting itself as a final source under the same genus can produce structurally different EPS of nutrition for the producer bacteria at extreme nutri- even if the culture condition remains unchanged (Celik et al. tional scarcity. 2008). Therefore, various attempts have been made to select Structurally, such metabolites are polymeric in nature and explore the capability of different species of a particular comprising repeating units which are, at large, polysac- bacterium having differences in its genetic configuration as charides in nature. Such extracellular polymeric sub- well as to study the role of different culture conditions under stances are henceforth coined as Exopolysaccharide or different physico-chemical parameters to optimize the yield of EPS. Apart from EPS, extracellular proteins are also a bacterial EPS so as to make such efforts economically viable produced as bacterial metabolites. The discovery of the and sustainable. first bacterial exopolysaccharide (EPS) dextran, pro- Eventually, it has come up to be a valid question whether duced by Leuconostoc mesenteroides, goes back to the the genetic composition of the bacterial species or the specific mid nineteenth century (Nwodo and Okoh 2013). It culture condition of the media plays a more distinctive or pioneered a field of an extensive research on the char- decisive role in the optimal production of a desired bacterial acter, structure, function, and potential applications of EPS. This review encompasses the significance and impor- bacterial EPS. tance of controlling both culture condition and bacterial genes With the advent of time, so many bacterial EPS like algi- on better EPS production in both qualitative and quantitative nate, cellulose, xanthan, gellan, levan, succinoglycan, and aspects. curdlan have been discovered, isolated, and studied in detail. They have diverse applications in human welfare like in food, oil, cosmetic, pharmaceutical, and other industrial sectors. For Why do bacteria produce EPS? example, bacterial EPS like dextran, levan, acetan, and xanthan have been widely used as viscosifier and thickening Owing to a gradual decline in natural resources, each agent in food industry; alginate has been established in wound and every life form on the Earth tends to exhibit a dual healing; colonic acid is widely used in cosmetic industry; trend for its survival. On one hand, during favorable emulsan has been identified for its propensity in the recovery conditions, there is a constant competition to outnumber of crude oil (Patel and Prajapati 2013); and so on. the other and, on the other hand, in adverse and ex- The isolation of EPS in the laboratory generally involves a treme situations, there is a tendency to colonize and stepwise procedure (Paulo et al. 2012). Sometimes this in- stay united to mitigate the hard time. volves a substantial time span for bacterial incubation such This dual behavior is well marked at bacterial level also. that the organism enters into the stationary phase of growth Based on such contrasting environmental conditions, they where the production of such secondary metabolites may be at synthesize several substances like toxins or endospores for its peak (Karuppiah et al. 2014). Often, the values of favorable sustenance and adaptation. Bacteria secrete polymeric sub- factors (like temperature, pH, and other culture conditions) for stance to their extracellular niche gradually resulting in the maximum production of bacterial metabolites differ signifi- formation of a colony of microbes often termed as biofilms cantly from those responsible for maximum growth of the or slime (Quiñones et al. 2005). Owing to the diversity of bacteria. This is really a matter of concern. Hence, intensive microbes, such metabolites produced have a diverse role in a research works are being contemplated to find out which fac- wide spectrum of research works and applications. tor(s) is/are primarily influencing the desired optimal yield of We often find that bio-films or pellicles are formed at the a bacterial EPS, whether it is primarily related to the type of a air-water interface composed of several macromolecules that bacterial gene itself or simply to the nature of the culture connect a single type of microorganism or a mixed population condition. Appl Microbiol Biotechnol (2018) 102:1587–1598 1589

A glimpse on the diversity of bacterial EPS Quorum sensing and EPS production

No less than 86% of the entire multitude of various species of Bacteria can often sense the density of their immediate local living organisms dwelling on the Earth are yet to be identified population by the mechanism of quorum sensing. This process or explored (Watson 2011). It is presumed that there might be basically involves the transmission of signaling molecules or one trillion microbial species in this planet of which more than inducers (like pheromone) emitted by organisms in a popula- 99% still remain undiscovered (Bakalar 2016). Oceans cater tion to the recipients so as to establish their presence. The the largest and the most diverse nutritional platter for some expression of such inducers (e.g., N-acetyl homoserine in millions of marine species and also remain as the largest res- Gram-negative bacteria) involved in quorum sensing is pro- ervoirs of carbon. Several marine bacterial species have been portional to the local population density and follows a positive identified to produce a high yield of EPS. Besides synthesiz- feedback loop. Quorum sensing is successfully described in ing EPS, the association between marine bacteria and other the marine organism Vibrio fischeri. The organism produces marine life forms might also trigger the bacteria to tailor struc- cell-density dependent auto-inducer signal (as N-homoserine turally complex architecture of different polymeric substances lactone) which is related to the bio-luminescence imparted by in marine ecosystem (Lema et al. 2012; Fuentes et al. 2016; the organism for inter-cellular communication (Schaefer et al. Ramanan et al. 2016; Kouzuma and Watanabe 2015). 1996). In the nitrogen fixing bacteria Sinorhizobium meliloti, Chemical analysis of dissolved organic carbon (DOC) of the inducer molecule for quorum sensing, N-acetyl ocean water suggests that polysaccharides are one of the major homoserine lactone (AHL) is produced by AHL synthase components which accounts for up to 50% of DOC of surface which is expressed by sinI gene. The expression of this gene water and 25% of that of deeper water. The sources of such is regulated by transcription regulators sinR and expR. polysaccharides happen to be mostly bacterial and phyto- Mutation in the sinI, sinR, and expR gene results in inhibition planktonic (Zhang et al. 2015). of production of bacterial exopolysaccharide (Gao et al. EPS produced by a certain bacterium is usually found to be 2012). unique and specific to that particular bacterial species. But, In bacterial community, quorum sensing is often a phenom- sometimes, it is also found that a particular bacterial species enon correlated to the formation of biofilm. Since formation of owns the capability to produce several EPS having diversity in biofilm basically involves the consolidation and maximum their structures and characters. For example, a specific strain utilization of space by a bacterial population, quorum sensing of Lactobacillus johnsonii FI9785 produces two structurally happens to be a pre-requisite for the participating bacteria. different types of exopolysaccharides—EPS-1 and EPS-2— Any impairment in the structural or regulatory genes in- which have basic differences in the branching of their respec- volved in quorum sensing or biofilm production renders the tive constitutional sugar moiety. Also, they differ in the pres- microorganism susceptible to such environmental atrocities. ence of substituent groups occupied by the sugar backbone. In For example, in Pseudomonas syringae-mediated AHL- EPS-1, it is 1-phosphoglycerol as well as o-acetyl group, dependent quorum sensing, mutation of the structural and reg- while in EPS-2, it is only a single o-acetyl group (Dertli ulatory genes concerned with quorum sensing shows signifi- et al. 2013). Similar is the case of Lactobacillus pentosus cant impairment in the production of alginate. Furthermore, LPS26 which also produces two different EPS whose individ- the mutant becomes more susceptible to hydrogen peroxide in ual production depends on the culture condition (Sánchez the local environment as compared to its wild type (Quiñones et al. 2006). et al. 2005). The maximum quorum sensing activity is observed at the late-exponential phase or at the onset of stationary phase of bacterial growth. In Halomonas sp., EPS is produced at the Genetic factors influencing EPS production stationary phase of growth (Quesada et al. 2004). So, it is quite logically predictive that there is an inter-relation between quo- The structural integrity of a bacterial metabolite is largely rum sensing and production of EPS. Both the processes seem dependent on the genetic constitution of the bacterial DNA. to be genetically controlled under the same signaling cascade In most of the bacterial species, we find a genetic locus in their and also might be dependent on the population density of their chromosome or plasmid where a particular gene or a genetic local environment (Llamas et al. 2005). cluster is primarily responsible for the production of EPS. Sometimes, they are also involved in other activities of the Genes concerned with EPS production bacterial life cycle. Such pleiotropic nature of EPS genes is often studied by techniques that generally involve the muta- Bacterial genes responsible for production of EPS that pro- tion of such genes and their consequences (Wang and mote various secondary functional features to the bacteria are Kuramitsu 2006;Wangetal.2011a, b). widely being identified and explored. In Bacillus subtilis,a 1590 Appl Microbiol Biotechnol (2018) 102:1587–1598 series of genes designated alphabetically as epsI to epsO are presence of EPS inhibits autophosphorylation (inactivation) of concerned with bacterial pellicle formation and swarming mo- the bacterial tyrosine kinase and promotes phosphorylation of tility. When mutated, the mutation in epsJ to epsN results in glycosyltransferase which stimulates prolonged production of detrimental effect on bacterial pellicle formation while muta- EPS. This phenomenon has been demonstrated as an example tion in epsO (eps operon gene) does not show such effect of positive feedback loop (Elsholz et al. 2014). This is likely to (Nagorska et al. 2010). Such studies help specific identifica- be a common phenomenon for regulation of such secondary tion of genes that are crucial in the production process of metabolites by bacterial cell. bacterial EPS. The manipulation of such related genes and Apart from chromosomes, EPS-coding genetic elements their subsequent over-expression seems to maximize the yield are also located in plasmids. Techniques like endonuclease of bacterial EPS. treatment, gel electrophoresis, polymerase chain reaction, In some bacterial species, yield of EPS markedly increases and Southern blotting are often implemented in figuring out by over-expression of the bacterial regulatory genes. In such the location of such intra-plasmid genetic elements. In cases, the nucleotide-sugar precursors and the related enzymes Lactobacillus lactis, an intraplasmic 12-kb region containing play a pivotal role. Enzymes like UDP galactose 4-epimerase, 14 genes has been mapped to show sequence homology with UDP glucose pyrophosphorylase,andphosphoglucomutase the genetic products involved in capsular polysaccharide, are associated with elevated levels of bacterial EPS (Degeest EPS, lipopolysaccharide, and teichoic acid biosynthesis. and De Vuyst 2000). Genetic engineering involving over- However, by single gene disruption technique, the epsD gene expression of such associated genes is constantly under is pointed out to be the one responsible for production of EPS progress. in the microorganism (van Kranenburg et al. 1997). With the advent of recombinant DNA technology, the op- From all such diverse observations and studies, it is quite timization of yield of bacterial secondary metabolite can be evident that under the influence of different intrinsic or extrin- programmed. Nox (NADH oxidase) gene cloned from sic factors, bacterial genes, which have got a specific propen- Streptococcus mutans was over-expressed in Lactobacillus sity for EPS production, often play a definitive role in control- casei LC2W where the resultant recombinant strain ling the yield of EPS. However, this propensity seems to be Lactobacillus casei LC-Nox shows over-expression of very species-specific, complex, unpredictable, and difficult to NADH oxidase, conferring improvement in the production assess beforehand. So, it is yet a long way to go until we can of bacterial EPS by 46% by the recombinant strain as com- evaluate the exact role of bacterial genes influencing EPS pared to the wild type strain (Li et al. 2015). This clearly production so far the practical application of EPS is concerned indicates that inter-bacterial gene cloning and recombination in different industries of human interest. often prove to be beneficial for increasing the yield of EPS. Production of bacterial EPS is also indirectly related to the presence of other substances which are genetically controlled. In Acinetobacter oleivorans, increased endogenous produc- Physico-chemical factors influencing EPS tion of hydrogen peroxide in ahpC mutant strain of the bacte- production rium in enriched media promotes better biofilm formation. The deletion of ahpC gene promotes increased EPS produc- The role of external factors that affect the production of bac- tion by an early expression of the EPS gene in the bacteria terial EPS is quite variable. In certain cases, it has been in- (Jang et al. 2016). Thus, it can be speculated that the formation ferred that production of bacterial EPS is a result of stressful of biofilm in the bacterial species is linked to the defense culture condition while in the other it has been observed that mechanism of the bacteria towards hydrogen peroxide. the enrichment of media influences the production process of Genes involved in some other activities, when mutated, EPS. However, it has been understood that bacterial EPS is sometimes show alteration in production of bacterial EPS or produced at maximum during the stationary phase of bacterial formation of biofilm. For instance, in Streptococcus mutans, growth. Sometimes, it has been noticed that the same bacterial mutation of a transcription regulator Frp decreases transcrip- species produces structurally different EPS when subjected to tion of several related genes in a cascade and eventually in- any alteration in the culture conditions. The basic parameters hibits sucrose-induced biofilm formation and production of in most of the studies pertaining to EPS production generally EPS (Wang and Kuramitsu 2006). Similarly, characterization include the following: variation in carbon source, nitrogen of ybjN mutant in Escherichia coli results in increased bacte- source (Degeest et al. 1999), concentration of oxygen rial motility, fimbriation, EPS production, and biofilm forma- (Thompson and Leps 1986; Lawford and Rousseau 1989), tion while over-expression of ybjN results in reverse pleiotro- pH of medium (Torres et al. 2012), incubation temperature, pic effects (Wang et al. 2011a, b). enzymes (Pham et al. 2000), ions (Qurashi and Sabri 2012), It is interesting to note that besides genetic factors, bacterial stresses (Sandhya and Ali 2015), and other fermentation con- EPS can also act as a self-regulator. In Bacillus subtilis,the ditions (Vaningelgem et al. 2004). Appl Microbiol Biotechnol (2018) 102:1587–1598 1591

Effect of stress Often we find a structural relatedness of the bacterial EPS with the carbon source used as the substrate for the bacterial Stress plays a major role inducing production of various mi- growth and production of EPS (Audy et al. 2010; Looijesteijn crobial EPS of potential interest (Sandhya and Ali 2015). et al. 1999). When we consider a simple sugar instead of a Since marine bacteria are always subjected to stress due to complex one as the sole carbon source in a minimal medium, frequent changes in their niche or habitat, they are always we usually find that the yield in EPS is relatively higher (Celik prone to readjustment. Also, the ongoing variation in the con- et al. 2008). In most cases, the more complex is the carbohy- centration gradient of saline water imparts changes in osmotic drate substrate in the medium, the more difficult it becomes pressure which again acts as an important mediator for pro- for the bacterium to break the intermolecular linkages to uti- duction of bacterial metabolites (Wood 2015). The saline wa- lize the sugar for its growth and subsequent production of ter acts as a hypertonic medium, and in order to maintain the secondary metabolites at the stationary phase. Furthermore, tonicity for survival, the bacterial cells readily exert to synthe- the bacterial enzymes often play significant role in the cleav- size polymeric substances that would surround them like an age of the glycosidic linkages of the complex sugars. Here, the armor for protection. Such marine bacterial species include relation between the role of glycolytic enzymes and the bio- Pseudoalteromonas sp., Alteromonas macleodii, synthesis of EPS in Lactobacillus lactis is worth mentioning Thermococcus litoralis, Geobacillus sp., Bacillus (Ramos et al. 2001). thermodenitrificans, and Bacillus licheniformis (Poli et al. The judicious treatment of the substrate before inoculation of 2010). the bacterial culture for growth and production is necessary and The fact that various stressful conditions trigger bacteria to often proves to be helpful in increasing the yield of a secondary accentuate their production of EPS is again supported when metabolite. So for increasing the final yield of a potential bac- Pseudomonas putida is subjected to environmental challenges terial metabolite, pre-treatment of the complex sugar substrates like very hot and dry climate during drought. The organism’s or raw materials by heat or microwave or acid hydrolysis or capacity to withstand such stress up to a certain limit by an with enzymes like amylases might be quite effective. enhanced production of EPS has been well substantiated. The structural configuration of bacterial EPS utilizing car- Here, the EPS produced is likely to induce the necessary os- bohydrates exhibits varied properties that might have potential motic and thermal tolerance to the bacterial species. Thus, it is impact on pharmaceutical, industrial, commercial, as well as quite evident that EPS plays a distinctive role in stress toler- environmental fields. EPS display varied rheological proper- ance and water holding capacity for the microorganism ties and sometimes even act as potent surface-active agents (Sandhya and Ali 2015). (Liang et al. 2014). Such bio-surfactants vary in molecular In contrast to the marine microbiota of aquatic environ- weight as well. The low molecular weight biosurfactants are ment, Cyanobacteria are found to behave as colonizers in often glycolipids in nature while the high molecular weights constrained environments like desert soils and lithic environ- are complex heteropolysaccharides or polysaccharide-protein ment. It has been observed that the colonization of complexes (Nwodo and Okoh 2013). Cyanobacteria and their subsequent production of EPS mark Apart from sugar substrates, lipid- or oil-based substrates the onset of a naturally stable and hydrated environment also represent a potential carbon based substrate for the pro- (Rossi and De Philippis 2015). duction of bacterial metabolites. Such bacterial metabolites often prove to be effective bioemulsifier with the propensity to minimize the repulsion between oil-water interfaces Effect of carbon source (Bhattacharya et al. 2014). Such bioemulsifiers might show changes in the bioemulsification or other rheological proper- The carbon source in bacterial medium has profound effect on ties based on their structural domain. The chain length of the the yield of EPS in both qualitative term, i.e., on the structural fatty acid is often a determining factor of the propensity of integrity of an EPS as well as in quantitative term, i.e., on the emulsifying property. Moreover, branching of the constituent overall yield of EPS (Molina-Ramírez et al. 2017). long chain fatty acids might further result in an additive effect Given a number of different sugar substrates, a spe- to such properties. Also, the orientation of the hydrophilic and cific bacterial strain is found to have a selective affinity the lipophilic region of such bioemulsifiers and their respec- for a particular substrate to produce an appreciable yield tive ratios determine the applicability of such potential bacte- of EPS (as enlisted in Table 1). For instance, rial metabolites (Nerurkar et al. 2009). Lactobacillus casei CRL87 gives higher yield of EPS when grown in galactose as compared to that in glucose Effect of nitrogen source (Mozzi et al. 2001). Similar is the case of Lactobacillus lactis that produces higher yield when grown on glucose Nitrogen source has also been found to play a significant role substrate compared to fructose (Looijesteijn et al. 1999). in the production and optimization of bacterial EPS. The 1592 Appl Microbiol Biotechnol (2018) 102:1587–1598

Table 1 Choice of carbohydrate substrates for production of commercially important EPS

Name of EPS Producer bacteria Composition Substrate of choice References for EPS production

Alginate Pseudomonas sp.,Azotobactersp. Guluronic acid, mannuronic Glucose (for Schmid et al. (2015); Sengha acid with acetyl group Pseudomonas sp.), et al. (1989); Ma et al. (1997) sucrose (for Azotobacter sp.) Cellulose Acetobacter, Azotobacter, Rhizobium, Glucose Glucose Schmid et al. (2015); Pseudomonas, Salmonella, Pourramezan et al. (2009) Alcaligenes, Sarcina ventriculi Dextran Leuconostoc mesenteroides, Glucose Sucrose Schmid et al. (2015); Sarwat et al. Streptococcus mutans (2008); Guimarães et al. (1999) Curdlan Agrobacterium sp., Paenibacillus Glucose Glucose Schmid et al. (2015); Rafigh et al. polymyxa, Pseudomonas sp. (2014); Yang et al. (2016) QL212 Diutan Sphingomonas sp. Rhamnose, glucose, glucuronic Malt extract Schmid et al. (2015); Coleman acid et al. (2008) Colanic acid Escherichia coli Fucose, glucose, glucuronic Glucose Schmid et al. (2015); Ren et al. acid, galactose, with acetyl, (2016) pyruvate group Levan Zymomonas mobilis Fructose, glucose Sucrose, glucose, Schmid et al. (2015); Oliveira cane sugar et al. (2007); Ananthalakshmy and Gunasekaran (1999) Succinoglycan Sinorhizobium meliloti Glucose, galactose with acetate, Sucrose Schmid et al. (2015); Jones succinate, pyruvate groups (2012) Welan Alcaligenes sp. Rhamnose, glucose, glucuronic Glucose, corn starch Schmid et al. (2015); Li et al. acid, mannose with acetate (2010); Roca et al. (2015) group Xanthan Xanthomonas campestris Glucose, mannose, glucuronic Sucrose, molasses Schmid et al. (2015); Kalogiannis acid with acetate, pyruvate et al. (2003) group Gellan gum Sphingomonas paucimobilis Rhamnose, glucose and Sucrose, glucose, Roca et al. (2015); Sa-Correia glucuronic acid with acetyl whey, soluble starch, et al. (2002) and glyceryl group molasses Clavan Clavibacter michiganensis Fucose, glucose and galactose, Glucose Roca et al. (2015) with acetyl, pyruvyl and succinyl group FucoPol Enterobacter A47 Fucose, glucose, galactose, Glycerol, glucose, Roca et al. (2015) glucuronic acid with acetyl, xylose pyruvyl and succinyl group Hyaluronic Streptococcus sp. Glucuronic acid and acetyl Glucose Roca et al. (2015) acid glucosamine

judicious use of nitrogen in the bacterial culture media is a concentration plays a positive or beneficial effect on the quan- principal prerequisite for the optimum yield. While little addi- titative yield of the bacterial EPS (Pal and Paul 2013)whilein tion of nitrogen source in media might be of some significance another situation, in Streptococcus thermophilus,wefindthat in the yield, excess of nitrogen might cause detrimental effects a gradual increase in the nitrogen complex in the media results in the production of EPS. So the optimization of nitrogenous in a shift of the produced bacterial EPS from higher to lower compound(s) in the media is necessary for a significant yield molecular mass (Degeest and Vuyst 1999). in EPS. Here, the role of nitrogen derivatives like sodium Other factors remaining constant, in order to understand the nitrate, yeast extract, urea, ammonium sulfate, amino acids effect of nitrogen in the production of bacterial EPS, it will be like glycine, glutamic acid, aspartic acid, phenylalanine in more reliable and practicable to optimize the C/N ratio of the the culture media, and its effect on the production of EPS is medium. The choice for suitable carbon and nitrogen source in also worth mentioning. the culture media for metabolite production often becomes a The role of nitrogenous compound(s) in a media is quite matter of concern. Moreover, figuring out the optimum con- complex. For example, we find that in case of the centration of each is equally important. In view to standardize rhizobacterium Cupriavidus pauculus, increased nitrogen a definite C/N ratio for a bacterial culture for maximum EPS Appl Microbiol Biotechnol (2018) 102:1587–1598 1593 production, the process of variation of different carbon gene. This is a symbiotic efficacy of the bacteria for sources with a constant nitrogen source and vice-versa might production of EPS (Bonomi et al. 2012). be quite helpful (Farrés et al. 1997). The perfect balance be- tween carbon and nitrogen in the culture medium is an essen- Effect of radiation tial criterion for maximization or optimization of yield. Bacillus thuringiensis produces significantly high level of Xanthomonas campestris, which produces a commercially EPS when cultured in 2% maltose and 1% peptone media important EPS xanthan gum, has been exposed to two differ- compared to other sources of carbon like glucose, sucrose, ent microwave powers—90Wfor6minand450Wfor3min or lactose or other common nitrogen sources like ammonium followed by plating of the resultant colonies. The EPS pro- salts, beef extract, or peptone. So the proper selection of the C duction happens to be 43.75 and 50% more respectively for and N sources is a primary prerequisite in the process (Wang exposed colony compared to the control. In both the cases, it is et al. 2011a, b). found that there is no further noticeable bacterial growth after the respective durations while the rate of xanthan production Effect of variation in temperature and pH changes. So, we can infer that the bacterial growth and the production of xanthan, when subjected to microwave treat- The temperature and pH required for the optimum growth of a ment, are independent of each other (Kothari et al. 2014). bacterium usually vary from those required for the optimum On exposure to microwave for limited duration, production of EPS by the same bacterium. For example, the Streptococcus mutans and Xanthomonas campestris showed required temperature and pH for optimum growth of increase in growth but in terms of production of EPS, such Gluconobacter hansenii is 30 °C and pH 4 while a tempera- treatment was detrimental for S. mutans while beneficial in ture below 30 °C with pH 5 results in the highest yield of EPS case of X. campestris (Kushwah 2013). This exemplifies the by the same bacterial species (Valepyn et al. 2012). Klebsiella varied capacity of bacterial strain in withstanding such treat- pneumoniae grown at a range of different temperatures from ment and the respective variation of their yield in EPS. 10 to 60 °C shows best production in EPS at circa 30 °C followed by that at 40 °C (Sivakumar et al. 2016). Effect of magnetic field A chemically defined medium (CDM) containing mainly lactose and glucose along with other essential salts promotes The effect of magnetic field on the bacteria Bacillus cereus the lactose fermenting bacteria, Lactobacillus delbrueckii and its production of EPS are worth mentioning. Magnetic subsp. bulgaricus, to produce EPS. When CDM is compared field up to a certain limit (6.0–10.0 mT) promotes the strain with milk as the substrate for bacterial growth and production to produce EPS while further increase in magnetic field (up to of EPS, a lower yield of bacterial EPS takes place in CDM 17.4 mT) increases the negative charge of the EPS resulting in compared to that in milk at neutral pH. However, when the pH bio-adsorption of cations. Also, the magnetic field energy is is maintained at 6.0, yield of bacterial EPS in CDM is the capable of influencing the hydroxyl group and carboxyl group same as that of milk (Petry et al. 2000). Thus, pH plays a by forming hydrogen bond (Xu et al. 2014). Thus, in certain distinct role in the rate of production of EPS. strains, magnetic field energy possesses the potential to alter Over a range of pH of 4.5, 5.5, and 6.5, there is a significant the structural integrity of the bacterial metabolite and thereby change in the production of EPS for Streptococcus affects the functional prospect of bacterial EPS. thermophilus after 24 h of incubation. Generally, a pH of 6.2 is optimum for the viability of the bacteria but the highest yield of EPS is noted at a pH of 5.5. Thus, an acidic stress Better production of EPS—an emphasis often plays a vital role in the production of EPS by certain bacterial strains (Zisu and Shah 2003). Modern day microbiological research works, reinforced by latest biotechnological techniques, are providing a lot of new Effect of light information about the isolation, optimization, characteriza- tion, and industrial application of different bacterial metabo- Light plays a definite role especially in photosynthetic and lites especially exopolysaccharides (EPS) which are likely to symbiotic bacterial population. Light regulates the production be produced under the influence of different intrinsic or ex- of EPS by the nitrogen fixing symbiotic bacteria, Rhizobium trinsic factors. leguminosarum, by altering the effect of a sensor histidine As much as a bacterial growth in a culture medium is given kinase which contains a LOV (light-oxygen-voltage) do- importance, the production of its metabolites at late- main and is encoded by the bacterial gene lov. The exponential phase or during stationary phase of growth by effect of light on the production of EPS by the bacterial following a metabolic pathway is also deemed to be a phe- strain is basically governed by the expression of the lov nomenon of equal significance (Ramos et al. 2001). This is 1594 Appl Microbiol Biotechnol (2018) 102:1587–1598 because the production of metabolite acts as a productive modulations in the quantitative yield of the desired bacterial marker for determining the sensitivity of the organism to its EPS (Degeest et al. 2003; Vaningelgem et al. 2004;Zhang relevant culture medium or its immediate vicinity (Sandhya et al. 2011). It is interesting to note that besides standard phys- and Ali 2015). ical parameters like temperature or pH, other physical param- The quantitative yield or qualitative nature of such second- eters like light, magnetic field, and radiation also possess some ary metabolite seems to be primarily based on the inherent modulatory effects on bacterial growth and production of EPS genetic constitution of the bacteria which may be of chromo- (Bonomi et al. 2012; Xu et al. 2014; Kothari et al. 2014). somal or plasmid origin. Accordingly, the nature of metabolite Recently, some specific frequencies of sound have also been is also a determinant (Zivkovic et al. 2015). It is well defined reported to stimulate microbial growth and production of cer- that bacterial regulator gene of the bacterial operon system tain metabolites (Sarvaiya and Kothari 2015;Shahetal. governs the expression of certain enzymes that directly or 2016). indirectly act as precursors in the production of the basic units Owing to a great diversity in bacterial metabolites, various of such polymeric metabolite (Huang et al. 1995;Dertlietal. upgraded and intricate methods involving UV-Vis spectrosco- 2016; Elsholz et al. 2014). py, FTIR spectroscopy, gas chromatography, mass spectros- With a constant change in the environment, bacterial pop- copy, HPLC (Madhuri and Prabhakar 2014; Sun et al. 2015), ulation also undergoes changes in terms of their genetic con- NMR, or MALDI-TOF spectroscopy (Gonzalez-Gil et al. stitution (Mendrygal and González 2000; Velasco et al. 2006). 2015) have come up to help determine the structural identity Eventually, the production of bacterial metabolites also shows of bacterial EPS more precisely. some qualitative or quantitative alterations. Genetic engineer- So we can well understand that in order to get a clear ing and gene cloning have paved the way for definitive ma- concept about various factors responsible for better production nipulation of such bacterial genetic framework in order to of bacterial EPS, a detailed knowledge is necessary about the modulate the optimum production of the desired metabolites influence of gene and its manipulation which is to be rein- that are industrially important (Schmid et al. 2015). forced by a synchronized support of physical as well as chem- In spite of how much we do speculate about the relevance ical parameters responsible for production of both qualitative- of bacterial gene on the production of a specific type of sec- ly superior and quantitatively maximized yield of the desired ondary metabolite, we cannot ignore the choice of an appro- EPS. Thus, in a nutshell, an amalgamation of precise manip- priate substrate which is to be utilized by the concerned bac- ulation of bacterial genes and proper selection of appropriate teria for sole purpose of production of the metabolite (Gamar- physico-chemical condition can be orchestrated in better pro- Nourani et al. 1998; Ruiz et al. 2010; VanderMolen et al. duction of a desired EPS. 2013). A definite knowledge of such suitable substrates for culturing the bacteria is often attained by culturing them in Acknowledgements The authors remain grateful to the Department of several pure carbon-sourced media (like glucose alone or ga- Chemical Technology, University of Calcutta and also to the Department of Science and Technology, Government of West Bengal lactose alone) so that we may ascertain the definite structural for the necessary support to contemplate this study. concept of the EPS produced (Mozzi et al. 2001; Yuksekdag and Aslim 2008). As stated earlier, we often find quantitative Funding information This study was funded by the Department of alterations regarding the yield of EPS by using different pure Science and Technology, Government of West Bengal (WB-DST). carbon-sourced substrates. Nitrogen sources or other inorgan- Compliance with ethical standards ic salt sources may also augment or inhibit such yield (Degeest et al. 1999; Rinker and Kelly 2000; Khani et al. Conflict of interest The authors declare that they have no conflict of 2016). So a judicious evaluation of a substrate to a bacterial interest. strain before a large-scale production of EPS often becomes imperative. Ethical approval This article does not contain any studies with human Thus, it appears no less important to incur a sound knowl- participants or animals performed by any of the authors. edge about the basic constituents of the bacterial culture media for maximizing the production of the desired EPS. 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