Effect of carbon and nitrogen sources on exopolysacharide production by rhizobial isolates from root nodules of Vigna trilobata

Twenty five (25) rhizobial strains were isolated from root nodules of Vigna trilobata cultivars grew in soils collected from all districts of Andhra Pradesh, India. Five out of 25 rhizobial strains which produced copious amount of Exopolysaccharides (EPS) on Yeast Extract Mannitol Agar (YMA) medium with congo red were identified by sequencing of their 16S rDNAs. The amount of EPS produced by these five strains increased during the first 72 h of incubation but it declined afterwards. The amount of EPS produced correlated positively with increase in mannitol concentration from 1 to 3% (m/v). However, there was a decrease in EPS production when mannitol concentration was equal or higher than 4%. All the five strains studied preferred mannitol and sodium nitrate as the best carbon and nitrogen sources for EPS production. Sinorhizobium kostiense MRR104 produced maximum levels of EPS 892 mg/100 ml when mannitol was used as carbon source, while S. xinjiangense MRR110 produced maximum levels of EPS 377 mg/100 ml when sodium nitrate was used as nitrogen source. Variation among the rhizobial strains in utilization of carbon and nitrogen sources for EPS production was clearly evident from this study. All the strains analysed in the present study can be exploited for production of commercial biopolymer (gum), due to their non pathogenic nature and their ability to produce copious amounts of EPS when compared to strains studied in earlier reports.


INTRODUCTION
Rhizobium spp.are known to synthesize a variety of cell surface polysaccharides, including exopolysaccharides (EPS), lipopolysaccharides (LPS), and cyclic glucans (CG).In addition, Sinorhizobium spp.produces K-antigen polysaccharides (KPS).All these four polysaccharides are important for bacterial performance in both free life and symbiosis (Margaret et al., 2011).Rhizobial exopolysaccharides (EPS) play an important role in plant root invasion during nodule formation and promotes growth of the plants by chelating various metal ions.EPS helps in creation of near anaerobic conditions in the microenvironment surrounding the rhizobial cell surface, before infecting the root hair, to protect the nitrogenase enzyme (Gupta et al., 1982) and enhances nodulation (Olivares et al., 1984).EPS protects the producing organism against desiccation (Sayyed et al., 2011) toxic compounds, and osmotic stress, helps in the formation of biofilms (Kucuk and Kivanc, 2009) and serves as energy source to be catabolised during nutrient deficiency.Apart from its role in symbiosis and plant growth, the EPS synthesized in culture have importance in many industries including food, oil and pharmacy.Microbial EPS have been commercialized as possible future industrial commodities for food as thickening agents and in agriculture for the encapsulation of somatic embryos, which offer a greater feasibility for precise delivery of plant growth regulators, fungicides and pesticides (Mathur and Mathur, 2001).EPS has been proved to be a potential biopolymer used as emulsifier in the degradation of hydrocarbons, stabilizers, binders, coagulants and separating agents in a variety of industries (Staudt et al., 2011).Bacterial EPS exhibit antioxidant activity (Mahendran et al., 2013).Recently rhizobial EPS were patented as best bioproduct for skin treatment (US Patent 20,130, 302, 261-2013).EPS produced by bacteria are commercially exploited so far, however, much of the present day research is concentrating on the industrial application of rhizobial EPS.Rhizobial biopolymers are preferred in the food industry by virtue of their non pathogenic nature and copious production (Bomfeti et al., 2011).Rhizobial EPS are species or strain specific hetero polysaccharides consisting of repeating units of sugars (Mandal et al., 2007) the majority of which are hexoses and uronic acids as well as non carbohydrate substituents such as acetate, pyruvate, hydroxyl butyrate and succinate (Aman et al., 1981;Cunningham and Munns, 1984).However, the most common types of EPS produced by Rhizobia are EPS1 and EPS2.EPS1 type are high molecular weight succinoglycans and EPS II are low molecular weight galactoglucans (Oliveira et al., 2012).Several factors influence the production of EPS by rhizobial strains, such as carbon and nitrogen sources as well as incubation period (Duta et al., 2004(Duta et al., , 2006)).
Vigna trilobata commonly called Pillipesara, was mainly cultivated as short term pasture and green manure crop in India, Pakistan, Indonesia and Sudan.Though nodulation in Vigna trilobata was first reported in Japan by Asia and in India by Raju in 1936 (Allen and Allen, 1981) the comprehensive studies on rhizobial symbiont characterization and other related studies are meagre.The present investigation was aimed to study the diversity of rhizobial strains producing exopolysaccharide.Although much work was published on EPS production, characterization and factors effecting EPS production by rhizobial strains from different host legumes, our study is the first of its kind on rhizobial strains isolated from nodules of V. trilobata.In this paper, for the first time, rhizobial species nodulating V. trilobata have been identified by 16S rDNA sequencing.

Isolation of rhizobia
Soil samples were collected from agricultural fields under the cultivation of V. trilobata from all the 25 districts of Andhra Pradesh.Certified seeds of V. trilobata were purchased from the National Seed Corporation (NSC) Guntur.Plants were grown in earthen pots filled with these district soils and were maintained properly in the Botanical garden of Acharya Nagarjuna University.After 90 days of germination, healthy root nodules from gently uprooted plants, surface sterilized with 0.1% mercuric chloride and 70% alcohol and washed thoroughly by sterile distilled water were used for isolation (Vincent, 1970).Rhizobial strains were isolated from root nodules of V. trilobata plants, using selective medium Yeast Extract Mannitol Agar (YMA) with congo red and pure cultures were maintained after sub culturing on the same medium.Pure cultures of all the 25 isolates were authenticated as rhizobia by performing the appropriate biochemical tests (Somasegaran and Hoben, 1994) and nodulation ability on homologous hosts by plant infection tests (Vincent, 1970).Out of the 25, the five strains which produced higher amounts of EPS were further identified up to the species level through 16S rDNA sequencing (Macrogen, South Korea) and the sequences were deposited in the gene bank.The strain names with allotted accession numbers, used in this study are Rhizobium sp.MRR103-JX576499 (isolated from Guntur district soil); Sinorhizobium kostiense MRR104 -KC428653 (isolated from Chittor district soil); Sinorhizobium xinjiangenseMRR110 -KC415691 (isola-ted from Kadapa district soil); Rhizobium sp.MRR 123 -KC503884 (isolated from Nellore district soil); Ensifer sp.MRR125 -KC503885 (isolated from Mahaboobnagar district soil).

Exopolysaccharide (EPS) production
For production of EPS, all the five strains were inoculated into Erlenmeyer flasks (250 ml) containing 100 ml of YMB supplemented with 1% Mannitol (m/v).The flasks were incubated at room temperature on an orbital shaker at 200 rpm for 72 h.After incubation, the broth was centrifuged at 3000 x g and the culture supernatant was mixed with 2 volumes of chilled acetone.The crude polysaccharide precipitated was collected by centrifugation at 3000 x g for 30 min.The EPS was washed with distilled water and acetone alternately, transferred into a filter paper and weighed after overnight drying at 105C (Damery and Alexander, 1969).The strain that produced maximum amount of EPS, S. kostiense MRR104 was used in the studies on optimization of the conditions for maximum EPS production, like the incubation period and the concentration of mannitol (carbon source in the YMA medium).

Effect of mannitol concentration
To optimize the concentration of Mannitol for maximum production of EPS production, the test cultures were inoculated with five different concentrations of Mannitol (1, 2, 3, 4 and 5 %) including the concentration prescribed in the original YMB medium (1%).All the inoculated flasks were incubated for 72 h at room temperature on an orbital shaker at 200 rpm and the amount of EPS produced was estimated by the method described by Damery and Alexander (1969).

Effect of incubation period
For the study of EPS production at different incubation periods, the test culture was inoculated into YMB medium (1% mannitol) and incubated at different periods from 12 to 84 h with 12 h intervals.The  Datta and Basu (1999a) described that EPS production by Rhizobium sp.isolated from C. cajan reached its maximum at 4% (m/v) of mannitol with an incubation period of only 65 h.All the five rhizobial strains utilized the 10 different carbon sources and produced significantly high amount of EPS than when a control medium without carbon source was employed (Table 1).Though all the carbon sources supported EPS production, maximum production was observed when sugar alcohol -mannitol was used as carbon source indicating that mannitol supported maximum EPS production by all the strains of V. trilobata.This is in agreement with previous reports by Ghosh et al. (2005a) in Rhizobium sp. from Dalbergia lanceolaria, Kucuk and Kivanc (2009) in R. ciceriRc5 and Mandal et al. (2007) in Rhizobium sp. from Vigna mungo.However, it has been also described that other rhizobial species show maximum EPS production using different carbon sources.Glucose was preferred as carbon source for maximum EPS production by rhizobial sp.from Crotalaria saltiana (Mukherjee et al., 2011) and rhizobial sp.from Melilotus alba (Datta and Basu,1999b) while xylose was preferred by isolates from C. cajan (Fernandes et al., 2011) and galactose by Rhizobium sp.SS5 from Sesbania sesban (Sridevi and Mallaiah, 2007).Ghosh et al. (2011) reported that sucrose (1.5%) induced the maxi-mum EPS production though maximum growth was observed with mannitol by Rhizobium sp. from Phaseolus mungo.Among the monosaccharides tested, after manni-tol, glucose was preferred by most of the strains in the present study while Nirmala et al. (2011) reported that sucrose was preferred next to mannitol by Rhizobium sp. from V. mungo.Among the disaccharides used, maltose was preferred next to glucose by MRR 104, MRR 123 and MRR 125 strains.From the present study, it is evident that rhizobial strains isolated from V. trilobata preferred sugar alcohol, mannitol for maximum EPS production and gave second preference to monosac-charide and less to the disaccharide carbon sources.Among the five strains studied, S. kostiense MRR104 produced maximum EPS of 892 mg/100 ml followed by Rhizobium sp.MRR 123 with 816 mg/100 ml.

Fig
Variation in EPS production by Aschenomenon aspera isolates when different carbon sources used was previously reported by Ghosh et al. (2005b).Similarly, significant variations in the amount of EPS produced among the different carbon sources, among the strains and also within the strains were recorded in the present study with the strains of V. trilobata.
Statistically there were significant differences between the carbon sources used and rhizobial strains in the production of EPS (p = <0.05).The Duncan test reveals that the highest EPS production occurred for S. kostiense MRR104 when mannitol was used as carbon source.
Effect of different nitrogen sources was studied by replacing 0.1% Yeast extract of the original YMA medium with five different nitrogen sources.All the strains studied efficiently utilized those different nitrogen sources and produced high amount of EPS over the control (Table 2).This clearly shows the significant role played by the nitrogen source on EPS production.Sodium nitrate was preferred for maximum production of EPS followed by potassium nitrate by all the strains studied.Similar results were reported previously by Kucuk and Kivanc (2009) in R. ciceri, Sridevi and Mallaiah, (2007) in Rhizobium sp.SS5 from Sesbania sesban.In contrast, potassium nitrate was preferred by Rhizobium sp. from V. mungo (Nirmala et al., 2011), rhizobial sp.from Melilotus alba (Datta and Basu, 1999b) and Rhizobium sp. from Dalbergia lanceolaria (Ghosh et al., 2005a).
The maximum EPS production of 377 mg/100 ml was recorded with strain S. xinjiangense MRR110 followed by 325 mg/100 ml by Ensifer sp.MRR125.Significant differences (P = <0.05) between the nitrogen sources on EPS production were recorded in the present study.The Duncan's test reveals that strain S. xinjiangense MRR110 produced maximun EPS when sodium nitrate was used as nitrogen source.Glycine and L-asparagine were less preferred by the strains in the present study.In contrast, Rhizobium sp. from Phaseolus mungo preferred glycine (Ghosh et al., 2011) and Rhizobium sp. from Vigna mungo preferred L-asparagine (Mandal et al., 2007) for maximum production of EPS.In the present study, S. xinjiangense MRR110 proved to be a more efficient EPS producer than those described in earlier reports of Sinorhizobium TR1 from Trigonella foenum-graecum (Tank and Saraf, 2003) which produced only 20 µgml -1 .Thus, the fact that rhizobial strains exhibit high variations in utilization of carbon and nitrogen sources for the production of EPS was proved among the strains isolated from V. trilobata cultivars by the present study.This variation can be attributed to the strain adaptability to the different environmental conditions that prevailed in the soil at geographically different areas from which they were isolated.From the present investigation, it is evident that the rhizobial strains from V. trilobata with high EPS production definitely could be added to the list of very few rhizobial strains -Mesorhizobium pluriforuium BR3804 with 1.44 g/l and Rhizobium tropici CIAT899 with 3.0 g/l EPS production, which were identified as most suitable for commercial production of gum (Bomfeti et al., 2011).

Table 2 .
EPS (mg/100 ml) produced by rhizobial strains in YMB supplemented with different nitrogen sources.