Screening and characterization of polymer producing strains:
In the present study 20 strains were isolated from the leaves of the plants that were blighted and produced yellow pigment demonstrating the occurrence of the species of the family Xanthomonadaceae. All the isolated plant pathogens were able to produce viscous polymer on nutrient agar and production media. However, only the yellow pigment producers were selected and recultured as Xanthomonadaceae family produce yellow pigmentation on nutrient agar and growth media (Borges et al., 2008, Candia & Deckwer, 2008, Freitas, et al., 2009, Kumar, et al., 2018). Among the screened cultures, strain X2 showed the highest production and was selected for biopolymer production optimization studies (Fig. 1A).
Biochemical characterization:
Through phenotypic profiling and biochemical characterization, 8 strains were found to be gram negative and showed quite similar characteristics and were studied further for biochemical test analysis. However, some of the strains were slightly oxidase positive and H2S negative unlikely as compared with the past literature of strains belonging to Xanthomonadaceas (Naqvi et al. 2012 and Arshad et al. 2013).
Table 2
Biochemical characterization of biopolymer producing plant pathogens
Strain
|
Strenotrophomonas maltophilia strain X2
|
Bacillus cereus strain X4
|
Strenotrophomonas maltophilia strain X7
|
Alcaligenes faecalis strain X10
|
Strenotrophomonas sp. strain X12
|
Bacillus cereus strain X14
|
Bacillus sp. strain X15
|
Strenotrophomonas maltophilia strain X22
|
Genbank Accession
|
MK422148.1
|
MK775363
|
MK775463
|
MK775526
|
MK775529
|
MK780189
|
MK780182
|
MK782051
|
Gram's reaction
|
–
|
–
|
–
|
–
|
–
|
–
|
–
|
–
|
Citrate utilization
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
Catalase
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
Glucose
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
Lactose
|
–
|
–
|
+
|
+
|
+
|
+
|
+
|
+
|
Maltose
|
+
|
–
|
+
|
+
|
+
|
+
|
+
|
+
|
Starch hydrolysis
|
+
|
+
|
+
|
+
|
+
|
+
|
-
|
+
|
Tween 80 hydrolysis
|
+
|
+
|
–
|
–
|
+
|
-
|
+
|
–
|
Urea utilization
|
+
|
+
|
–
|
–
|
–
|
+
|
+
|
–
|
Nitrate reduction
|
–
|
+
|
+
|
–
|
+
|
+
|
+
|
–
|
H2S production
|
–
|
–
|
–
|
–
|
–
|
-
|
-
|
–
|
Indole
|
–
|
–
|
–
|
–
|
–
|
+
|
+
|
–
|
Motility
|
+
|
+
|
+
|
+
|
+
|
-
|
-
|
–
|
MR
|
–
|
–
|
–
|
–
|
–
|
+
|
+
|
+
|
VP
|
–
|
–
|
–
|
+
|
–
|
-
|
-
|
–
|
KOH
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
Bioinformatics analysis:
All the strains were subjected to ribotyping and found to be belonging to different genera of bacterium (Table 2). Owing to high biopolymer production, Bacterial strain X2 was selected for further study on biopolymer production parameters rest of all strains were glycerol preserved for future studies. The strain X2 was characterized as Stenotrophomonas maltophilia belonging to family Xanthomonadaceae. 16S rDNA nucleotide sequence was compared with other related species sequences by using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). It showed that the sequence is novel and similar to Stenotrophomonas maltophilia after multiple alignment. Nucleotide sequences of all strains were deposited in GenBank database and received accession number as listed in Table 2. This sequence of Stenotrophomonas maltophilia X2 (MK422148) was further analyzed by phylogenetic tree construction (Fig. 3).
The S. maltophilia strain X2 is genetically related to the Stenotrophomonas sp. strain X22. Basically, the Stenotrophomonas sp. strain X22 is genetically subdivided into two phylogenic nodes (categories), one of which gives rise to the strain x12. And the other node gives rise to the clad that bears S. maltophilia strain X7 and S. maltophilia strain X9, which are genetically close to Stenotrophomonas sp. strain X12.
The other node (15) of Stenotrophomonas sp. strain X22 gives rise to the strain S. maltophilia strain X2, that are further subdivided to the strain A. faecalis strain x10, B. cereus strain X4, Bacillus sp. strain X15 and B. thuringienesis strain X14, on the basis of small genetic differences. In these sub divisions, Bacillus sp. strain X15 and B. thuringienesis strain X14 are likely similar to each other. Whereas, B. thuringienesis strain X14 and Bacillus sp. strain X15 are likely similar to each other. The ancestor of strain A. faecalis strain x10 is likely similar to strain S. maltophilia strain X2. On the other hand, B. cereus strain X4 is similar to the ancestor of Bacillus sp. strain X15 and B. thuringienesis strain X14.
Members of this species are known to display high genetic, ecological and phenotypic diversity, forming the so-called S. maltophilia complex (Smc). Heterogeneous resistance and virulence phenotypes have been reported for environmental Smc isolates of diverse ecological origin. According to the past literature regarding the phylogenic analysis of stenotrophomonas maltophilia, its genome is reported to have certain genetic matches with some species of bacillus and Geobacillus stearothermophilus strain KCB 2.
The evolutionary timeline of the Stenotrophomonas maltophilia X2 can be seen in Fig. 4 which reveals its presence in several ages and its biological features, including the fact that, this strain was first found 1733 MYA (Million Years Ago). The presence of this bacteria covered several geological periods comprising on, meso-proterozoic, neo-proterozoic, paleozoic and mesozoic periods. These geological timescales showed a great variation in the levels of oxygen, carbon dioxide and solar luminosity, that were acceptable for the growth of Stenotrophomonas maltophilia
Optimization of Fermentation parameters
Stenotrophomonas maltophilia X2 was studied for production and biopolymer recovery parameters. Physicochemical parameters such as incubation time, temperature, pH, shaking/static and substrate concentration were analyzed one at a time. Each parameter was performed in a triplicate set.
Effect of incubation time
Incubation time is an important variable for biopolymer production. By varying the incubation period from 24 hrs to 192 hrs, it was found that at 120 hrs X2 yield highest biopolymer production and then gradual decrease was observed (Fig. 5). In the initial hours, the nutrients are expected to be maximally utilized for biomass increment; the polymer production being the secondary metabolite increased in later phases on incubation. After 120 hrs, the polymer production was found to be decreased which could be attributed to utilization of polymer as sugar reserve by the bacterium (Candia & Deckwer, 2008).
Effect of sucrose concentration
Sucrose was used as carbon source in the media as reported in literature (Zohra et al., 2008). To investigate the appropriate amount of sucrose required to produce highest yield of xanthan, different concentrations were checked i.e. 3%, 5%, 7% and 9% respectively. The best biopolymer production by X2 was found at 5% and it was observed that the growth and biopolymer production started to restrict after 5% (Fig. 6). In the past literature sugar concentration higher than 6% had negative influence on xanthan production and viscosity and 2–4% of carbon source is preferred as high concentration of sucrose inhibits the bacterial growth (Caroline & Claire, 2007, Swamy et al., 2012, Arshad et al., 2015)
Effect of pH
Fermentation process for biopolymer is slightly acidic because of the metabolism of nitrogen and sugar sources which produce various acids as end product (Zohra et al., 2008). The growth of cells is dependent on pH, hence affecting the biopolymer production (Caroline & Claire, 2007). The pH 7 was found to be sufficient for biomass and biopolymer production (Fig. 7). However, the pH below and above 7 were insufficient for the biomass and biopolymer production rate (Letisse et al., 2001, Bajaj et al., 2007, Kerdsup et al., 2009, Gumus et al., 2010, Soudi et al., 2011, Ozdal & Kurbanoglu, 2018).
Effect of agitation of fermentation media on biopolymer production
It was concluded that, the bacterial cells were affected by mechanical agitation negatively and were best grown at static condition. The static incubation conditions for biomass and biopolymer production were found to be sufficient (Fig. 8). However, according to past literature biomass and biopolymer production increases within agitation fermentation condition (Freitas, et al., 2009). While in this study the agitation fermentation condition showed inverse relationship with the xanthan production using strain X2 (Caroline & Claire, 2007, Soudi et al., 2011, Swamy et al., 2012).
Effect of temperature
The effect of temperature on biopolymer yield and growth of the organism were studied by optimizing the temperature between the ranges 25°C to 35°C.Ideal temperature for biopolymer production from the results obtained was 30°C at which both the biomass and polymer yields were highest (Fig. 9). These results were in agreement with the past literature (Candia & Deckwer, 2008, Gumus et al., 2010, Shu & Yang, 2010).
Downstream processing of biopolymer:
Biopolymer produced by the strain in response to supplied nutrients and fermentation conditions can be recovered from various techniques like ultrafiltration, precipitating agent, and enzyme treatment. Because of the high viscosity of fermented medium it is difficult to separate xanthan gum from unwanted constituents. High viscosity of fermented medium leads to degradation of polysaccharide during centrifugation (Suresh & Prasad., 2005). To be use as food additives it is necessary for xanthan gum to be free of biomass and recovery agents. Including separation of unwanted debris, it is also important not to effect the properties of xanthan gum, which can be effected adversely by heat treatment processes (Garcia, et al., 20000). It is a standard procedure for xanthan gum recovery to precipitate out polysaccharide using precipitating agent such as isoprapanol, isobutanol (Nasr, et al., 2007). Other oragnic solvents can also be used like methanol, ethanol, t-butanol and acetone (Palaniraj & Jayaraman., 2011). Salts can also be used as precipitating agent in polyvalent forms(Pace & Righelato, 1981).
Biopolymer produced by X2 strain was recovered using microbial cell separation and alcohol precipitation technique. After removing the cells by centrifigutaion, chilled ethanol was added into the cell free fluid (CFF) in an optimized ratio of 3:1 (ethanol:CFF). The mixture was then kept at -18-20°C for efficient recovery of polymer.
Centrifugation and drying the pellet in dry heat oven separated the polymer from the cell free fluid + alcohol suspension. Figure 10 shows the recovered polymer. It was observed that this scheme of polymer purification led to 40% recovery of the polymer from the fermentation broth.