Isolation and screening of bacterial isolates
Initially, D-serine-producing microbes were isolated on an M9 minimal salt medium containing L-serine as a nitrogen source. In Total 9 potent different colonies (given as b-Ca2C3, c-Rm2C2, d-Rm2C3, e-A1C1, f-A1C2, g-Ca1C1, h-Ca1C2, i- G1, j-K1) (Fig S1) were isolated from different sites (Table S1) in the period between August 2018-October 2018. The average high/low temperature was recorded for Bilaspur 100°F/75.2°F, Hamirpur 98.6°F/75.2°F, Solan 69.8°F/62.6°F, Mohali 93.2°F/75.2°F, Srinagar 86°F/48.2°F. Among, all the isolates A1C1 showed maximum activity (O.D600 – 1.97±0.4x109 cells/ml) even after 48h of incubation at 37±0.5℃ (Fig.1). The assay was performed in triplicates (n = 3).
Hemolysis of blood cells
The pathogenic behaviour of the isolated strain was performed on the test medium supplemented with human blood 5% v/v and confirmed by the hemolysis of RBCs. Bacterial isolate was observed for 5 days of incubation at 37±0.5℃. No significant changes were observed during the incubation time.
Molecular, biochemical, and morphological identification
The 16s rRNA sequence analysis of the isolate A1C1 confirmed its close relationship to Bacillus tequilensis. BLAST analysis was conducted for a sequence of 1400bp length which showed a 99.93% similarity index to Bacillus tequilensis KCTC 13622(T) (accession no. AYTO01000043). The next closest homolog was Bacillus cabrales TE3(T) (accession no. MK462260) similarity index 99.86% and Bacillus inaquosorum KCTC 13429(T) (accession no. AMXN01000021) similarity index 99.86% (Table S2). To infer the evolutionary relationship between nearby homologs, a phylogenetic tree was also constructed which showed the sister descendant i.e., Bacillus tequilensis KCTC 13622(T) (accession no. AYTO01000043) Fig. 2.
Biochemical and physiological properties of A1C1 isolated from the soil of a pomace dumping site in Solan, H.P were also identified and summarised in Table 1. The temperature assay showed growth of the isolated bacteria on a temperature range from 20-50℃ and optimum growth observed at 30-40℃. The pH assay showed growth ranges from 4-10pH and optimum growth observed at 6-7pH, at 150 RPM. The isolated strain was recorded positive for the triple sugar iron test but no black precipitate was observed. A1C1 was found motile and able to ferment lactose, indole production, and grow in MRS broth. A1C1 was reported negative for starch hydrolysis, urease production, MR-VP test, citrate utilization, and cellulose hydrolysis. The isolated strain was also compared with standard strain and other reported Bacillus tequilensis strains. A1C1 showed maximum similarity to the standard strain Bacillus tequilensis 10bT.
Morphological characteristics of A1C1 are shown in Fig.3. The isolated strain was Gram-positive and tend to form spores. A1C1 was also viewed under Scanning electron microscopy (SEM) and observed as columnar. The cultured colonies were large, spreading, and irregular in shape. Combined with all the morphological, biochemical, and molecular characterization, A1C1 was designated as Bacillus tequilensis. In addition, 16S rRNA partial sequence was submitted to the GenBank with accession no. MZ337537 as Bacillus tequilensis A1C1 (Bacillus tequilensis strain A1C1 16S ribosomal RNA gene, partial sequence - Nucleotide - NCBI (nih.gov)).
Determination of enzymes and metabolites
The isolated strain showed hydrolysis in selected mediums depicted in Fig. 4. The hydrolysing potential of the strain was observed for up to 5days at 37±0.5℃. A1C1 was growing well on all the mediums but displayed a clear zone of hydrolysis surrounding the colony only in protease and gelatine medium. Which indicated A1C1 could secrete proteolytic and gelatine hydrolysing enzymes. A zone of hydrolysis has appeared after 14h of incubation at 37±0.5℃. A1C1 was unable to hydrolyse cellulose, pectin, phosphate, and IAA production.
Active secondary metabolites and cell wall degrading enzymes are present in many biological control agents. As a result, the isolated strain was also investigated for its potential to be used in the synthesis of antimicrobial agents. Bacterial cellular fractions were used to evaluate the antimicrobial potential of A1C1. The extracellular fraction was found to be more active against MTCC 40 (4.6±0.5 mm) and MTCC 11949 (2.3±0.5 mm) as compared to the intracellular fraction and is summarised in Table S3. The zone of inhibition of both the fraction is shown in Fig 5.
The zone of inhibition was observed 5mm against Escherichia coli (MTCC 40). The zone of inhibition was observed 2mm against Staphylococcus aureus (MTCC 11949). Further, from the visible clear zone, the sample was taken and streaked on another agar plate which showed that the extracellular fraction had bacteriostatic activity. Efficacy of the extracellular fraction was also compared with the standards and values enumerated in percentage (%). From the experiment, it was observed that against MTCC 40, the efficacy values of the test samples were given as 18% for ampicillin, 17% for penicillin, and 5% for the bacterial extracellular fraction. For MTCC 11949, the efficacy values of the test samples were given as 15% for ampicillin, 14% for penicillin, and 2% for a bacterial extracellular fraction.
Chromatographic analysis of the enantiomers
The method described here for TLC was applied successfully to separate the amino acids present in both the cellular fractions. The coloured spots obtained after spraying ninhydrin solution and the RF value of each spot are calculated and shown in Fig 6. The solvent system was designed carefully to maintain the pH and to separate polar amino acids. For TLC, L-serine was used as the reference standard and developed a purple-pink chromatographic band with RF value 0.51±0.015.
The intracellular fraction was separated into three different chromatographic bands b1(Rf -0.55±0.005), b2 (Rf -0.62±0.010), and b3 (Rf -0.73±0.017). The extracellular fraction was separated into three chromatographic bands c1(Rf -0.48±0.015), c2 (Rf -0.62±0.005), and c3 (Rf-0.65±0.011). Chromatographic bands b1 and c1 showed Rf values near to the standard l-serine chromatographic band. For HPLC analysis methodology described by Kubota et al., 2016 was adopted and used after modifications insolvent system. The obtained chromatograms were shown in Fig. 7.
Under the optimal reaction conditions, a linear calibration curve (peak area versus concentration) (Fig. 8) was obtained over a concentration range of 5nM to 30nM (Table S4) of the standard D-Serine containing 5, 10, 20, 30nM. The correlation coefficient was found to be 0.9895. The standards for the linearity curve were prepared by adding the derivatizing agent to the std. the solution as discussed above in methodology.
The bacterial sample was analyzed using the above-mentioned HPLC method. The quantification of the target analyte (D-Serine) was done using the linear curve equation (y=mx+c). The concentration was found out to be 0.919±0.02 nM. Further, the validation of the obtained results has been carried out by performing the recovery test. For this, the analyzed sample with the known concentration of D-Serine was spiked with different concentrations of standards i.e., 5nmol, 8nmol, and 10 nmol. The chromatogram for the spiked sample of D-serine by adding 8 and 10nM is given in Fig. S2. The obtained results were tabulated in Table 2. The recovery of the spiked samples varies from 85 to 90%.