Isolation and Screening of bacteria
Isolation of bacterial colonies was made by enrichment culture technique (Khatoon et al. 2019) with some modifications in mineral salt media (Table 1:- composition of mineral salt media).
Table 1
Composition of mineral salt media
Composition | Gram/litre |
NH4NO3 | 2.0 g/l |
KH2PO4 | 2.0g/l |
Na2HPO4 | 3.0g/l |
0.5% NaCl | 1.5 g/l |
MgSO4 | 0.2 g/l |
Na2CO3 | 0.1 g/l |
CaCl2 | 0.01 g/l |
Mn SO4 | 0.02 g/l |
FeSO4 | 0.01 g/l |
Soil sample were collected from agricultural field of nearby area of CUJ, Brambe (Lat:-23.441966; Longitude:-85.151209). Soil samples were collected from depth of 0–10 cm of the soil surface and stored at 4°C for further analysis. pH of soil sample was determined by method of Fulthorpe et al.(1996). Slurry was made by vortexing 1 gram of soil in 5 ml of deionized water for 1 minute. Then pH was determined by pH meter. Soil sludge was prepared by adding 1% diesel, petrol and mustard oil respectively in 3 different vials at regular intervals for two months. One is left as without addition of oil as control. 5g of soil sludge sample was mixed with 100 ml of Mineral Salt Medium (MSM) with 3ml of oil added to the medium in conical flask having capacity of 250 ml, as the carbon sources and then incubated for 72 hours at 30°C. The sample was re-enriched for 24 hours in MSM media by transferring 5 ml culture broth into other 250 ml conical flask containing 100ml MSM. After serial dilution of enriched sample 100 µl aliquots of 10− 6 time dilution was spreaded on to nutrient agar plate by glass rod spreader.The plate was inverted and incubated at 37° C, for 72 hours. After incubation, morphologically distinct colonies were selected and purified for further studies. CFU was determined. Mineral salt media is used for enrichment culture and Nutrient agar slants were used for maintainance of pure culture. Pure culture was stored at 4oc in nutrient agar slants. Prior to the screening for biosurfactants, the isolates were inoculated into 10ml of nutrient broth medium each and then incubated at 37oC for 72 h shaking at 150 rpm, which were used as mother inoculums. Mineral salt minimal medium was prepared and pH of the medium was adjusted to 7.0 ± 0.2 by using pH meter. Carbon sources were added separately. All Media and Glassware were sterilized in autoclave at 121°C with 15 lbs pressure for 20 minutes. Bacterial pure Cultures were inoculated and incubated at 37°C, at shaking conditions for fixed period of time. After incubation period culture broths were centrifuged at 6000 revolutions per minute (r.p.m.) to eliminate the bacterial cells for 15 min at 4°C. The Cell free supernatant was collected and used for the various biosurfactant screening tests or assays comprise: E24 activity, oil displacement test, blood hemolysis test and drop collapse assay.
In oil displacement test, 20 ml of distilled water was poured to a plastic petriplates followed by addition of 0.5 ml of oil to the center of surface water. 0.1 ml of the cell free culture broth was then added to the oil surfaces.. If the oil displaces and clear zone forms then it shows the presence of biosurfactant. Formation of halo supports the presence of surfactants and their ability to displace
hydrophobic compounds (Morikawa et al. 2000).The displaced diameter is measured after 30 second (Rodrigues et al. 2006). De-ionized water and triton X-100 was used as negative and positive control respectively. Emulsification assay (Cooper and Goldenberg, 1987) was done by mixing 2ml kerosene oil with 2 ml supernatant, obtained after the centrifugation of bacterial culture, and vortexes for 5 minutes to confirm mixing of both the liquids. The emulsification activity was observed after 24 hours and it was calculated by using the formula (Barakat et al. 2017):
$$E24=\frac{\text{H}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{t}\text{h}\text{e} \text{e}\text{m}\text{u}\text{l}\text{s}\text{i}\text{f}\text{i}\text{e}\text{d} \text{l}\text{a}\text{y}\text{e}\text{r} \left(\text{c}\text{m}\right) }{\text{T}\text{o}\text{t}\text{a}\text{l} \text{h}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{t}\text{h}\text{e} \text{c}\text{o}\text{l}\text{u}\text{m}\text{n} \left(\text{c}\text{m}\right) } \times 100$$
The E24 index gives the measure of the strength as well as the quantity of the biosurfactant. The hemolytic assay for screening of biosurfactant production by bacteria was performed by method of
Youssef et al. (2004). The bacterium was streaked on blood agar plate and incubated for 24 h at 30°C, and then the hemolysis zone was observed around the spot. Drop Collapse assay was done by placing a drop of culture supernatant onto the surface of the parafilm strip, and the shape of the drop was observed. If the culture producing biosurfactant,interfacial tension was reduced between the parafilm strip and drop of supernatant and then the drops on the film would collapse. The uninocuated culture supernatant served as a negative control.
Characterization of bacterial isolates
Morphological and biochemical characterization of bacterial isolates has been done by analyzing colonial morphology, gram reactions, catalase production, MRVP reaction, indole production, starch hydrolysis, citrate utilization, lipid and gelatine hydrolysis. Molecular characterization has been done by 16S rRNA sequencing analysis and BLAST report has been generated. The bacterium was identified by molecular technique using sequence analysis of 16S ribosomal DNA gene. Genomic DNA was extracted by using HiPurA Bacterial DNA purification spin column kit (MB505-250PR, HiMedia, India) and checked on 1% agarose gel electrophoresis.F27 (5 ’AGAGTTTGATCMTGGCTCAG 3’) and 1492R (5’ GGTTACCTTGTT ACGACTT 3’) primers were used for PCR amplification(Jill E. Clarridge, III ,2004). PCR amplification was performed using Applied Biosystems Veriti Thermal Cycler as follows: denaturation at 94°C for 5 min followed by 34 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1.30 min and a final cycle at 72°C for 7 min. The 16S ribosomal DNA gene sequence was proofread and aligned with bacterial 16S rRNA database available at NCBI using BLAST to determine the similarity and identification of the isolated bacterium.
Growth kinetics of bacterial isolates
Growth curve has been prepared for selected isolates (≥ 40% BS production) for seven days of incubation period. Growth curve has been studied by following the methods of Zhou et al. (2015) with slight modifications. For growth kinetics study mother culture was prepared as by inoculating bacterial culture in 10 ml nutrient broth.2% of mother inoculum is inoculated in mineral salt media incubated at room temperature in shaking condition for 7 days. For growth curve analysis, cultures were taken out at regular intervals at 24 hours and optical density has been measured at 600 nm by UV spectrophotometer and screening of biosurfactant production has also been done.
Selection of parameters for optimization to get highest yield of biosurfactants
For optimization different factors have been choosen following the method of Bibi et al.(2018).The factors chosen for optimization to obtain higher productivity of the biosurfactants were media constituents such as: carbon source (C), nitrogen source (N) and pH. The first variable chosen for optimization process are carbon source with fixed compositions of other media constituents. The carbon sources selected were monosaccharides (glucose), disaccharide (sucrose), polysaccharide (starch) and other water soluble such as glycerol and water insoluble sources such as olive oil and PAH. The optimization experiments were set up in with five different carbon sources in incubator shaker for 3 days. 2% (v/v) concentration of carbon source was supplemented to the growth media. After incubation period each flasks were taken out, centrifuged and supernatant collected for further studies. Optical density of pellets is measured at 600nm.Screening of biosurfactant production has also been assessed with different carbon sources. The second parameter for optimization was nitrogen source. The minimal media already contained NH4NO3 as nitrogen source. To study the effect of other nitrogen sources NH4NO3 was replaced by NaNO3, peptone, yeast extract, NH4SO4 and urea. The culture media was supplemented with a single nitrogen source at a time and incubated in incubator shaker for 3 days. After incubation period each flasks were taken out, centrifuged and supernatant collected for further investigations. Optical density of pellets is measured at 600nm and screening for biosurfactant production has also been done. The range of pH selected for production of biosurfactants was from pH 5- pH 9. pH of the broth is adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0 in different flasks using 1NHCl and 1NNaOH and sterilized. Different sets of experiments were set up with different pH. The media with the culture were incubated at 37°C for fixed period of time. Biosurfactant produced with different pH was detected by the preliminary methods of detection described earlier. Optical density at 600 nm has been measured and screening for biosurfactant production has also been done.
Growth of isolates at various concentration of anthracene (polyaromatic hydrocarbon)
MSM media was prepared and autoclaved, after that different concentration of anthracene as 0, 0.5, 1,2,3,5 and 10 percent are added as a carbon sources and inoculated with mother inoculums. After 3 days, growth of isolates at different concentration of anthracene has been measured spectrophotometerically at 600nm.
Statistical analysis
The assays were conducted in triplicates (n = 3) and the mean value of those three parallel investigations was calculated. The result was then presented as the mean ± standard deviation (± SD) of triplicates using Prism Graph Pad 5.0.0 statistical analysis software. The differences among the factors were assessed using Two-way ANOVA. P-value less than 0.0001 were regarded as statistically significant. The average of emulsification activity and biomass values was calculated and expressed as mean standard deviation.
The 16s rRNA sequence of isolated bacterial strains has been submitted to NCBI database under the accession number PP434501, PP434812, PP436389 for PS1,DS2 and MS4 respectively.