Optimization and Molecular Characterization of Lipase Producing Bacillus subtilis strain Rcps3 and Bacillus fumarioli Strain Rcps4 from Oil-contaminated Soils of Warangal

Microbial sources are regularly used as reliable biocatalysts sources which are often used in the process and production industry. Demands for such organisms with greater capacity of intended enzyme production are on the rise. Lipase is important enzyme used in the biotechnological process of hydrolysis of fats in almost all the relevant industries We have utilized the local oil-contaminated soil resources to search for efficacious bacterial strains that have excellent lipase activity. We were successful in identifying two such bacterial sources, namely, Bacillus subtilis strain RCPs3 and Bacillus fumarioli strain RCPS4, responsible for lipase production from oil effluent contaminated soil of Telangana. This is the first report of these two strains from this part of India that are involved in lipase production. The strains were isolated, optimized, and purified using standard microbiology protocols and were characterized at the molecular level using the biomarker 16s ribosomal RNA genes of the strains. The identified and isolated bacterial strains were confirmed as Bacillus subtilis strain RCPs3, and Bacillus fumarioli strain RCPS4 through molecular and computational characterization.

number (EC number) of EC 3.1.1.3 5 . Sustainability, green production, and application of lipases can induce the lipase enzyme production further from the microbial sources 6 . The global food and feed industry enzyme market has usage of almost 10% lipase for various purposes 7 which is having almost 8% annual growth 7 . Thus, lipase sources, production are the present industry requirement 8 . Searching novel enzyme sources will always aid in the further development of the industry that extensively uses lipase as a biocatalyst 8 . Several other literature sources have extensively documented the industrial need, applications, and effectiveness of using lipase [9][10] .
Urban waste management has become another challenge for our societies. The evergrowing population in the urban area, poor sanitation, lack of planning, and limited resources are fueling the poor management of urban waste. In such conditions, utilization of waste resources, especially for industrial production could serve as a great solution. Therefore, effluents, specifically oil contaminated waste, and waste volatile substances, could be an excellent source that can be utilized 11 . Different waste sources and bacterial origins have been used for better production of lipases, such as agricultural industrial waste and Aspergillus niger 12 . Scaling-up of the lipase production to meet the industry requirements is another important aspect that should be addressed with different technical and feasible approaches. Proper media sources may serve as a solution 13 . Solid fermentation technology and fungal sources have been also utilized for better lipase enzyme production 14 . Therefore, attention should be given to the isolation process, purification, and characterization of the enzyme production [15][16] . Presently, scientific research and investigations in this direction have turned into interdisciplinary and better outcomes are being drawn from computational approaches, chemical engineering, application microbiology and biotechnology, genetic engineering, and other disciplines.
In the present study, we have isolated, optimized, and characterized lipase enzymes from thermophilic Bacillus subtillis and Bacillus fumarioli that were collected from oil-contaminated soils of Warangal district.

Sample collection
Different oil-contaminated regions around the Warangal district were surveyed and various soil samples were collected that were contaminated with oil effluents.

Isolation of lipase producing bacteria
Isolation of lipase producing bacteria was done by the spread plate method using the serial dilution process on the nutrient agar medium. Bacterial colonies were grown and isolated by streaking over the Tributyrin and Rhodamine-B agar medium. Halos formed around the hydrolytic zones and the bacterial colonies with positive results were identified and separated. Such colonies were subcultured again for future preservation and use. The positive colonies were subcultured in the TBA (Tributyrin agar medium) medium in test tubes by preparing slants. These slants were inoculated with positive colonies and incubated for 24 hours. After incubation these slants were preserved at 4°C for further experiements.

Screening of bacterial strains
Primary screening of the bacterial strains was done by the method suggested by Limpon et al. (2006) 17 . Following the screening of the bacterial strains, the lipolytic activity of the isolated strains was assessed.

Lipase assay
The lipase activity was measured by the titrimetric method 18 . The isolated strains that showed positive results for lipase production, were further isolated for the production process.
Inoculum medium was prepared using 10 gm peptone, and 10 gm sodium acetate (CH 3 COONa). Other component used were 3H 2 O,10 gm yeast extract, 20 gm glucose, 0.03 gm manganese sulfate (MnSO 4 ), 0.09 gm magnesium sulfate (MgSO 4 ), copper sulfate 1.5 gm (CuSO 4 ), 0.5 gm Potassium chloride (KCl), 5 ml Olive oil, and 1000 ml of distilled water. The pH was set to 10.8. Following the stock preparation, 15 ml of prepared inoculum was used for the inoculation of the isolated culture. Later, the inoculated flasks were incubated in the rotary shaker overnight at 120 rpm and 37°C temperature.
The production medium was prepared using 0.24 gm MgSO 4 , 2.5 gm Sodium chloride (NaCl),1 gm Ammonium dihydrogen phosphate (NH 4 H 2 PO 4 ), 2 gm Peptone,0.24 gm Calcium dichloride hydrate (CaCl 2 H 2 O), Olive oil 12 ml, and 2-3 drops of tween 20 for every 1000 ml of water. Further, 1 ml of the inoculated culture was added to the Erlenmeyer flasks containing 100 ml of production medium and incubated for 24 hours on a rotary shaker at 120 rpm, and the temperature was maintained at 37°C (temperature for wild type) 19 .
For every 24 hours, the inspection was done for consecutive 5 days. Every day, 1 ml of culture from the production medium was centrifuged at 8000 rpm for 60 mins. The centrifuged supernatant of cell-free culture was used as the crude enzyme source of lipase. The lipolytic activity was determined by the titrimetric method mentioned earlier.

Lipase activity
The lipase activity was measured by the titrimetric method using olive oil as substrate. To 1 ml of culture supernatant, 1ml of olive oil and 2 ml of phosphate buffer was added and incubated for 60 mins at 37°C temperature. After completion of incubation, the reaction was halted by adding 1 ml of acetone-ethanol solution that was prepared in 1:1 proportion. Later, the amount of acid released was determined by titration with 0.05M sodium hydroxide (NaOH) at pH 7.0 and the temperature was maintained at 30°C.

Assessment of Carbon sources
Different types of sugars were added as carbon sources and were optimized. Sugars such as Lactose, Maltose, and Dextrose hindered the lipase production, while starch enhanced of the lipase production. The best carbon source was identified as starch for lipase production in the present study. In many cases, reduction in the production of lipase was noted in the presence of sugars that were used as carbon sources, this might have happened due to catabolite repression as the carbon sources were already available in the medium.

Nitrogen sources
The effect of various organic and inorganic nitrogen sources was examined for better lipase production. Organic sources that were used were tryptone, yeast extract, beef extract, protease, and peptone. Considered inorganic nitrogen sources were ammonium nitrate and calcium nitrate. These nitrogen sources were added to the final concentration of 1% (w/v) to the medium and incubated for 24 hours in a rotary shaker of 150 rpm at 36°C. effect of temperature, ph, and agitation on the lipase production The effect of temperature, pH, and agitation was examined for lipase production by altering the pH of the medium between 05.0 and 10.0. Similarly, the temperature was also altered between 20°C and 50°C. Moreover, the effect of agitation was tested by incubating the medium with culture on the orbital shaker with different agitation speed adjustments, ranging from 110 rpm to 200 rpm, for 24 hours.

Screening
The oil spilled soils that were collected from different areas of Warangal oil mills were used as a source for bacterial samples. These soil samples were serially diluted and then spread on the TBA plates by spread plate method. A total of 100 colonies showed hydrolytic zones that were displaying clear hallows around the colonies. Among the 100 colonies, we found 20 colonies having lipase positive bacterial strains. Upon screening, these 20 isolates were identified with the highest lipolytic activity. Further, the molecular characterization was done for these isolates.

Molecular identification of the isolated bacterial strains
To characterize the isolated strains further, DNA was isolated from the culture and the quality of the isolated DNA was evaluated using 1.0% agarose gel, and a single band of high molecular weight DNA was observed. Later on, the fragment of the 16S rDNA gene was used as a biomarker, hence, amplified by 27F and 1492R primers. A single discrete PCR amplicon band of 1500 bp was generated. Next, the PCR amplicon was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with forward and reverse primers using the BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer.
The consensus sequence of the 16S rDNA gene was generated from the forward and the reverse sequence data using Aligner software. In the next stage, bioinformatics analysis was conducted. The 16S rDNA gene sequence was used to carry out BLAST with the NCBI GenBank database. Based on the maximum identity score, the first ten sequences were selected and were aligned using multiple sequence alignment program ClustalW. The distance matrix was generated and the phylogenetic tree was constructed using MEGA7.

ReSuLTS AND DISCuSSIONS Survey and soil sample collection
A detailed survey of oil-contaminated regions was done before the sample collection. Possible collection spots were identified and soil samples were collected from the designated places. The collected samples were used as potential bacterial sources that may have better lipase activity. The samples were containing oil and effluent mixed soil which were dry in nature.

Isolation and screening of bacterial strains
As mentioned in the methodology section, the bacterial samples were isolated applying serial dilution and spread-plate technique. Nutrient agar was used initially, and the Tributyrin agar medium was used later. A total of 70 bacterial colonies were obtained, out of which 30 were positive. From these 30, further we have chosen 10 positive colonies. The strains were morphologically circular, white, smooth, gram negative, cocci, and motile in appearance.
The obtained colonies are represented in Fig. 1. Different colony formation is presented in Fig. 1(A) and Fig. 1(B). The Figs. display the efficient lipase activity of some of the colonies.
Positive colonies were detected for further use through the Rhodamine-B agar medium as mentioned in the prior section. Fig. 2 presents the results obtained from the assessment of the colonies using the Rhodamine-B medium. Detection of the colonies is shown in Fig. 2(A) and the formation of the halos is shown in Fig. 2(B).

Production medium for growing isolated bacterial strains
The increased production of the isolated strains from the colony was important to estimate the lipase activities of the strains. Hence, they were systematically grown in the production medium. A detailed description of the growth of the bacterial strains in the production medium is shown in Fig.  3. In Fig. 3, it is evident that the sample 1 and sample 2 have slight growth, whereas sample 3 and sample 4 are showing yellow color due to lipolysis. Besides, sample 5 is showing the color of light greenish-yellow due to the slow or reduced pace of lipolysis. Sample 6 presents no growth and was white as the fresh production medium. A total of 6 samples were inoculated in 6 flasks and   incubated for lipase activity . The flasks 3, 4 and 5 showed lipolytic activity.

Assessment of Lipase activity by the bacterial strains
The lipase activity was assessed with extreme care for the selected bacterial strains. The observed results are presented in Fig. 4. Fig. 4 (A) presents the lipase activity of the sample where activities were measured in µl/ml. The maximum lipase activity value observed was 4.65 µl/ml whereas the lowest lipase activity recorded was 1.3 µl/ml.

Optimization of Lipase activity
Along with lipase activity assessment, influencing factors were examined to optimize the lipase production capacity of the selected bacterial strains. Therefore, pH (Fig. 4B), temperature (Fig.  4C), and agitation (Fig. 4D) were tested extensively. The optimum pH was pH 8.0 with 4.6 µl/ml lipase activity. Similarly, the optimum temperature was observed at 36°C with 24.5 µl/ml lipase activity. Agitation was decided through the rpm as shown in (Fig. 4D), 160 rpm was found optimum with 24 µl/ml lipase activity.

Optimization of nutrient resources
Nutrients resources play a vital role in bacterial growth. Therefore, optimization of the nutrient resources, specifically the carbon and nitrogen sources are important to optimize the lipase production and activity. Various nitrogen and carbon sources were considered and comparative growth of the selected strains was assessed. Fig. 5 presents the comparative estimation of growth in the presence of different nitrogen sources (Fig. 5A) and also in the presence of various carbon sources (Fig. 5B). Peptone was the optimum nitrogen source for both Bacillus subtilis and Bacillus fumarioli strains. Tryptone was a good source for Bacillus subtilis and yeast extract was effective for Bacillus fumarioli strains. Similarly, starch was found an excellent carbon source for both Bacillus subtilis and Bacillus fumarioli. Unfortunately, other carbon sources did not provide better outcomes.

Molecular characterization
B e tte r re s u l t s i n t h e p re v i o u s experimentations inspired us to further characterize and identify the isolated bacterial strains through molecular approaches.16SDNA based marker was used in this experimentation as mentioned in the method section. The primers used were, 27F and 1492R primers ((5→3´) for forward strand: "AGAGTTTGATCMTGGCTCAG", and (3´→5´) for reverse strand: "AAGTCGTAACAAGGT"). The PCR amplicon band was developed in Agarose gel. Two different bacterial strain samples (Bacillus subtilis (sample SOSS_7_4; Accession Number: (MN173874), and Bacillus fumarioli (sample POSS_8_2; Accession Number: MT645092) that had maximum lipase activity were used for final molecular characterizations. Fig. 6 and Fig. 7 presents the results of the molecular characterizations.
After DNA isolation, purification, and PCR amplification, the samples were assessed in the presence of the 100bp ladder marker (Promega, USA) in an agarose gel (Fig. 6A, Fig.  7A). Further, eluted DNA was sequenced for species identification. The obtained sequences are represented in Fig. 6B and Fig. 7B for both the samples.  All these 10 sequences for both the query sequences were collected and multiple sequence alignment was developed for individual cases, respectively. Next, the phylogenetic tree was developed using the MEGA7 tool to understand their respective phylogenetic distances as presented in Table 3 and Table 4.
Our molecular and bioinformatics analysis confirmed that the isolated, separated, and characterized bacterial strains were Bacillus subtilis  (sample POSS_8_2), and Bacillus fumarioli(sample SOSS_7_4). Further, it was confirmed that the strains were thermophilic and they were Bacillus subtilis strain RCPS3 and Bacillus fumarioli strain RCPS4 (Accession Number: MT645092).
Many studies have been conducted on novel lipase producing bacteria identification where oil spilled and petrol spilled soils were used [20][21][22][23] . Successful isolation of bacterial strains requires further optimization of lipase production. Assessment of the enzyme production activity in the laboratory, followed by pilot-scale evaluation and industrial-scale quantification is necessary. The amalgamation of different advanced culture techniques 24-26 , production 27-29, and molecular techniques 30-32 , and advanced bioinformatics analysis [33][34][35][36] can provide the intended results with maximum accuracy and help in the identification of exact strains with all necessary information.
In the present study, we have isolated, assessed, and optimized novel species that are having excellent lipase production and activities from oilcontaminated soil sources in Warangal district of Telangana state, India. As a next step, pilot-scale and industrial-scale production can be attempted to use the identified and isolated organisms for the industry.

CONCLuSION
Identification of novel and cheap sources of efficient lipase producing bacterial strains is of pivotal importance considering the excessive industrial applications. Moreover, oil effluent sources can also help in managing environmental aspects. In the present study, we were successful in identifying, isolating, optimizing, and characterizing bacterial strains that are efficacious in lipase production and lipase  This work would help in further using the novel bacterial species that were found efficacious in lipase production for the industry. Necessary action should be taken for further optimization and advanced molecular techniques can be used to scale-up the activities of the strains.  Table 1 and Table 2 (sample POSS_8_2 and SOSS_7_4).