1. Introduction
Biosurfactants are a class of microorganism-formed compounds with surface activities. Biosurfactants are microbial substances that have significant surfactant and emulsifying properties. They are less toxic, structurally complex, extremely biodegradable, eco-friendly and highly substrate-specific [
1,
2]. Biosurfactants are often either anionic or acidic, and sugars, amino acids, phosphates, or any other compounds can be hydrophilic [
3]. Because of their variety, environment-friendly character, adaptability for large-scale production, and selectivity, they have mostly been used for ecological applications [
4]. Despite their promise and biological origins, only a few studieson their potential application in the biomedical sector have been conducted. Biosurfactant activity has been extended to cover future applications in several industries, such as food, agriculture, pharmaceuticals, petrochemicals and the paper industry [
5,
6,
7].
In addition, biosurfactants also play a vital function in the treatment of wastewater. The oil sector pollutes the environment, and has an impact on the balance of the ecosystem [
8]. Some biosurfactants are safe and effective therapeutic agents that can be utilized as an alternative to synthetic medications and antibacterial agents [
9]. As a new technique to minimize adhesion, the bio-conditioning of the surface viathe application of microbial surfactants has been performed. Wastewater from these agricultural and anthropogenic sources has an effectnot only on the health of the ecosystem, but also on human health, which causes numerous diseases. Toxicity has been treated using a variety of management measures, including biological, physiochemical, and thermal methods. Wastewater is known to contain multiple radioactive elements, along with dangerous microorganisms, which are released into the atmosphere and pose a danger to the local community [
10].
However, the difficulties ofbiosurfactant commercialization include the lack of efficient strains and effective identification routes, and the high processing cost [
11]. Several species of
Bacillus,
Alcaligenes,
Pseudomonas, and
Corynebacterium have been found to produce biosurfactants that aid in the degradation of wastewater. Compared to these techniques, biological treatment using these bio-surfactants helps us to reduce the interfacial surface tension, which, in turn, inhibits the mobility of organic compounds. lt can also inhibit the transformation of organic compounds, and therefore reduce the formation of toxic by-products [
12,
13]. In this case, biosurfactants are a good choice for the treatment and remediation of waste in the environment. Biosurfactants have also been shown to have antibacterial and antifungal effects. Keeping this in mind, ourstudyplanned to use microbial surfactants for the remediation of wastewater from agricultural and household operations, and the elimination of the involved dangerous microorganisms and toxic chemicals. The biosurfactant-mediated treatment of wastewater was further verified by an intergenomic approach [
14]. Metagenomic approaches are currently being used to analyze the structure of the microbial population during the remediation of wastewater by biosurfactants. This method providesaccurate knowledge related to the existence of microorganisms in wastewater [
15]. The sequencing of 16S rRNA genes with ahigh-performance metagenomic method, such as Illumina Mi-Seq, has beensuccessfully implemented in anaerobic digestion for the identification of different microbial communities. As a result, bacterial strains with the ability to create biosurfactants with improved oil-degrading abilitiesare suggested for use in achieving rapid crude oil degradation.
The metagenomic approach illustrates the efficacy of biosurfactant wastewater treatment by evaluating microbial wastewater populations, in view of the need to mitigate wastewater and troubling aquatic contamination. As a consequence, the objective of this study is to find the most effective biosurfactant-producer, optimize bacterial culture by RSM, and analyze experimental data using ANOVA. The wastewater isanalyzed for the parameters pH, EC, DO, COD and BOD, enabling us to characterize thebiosurfactants we make. We planned to treat wastewater with the help of Bacillus safensis (YKS2) surfactant. In addition, the efficacy of the surfactant against theharmful microorganisms present in wastewater was investigated by an antimicrobial and metagenomic method.
2. Materials and Methods
2.1. Sample Collection and Isolation of Bacteria
Soil samples were collected from Yercaud hills, Salem, India (11°50′44.61 N, 78°14′55.32 E). In the Yercaud hills, twenty separate soil samples were taken from five different places. Sub-soil leaflets were harvested, and the soil was collected using a sterile scoop (up to 10 cm deep) and sterilized polyethylene bags were transferred to the laboratory and kept at 20 °C for further investigation. Soil samples were serially diluted to 10−1- to 10−7-fold and spread plated on nutrient aga, which was incubated at 37 °C for 24 h. The colonies that were morphologically distinct were chosen, purified, and stored on nutrient agar slants for additional investigations.
2.2. Screening of Biosurfactant Producing Bacteria
The selected bacterial strains were screened for biosurfactant production by following the standard methods, and the strain with higher biosurfactant production was selected for further studies [
16].
2.2.1. Hemolytic Activity
The freshly prepared blood agar was streaked with pure bacterial strain culture and incubated for 48–72 h at 37 °C. The clear zone type found was used to report the results, i.e., alpha-hemolytic when the colony was surrounded by a greenish zone, β-hemolysis when the colony was surrounded by a clear white zone, and γ-hemolysis when the medium around the colony remained unchanged [
16].
2.2.2. Drop-Collapsing Test
The qualitative drop-collapse test was used to conduct biosurfactant output screening [
17]. In this test, crude oil was used. On the 96-well microplate covers, delimited to the well regions, 2 μL of oil was added. At 12,000 rpm, 5 μL of the 48 h culture was centrifuged for 5 min; the cells were removed and the supernatant was transferred to the oil-coated well areas, and with the use of a magnifying lens, the drop size was measured after 1 min. When the drop was flat, the effect was deemed beneficial for biosurfactant production, whereas cultures that produced rounder drops were considered negative. This indicated that the production of biosurfactants was lacking [
18].
2.2.3. Oil Spreading Assay
Oil displacement activity of surfactants was assessedin the oil spreading assay as per the method described by Maneerat and Phetrong [
18]. The concept of this method isbased on the biosurfactant’s ability to change the angle of interaction at the oil–water interface. The oil was displaced by the surface pressure of the biosurfactant. In this method, 10 μL of kerosene oil was added to the surface of the petri dish with 50 mL of distilled water. As a result, the oil forms a thin layer, in the middle of which is softly placed 10 μL of cultured supernatant. If the oil is displaced and a clear zone is formed, the presence of biosurfactants is suggested. The amount of surfactant forming 1 cm
2 of oil displacement area was specified as one biosurfactant unit (BS unit) [
19].
2.2.4. Blue Agar Plate (Bap) Method
Mineral salt agar media combined with glucose as carbon source (2%) and cetyltrimethylammonium ammonium bromide (CTAB: 0.5 mg/mL), and methylene blue (MB: 0.2 mg/mL) were used for the detection of anionic biosurfactant [
20]. Each methylene blue agar plate is filled with thirty microliters of cell-free supernatant produced with a cork borer (4 mm). At 37 °C for 48–72 h, the plate was then incubated. For the development of anionic biosurfactants, the presence of a dark blue halo surrounding the culture was regarded as favorable.
2.3. Identification of Biosurfactant Production Strain
2.3.1. Morphology and Biochemical Characterization
Gram staining and the biochemical activities of the biosurfactant bacterial strains, such as indole production, MR-VP test, catalase, oxidase and urease test, and motility and citrate utilization activities, were examined as per the method followed by Cappuccino-Sherman [
21].
2.3.2. Molecular Identification of the Biosurfactant Producing Bacteria
The genomic DNA of bacteria-producing biosurfactants was purified using the technique described by Barakat et al. [
22]. The 16S rRNA gene was amplified using universal primers forward (5′d AGAGTTTGATCMTGGCTCAG3′) and reverse (5′TACGTTATCCAGCCGCA 3′). The reaction mixture for PCR amplification was set for atotal volume of 25 µL with autoclaved deionized water (17.3 µL), 10X
Taq buffer (2.5 µL), forward primer (1 µM/µL) (1.0 µL), reverse primer (1 µM/µL) (1.0 µL), dNTPs (10 mM/µL)(2.0 µL),
Taq polymerase (3 U/µL) (0.2 µL) and genomic DNA template (1.0 µL). 16S rRNA gene sequencing was further characterized by the isolated bacterium as followed by Kumar et al. [
23]. For bacterial strain identification, the sequences were compared using BLAST (National Center for Biotechnology Information). The sequence was aligned using the cantor model of jukes. In the Molecular Evolutionary Genetics Analysis (MEGA 7.0), the phylogenetic analysis was performed using the neighbor-joining method and submitted to the GenBank.
2.4. Optimization of Bacterial Culture Conditions by RSM
Response Surface Methodology (RSM) is a statistical grouping technique that is practical for analyzing the special effects on the system response of more than a few autonomous variables, without the need for a given relationship between the function of the intention and the variables. To get the best results for bacteria growth and activity, the composition of the medium and the growth parameters were improved using four distinct culture conditions with the use of RSM. Temperature, pH, carbon sources (palm jaggery) and nitrogen sources were all used as input factors in the model’s developmen(glycerol). Codes were assigned to each parameter, and a variety of parameters wasemployed to simulate the biosurfactant [
24]. Culture samples were taken at regular intervals.The experimental data werethen analyzed using the Analysis of Variance (ANOVA) approach to determine which factors create the most effective interactions and help in the lowering of surface tension [
25].
2.5. Extraction and Purification of Biosurfactant
After 48 h, the biosurfactant was recovered from cell-free supernatant. By reducing the pH level to 2.0, acid precipitation was carried outon cell-free supernatants. At an acidic pH, the biosurfactant was precipitated. The precipitate was then recovered by centrifugation and the pH was corrected to pH 7.0 before being freeze-dried.
2.6. Characterization of Biosurfactant
The UVspectrophotometer is used either in the ultraviolet, visible, or near-IR spectral regions (200–800 nm) to measure the quantity of chemicals in the solution viathe chemical’s reflective or distribution properties depending on the wavelength of radiated light [
26]. In order to identify the chemical in the mid-infrared (MIR) range of 4000–400 cm
−1 of biosurfactants from samples isolated from bacteria, FTIR analysis was performed using the Perkin Elmer Spectrum. The scanning electron microscopic (SEM) study was conducted using the FEI QUANTA 200 FEG HR-SEM model with a working distance of 8 mm at 30 kV. A very small amount of the specimen was placed on the sample holder and thin films of the samples were prepared on carbon-coated paper. Using blotting paper, the extra solution was removed and the film on the SEM was allowed to dry by keeping it under a mercury lamp for 5 min. The GC-MS (Gas Chromatograph Mass Spectrometer) was used to analyze partly filtered biosurfactants. As the carrier gas, helium with a flow rate of 1.0 mLmin
−1 was used. The initial temperature of the column was 1000 °C for 1 min, which was then ramped up to 2700 °C at 300 °C, and eventually kept for 10 min at 2700 °C. The inlet temperature, transfer line, ion trap, and quadruple were 270, 280, 230, and 1500 °C, respectively. Using the DB 35-MS capillary standard non-polar column (30 Mts, ID: 0.25 mm, FILM: 0.25 μm), the GC analysis may be carried out. The analytical conditions that can be used are: The temperature of the injector oven of 50 ° C for 0.5 min increased to 235 °C at 65 °C/min for 12 min. Electron effect with a 50–450 Da scan range and an injection volume of 1 m Lat 70 eV. The temperature of the source was 200 °C and the temperature of the interface was 250 °C, with 70 even energy and EI + 50–700 m/z. At the South India Textile Research Group, Coimbatore, India, GC-MS analysis was carried out.
2.7. Biosurfactant-Mediated Wastewater Treatment and Microbial Community Analysis Using NGS Technique
The domestic wastewater samples were collected from the discharge sites of the Salem district and were used for treatment (Latitude of 11.664° N and Longitude of 78.146° E). The wastewater sample was collected using sterile containers and stored in arefrigerator at 4 °C until further processing. The wastewater treatment was performed by adding 1 gm of biosurfactant into 100 mL of wastewater and incubating at room temperature for 3 h. Physico-chemical parameters such as pH, EC, alkalinity, turbidity, dissolved oxygen (DO), chemical oxygen demand (COD) and biochemical oxygen demand (BOD) were analyzed in the wastewater before and after treatment. Treated (SWT) and untreated (SWC) wastewater samples were used to estimate the microbial communities by employing metagenomic sequencing. The wastewater samples were filtered to remove the floating particles before DNA extraction. A power water DNA extraction kit (MoBio, Carlsbad, CA, USA) was used to extract the total microbial DNA, which prevents humic acid interaction from the DNA. The recovered DNA was tested in Nano Drop to estimate the quantity and purity. Universal bacterial primers covering the V3–V4 regions were used for 16S rRNA gene amplification (34′F-CCTACG GGN GGCWGCAG; 805R GACTAC HVG GGTATCTAATCC) [
27]. The amplification reactions were performed as per the methods of [
28]. Using a next-generation DNA library preparation kit, the metagenomic library was prepared and sequenced using the Illumina MiSeq Next Generation Sequencer [
29].
2.8. Antibacterial Activity of Biosurfactant
The antimicrobial activities of the biosurfactant developed from the
B. safensis (YKS2) strain wereinvestigated against different pathogen bacterial strains. This was achieved by utilizing the agar well plate susceptibility method [
30].
2.9. Statistical Analysis
ANOVA and Duncan’s multiple range examination were used to analyzeall the results (three replicates of each treatment). Microbial enumeration data were was subjected to two-ways ANOVA (SPSS version 16.0, Inc., Chicago, IL, USA) for data processing.
4. Conclusions
The present study evaluated a B. safensis (YKS2) biosurfactant for use in wastewater treatmentand itsantibacterial activity. The biosurfactant was shown to have good surface tension-lowering and emulsification properties. The biosurfactant showed the potential for enhanced antimicrobial activity. The characterization analysis of theFT-IR and GC-MS results of the wastewater reveal that the biosurfactant generated by B.safensis YKS2 is similar to glycolipid, hexadecanoic, pentadecanoic, dodecanoic, tetradecanoic, and octadecanoic biosurfactants.This biosurfactant’s thermal stability, tolerance ofa wide range of pH values and salt concentrations, and lack of toxicity make it a viable choice for biotechnological, environmental, cosmetic, food and medicinal applications. It isconcluded that B. safensis YKS2, producinga glycolipid type of biosurfactant, has potential benefitsin wastewater remediation and can be utilized in the biosurfactant industry on a large scale. Future technologies, such as wastewater and various drone applications, are also seen to be compatible with biosurfactants. The fascinating stability and efficient emulsifying capability of the biosurfactant make it a promising economic alternative for various industrial and environmental applications.