Design of microbial consortium and exploring their hydrocarbon biodegradation ability
5.2. Materials and Methods 1. Chemicals
The chemicals and reagents with their source information have been mentioned in previous chapters.
The remaining chemicals i.e., Phenanthrene (RM2369-25G), Hexadecane (RM2238-100ML) were purchased from HiMedia, India.
5.2.2. Microorganism and growth conditions
Three bacterial genera were individually enriched in BH agar plates with 0.1 % (v/v) crude oil as the sole C source. The plates were incubated overnight at 25°C, and single colonies were picked using a sterile loop and inoculated in sterile NB followed by incubation in a shaker incubator maintained at 180 rpm and 25°C. Later, the bacteria were recovered using centrifugation and resuspended in sterile distilled water to an OD of 1 (1.4×107 CFU/mL) and then used as inoculum for biodegradation studies.
5.2.3. Microbial hydrocarbon biodegradation studies
HEX and PHE were chosen as model aliphatic and aromatic hydrocarbons. Three bacterial strains, namely Agrobacterium fabrum SLAJ 731, Pseudomonas aeruginosa P7815 and Bacillus subtilis RSL2, were selected based on their hydrocarbon degradation ability using crude oil as their C source in previous studies (Sharma et al., 2019; Sharma et al., 2018; Verma et al., 2020). The experiments systematically studied microcosm and individual bacterial growth and biodegradation ability using an individual hydrocarbon as the sole C source. In the next step, the mixture of both the hydrocarbons was tested for biodegradation using all three bacteria and their microcosm. A similar control experiment was performed without inoculum to consider the hydrocarbon degradation due to abiotic (physical and chemical) factors such as dispersion, evaporation, dissolution, sorption, photo-oxidation, and auto- oxidation.
The bacterial inoculum was inoculated at 10 % (v/v) of concentration to sterile BH broth containing an additional 50 ppm yeast extract with HEX or PHE or both and incubated at 25 °C 180 rpm for 7 days until the bacteria reached their death phase. The average total petroleum hydrocarbon concentration at crude oil contaminated sites has been reported to be approximately in the range of 2 to 50 ppm (Gaur et al., 2021; Kriipsalu et al., 2008; KURNAZ and BÜYÜKGÜNGÖR, 2016; Nganje et al., 2007); thus initial hydrocarbon concentration was selected as 50 ppm in the case of the solo substrate and 25 ppm of each in the binary mixture. The bacterial growth was estimated by dry cell weight (DCW) measurement during the incubation period. The bacterial culture was centrifuged at regular intervals, and the pellet was recovered, washed, and allowed to dry overnight. The bacterial death was inferred from the reduction in the dry cell weight of the biomass due to cell lysis leading to the release of intracellular content, which was observed as a loss in biomass gravimetric weight (Liu, 2017). The specific growth rate (𝜇) was calculated using equation 5.1. Here X is the DCW of bacteria expressed in mg/L at a given time 't' (h), and the slope refers to µ (h-1).
𝒍𝒏𝑿 = 𝝁𝒕 (5.1)
Similar to the aforementioned axenic biodegradation study, an equal volume of each bacterial strain at OD of 1 was used to prepare a 10 % (v/v) bacterial inoculum to study hydrocarbon degradation using microconsortium (MCM). The degradation kinetics was analyzed by analyzing residual hydrocarbon and degradative enzymes. The cell-free supernatant's surface tension was measured using a Du Nouy ring method using a tensiometer (Dataphysics, DCAT 11 EC) at 25 °C, as discussed in Chapter 3, section 3.2.4.2.
5.2.4. Estimation of residual hydrocarbon
The cell-free supernatant was used to estimate bacterial hydrocarbon biodegradation activity at regular time intervals. The residual hydrocarbon was extracted in an equal volume of chloroform and separated using a separating funnel. The extracted organic layer was investigated for the concentration of residual HEX concentration using Gas Chromatography-Flame ionization detector instrument (GC-FID, Varian 450) equipped with Sil-8 CB column (30 m × 0.25 mm × 2.5 µm). The program used for this measurement included an injector temperature of 250 °C and detector temperature of 280 °C. 1 µL of sample previously extracted in chloroform was injected into the column. The standard curve was prepared by dissolving commercial HEX in chloroform prepared in different concentrations (0 to 50 ppm). The initial temperature of the column oven was maintained at 60 °C, raised at ramping of 20
°C/min to 190 °C followed by ramping of 10 °C/min till 280 °C. Helium gas was used as carrier gas maintained at a 1:50 split ratio.
Similarly, the residual PHE concentration was determined using a High-performance liquid chromatography instrument (HPLC, Shimadzu LC300) equipped with a PFP-C18 column (ACE®) and UV detector (254 nm). The column oven was maintained at 30 °C. The analysis used the isocratic mobile phase containing methanol: water (90:10) with 0.1 % trifluoroacetic acid (TFA) at a flow rate of 0.6 mL/min. The standard curve was prepared by dissolving commercial PHE in chloroform prepared in different concentrations (0 to 60 ppm) with a sample injection volume of 20 µL. The samples were prepared in chloroform, and an acquisition time of 30 min was maintained for each sample analysis (Wang et al., 2010).
5.2.5. Kinetics of hydrocarbon degradation
Various literature suggested the first-order exponential decay model (Hajieghrari and Hejazi, 2020) and Monod's model (Chettri and Singh, 2019) for the hydrocarbon degradation analyses. For the present study, the concentration of residual hydrocarbon at the regular time was calculated using chromatogram peak area analyses. The abiotic hydrocarbon degradation was fitted to a first-order kinetic model (equation 5.2) as expressed below.
𝑪𝒕= 𝑪𝟎𝒆−𝒌𝟏𝒕 (5.2)
Ct and Co refer to residual hydrocarbon concentration at the time 't' (h) and at 't = 0' and k1 represented the abiotic hydrocarbon degradation rate. While for analyzing the biodegradation kinetics, Monod's equation was integrated with the first-order equation to represent bacterial and abiotic degradation, respectively (equations 5.3 and 5.4).
𝑪𝒕= 𝑪𝟏𝒆−𝒌𝟏𝒕+ 𝑪𝟐 𝒆−𝒌𝒅 𝑿 𝒕𝒌𝒔 (5.3) 𝑪𝟎= 𝑪𝟏+ 𝑪𝟐 (5.4)
Here, C1 and C2 refer to hydrocarbon (g/L) concentrations subjected to abiotic losses and microbial biodegradation, respectively. X is the concentration of biomass (g/L). kd and ks represent microbial hydrocarbon biodegradation rate and half-saturation constant, respectively. In the Monod equation ks
was assumed to be much greater than C (ks >> C). The 𝑘1 value was obtained by fitting abiotic data to equation 5.2. The microbial biodegradation data were fitted to equations 5.3 and 5.4 to determine the values of C2, kd and ks. The value of𝐶0 was 50 ppm for the degradation of individual hydrocarbons and 25 ppm in the case of a binary mixture. Further, the overall bacterial biodegradation percentage was calculated after 7 days of incubation period using equation 5.5. Here 𝐶0 and 𝐶𝑡 refer to the initial hydrocarbon concentration and residual hydrocarbon at day 7, respectively.
𝑂𝒗𝒆𝒓𝒂𝒍𝒍 𝒃𝒊𝒐𝒅𝒆𝒈𝒓𝒂𝒅𝒂𝒕𝒊𝒐𝒏(%) = (𝑪𝟎− 𝑪𝑪 𝒕 )
𝟎 × 𝟏𝟎𝟎 (5.5)
5.2.6. Degradative enzymes activity determination
The bacterial enzyme activity in the presence of different hydrocarbons was estimated by harvesting cells using centrifugation (8000 rpm for 10 min) at regular time intervals. The pellets were washed and resuspended in 20 mM Tris HCl (pH 7.4) in the case for aliphatic hydrocarbon-degrading enzymes estimation and 50 mM PBS buffer (pH 7.4) for aromatics degradative enzymes. Next, the cells were lysed using ultrasonication with a 20 kHz frequency (20 cycles with 10 seconds breaks). The required enzyme crude extract was then recovered using centrifugation at 11,400 rpm for 45 min at 4°C and used for further enzyme analysis.
The AH activity analysis was performed as mentioned in Chapter 3, section 3.2.6. Overall, 1 unit of enzyme activity was estimated as the enzyme responsible for the oxidation of 1 mM NADH per min . The activity of C23DO was analyzed by investigating the formation of 2-hydroxymuconic semialdehyde in the reaction mixture using catechol as substrate. It was observed as an increase in the absorbance at 375 nm. Each 1 mL reaction mixture contained 100 µL of 50 mM catechol and 100 µL of crude enzyme extract in 50 mM PBS buffer. As previously reported in the literature, the enzyme activity was expressed as a catechol (mM) concentration oxidized per minute (Elumalai et al., 2021).
5.2.7. Statistical analysis
The statistical significance of experimental data was estimated using the analysis of variance (ANOVA) technique using OriginPro 8.5 software. All biodegradation studies were performed in triplicates, and their mean value ± standard deviation is reported.
5.3. Results and discussion