The synergistic effect of biosurfactant production on the bacterial biodegradation efficiency of crude oil was investigated. Further disclosure of the effect of biosurfactants as a stimulating agent in the bioremediation of crude oil was investigated in the second part of the thesis.
LIST OF TABLES
Fitted kinetic parameters of hydrocarbon biodegradation using the integrated first-order exponential decay and Monod degradation model for the binary mixture of HEX and PHE as C substrate. Acronyms and Nomenclature AH Alkane Hydroxylase ANOVA Analysis of Variance B+C Biosurfactant + Cells BBD Box–Behnken Design.
Introduction
Objectives
An integrated strategy of hydrophobic surface-induced biosorption and microbial biodegradation for the improved purification of crude oil.
Thesis Outline
- Chapter 2
- Chapter 3
- Chapter 4
- Chapter 5
- Chapter 6
- Chapter 7
Biosorbent dosage was pre-optimized using RSM statistical tool and later inoculated with bacteria to explore their effectiveness in overall oil biodegradation. Further, using this integrated strategy, a fed-batch mode was selected to enhance the biodegradation ability of the bacterial oil.
Review of literature
Crude oil origin and characteristics
Average elemental and component composition of crude oil in relative percentage (Demirbas et al., 2015; National Academies of Sciences, 2016). Typical classification of crude oil based on variation in percentage of its constituents Adapted from Ref.
Sources of crude oil contamination and its hazard
Impaired organ metabolism and increased mortality have also been demonstrated as adverse effects of crude oil toxicity (Laurel et al., 2019). The toxicity is chronic due to the presence of various carcinogenic and mutagenic components in crude oil.
Current tools for the control of crude oil contamination
Many aquatic toxicological studies reported negative effects of oil dispersants on marine life, such as reduced feeding rate of Crassostrea virginia (Jasperse et al., 2018) bioaccumulation in bivalves (Durier et al., 2021; Scarlett et al., 2005). Dilutes thick oil particles into small oil particles, increasing oil surface area for microbial degradation.
Bioremediation of crude oil: a sustainable approach
- Mechanism of crude oil biodegradation
- Isolation of oil degraders and exploration of their biodegradation ability
This anaerobic pathway occurs through (a) addition of fumarate; b) hydroxylation under anoxic conditions; (c) carboxylation; (d) reverse methanogenesis; or (e) hydration (Laczi et al., 2020). The natural breakdown of contaminants from crude oil by the inherent microbes without human intervention is called natural degradation (Agnello et al., 2016).
Biosurfactant: an effective tool of bioremediation of crude oil
- Factors regulating microbial biosurfactant production
- Temperature
- Carbon (C) source
- Nitrogen (N) source
- C/N ratio
- Other factors
- Optimization designs for maximizing biosurfactant production
- One- factor-at-a-time (OFAT)
- Plackett burmann design (PBD)
- Taguchi model
- Response surface methodology (RSM)
This was due to poor assimilation of C source in N source limitation (Santos et al., 2016). Thus, increased biosurfactant production was achieved with an integrated optimization model (Biniarz et al., 2018).
Recent expansions in the bioremediation of crude oil
- Design of Microconsortium for effective crude oil bioremediation
- Criteria of designing microbial consortium
- Advantage of the microbial consortium over pure isolates
In contrast, the second approach focuses on "bottom-up" design, where each microbe in the consortium (does not have to be of the same country of origin) is selected with the desired traits and later enriched in the microbial consortium (Ibrar and Zhang , 2020). Three biosurfactant-producing strains (Joostella sp.. A53) were investigated for their hydrocarbon degradation ability in pure culture and in consortium. The authors highlighted the role of coordinated metabolic activity in the consortium that led to better outcomes (Kumari et al., 2018).
Various researchers have also reported increased catabolic and degrading enzyme activity in the presence of metabolically enriched consortia (Loureiro et al., 2020; Suganthi et al., 2018).
Integrated physiochemical techniques assisted crude oil biodegradation
- Photo-oxidation assisted bioremediation
- Electrokinetic-bioremediation
- Biosurfactant mediated biodegradation
The study showed that microcapsules removed 944.1 mg/kg of PAHs in the contaminated soil within 30 days (Wang et al., 2019). The resulting TPH removal rate was reported as having enhanced inherent microbial dehydrogenase and polyphenol oxidase activities (Zhen et al., 2021). A gradual breakdown of TDH into smaller units was reported in the presence of a BC-nZVI/PS-mediated biodegradation study (Zhang et al., 2020).
Thus, the biodegradability of hydrocarbons and the biodiversity of indigenous microbes increased in modified bagasse (Zhen et al., 2021).
Challenges associated with microbial bioremediation
- Research gaps
- Hypothesis
Recent in-situ remediation techniques include sparging-slurping followed by biostimulation with nutrients and biosurfactants (Akbari et al., 2018). High initial oil concentration or supersaturated oil concentration at the spill site inhibits the biodegradation process and extends the delay phase by 2-4 weeks. Based on these challenges, we hypothesize that crude oil bioremediation can be improved by isolation, screening and identification of inherent oil degradation agents and optimization of parameters such as pH, temperature and concentration of crude oil that limit their biodegradability.
Further immobilization of potential oil-degrading consortia in suitable carriers would prevent toxicity and enhance their growth, which would help improve their crude oil biodegradation capability.
Conclusions
Exploring the crude oil biodegradation and biosurfactant production abilities of isolated Agrobacterium fabrum SLAJ731
Introduction
The main problem in the biodegradation of spent crude oil is its hydrophobic nature, as it causes a decrease in its bioavailability for natural reduction. These profitable advantages encourage the optimization of biosurfactant production to enhance the biodegradation of crude oil. Furthermore, in addition to the emulsifying activity of biosurfactants, the rate of biodegradation of crude oil has also been attributed to the enzymatic activity of some oil-degrading enzymes such as alkane hydroxylase (AH).
Interestingly, this study investigates the synergistic effect of co-substrate glucose on the present strain's crude oil degradation and biosurfactant production capacity in terms of enzyme activity of AH.
Materials and methods
- Microbial strain and chemicals used
- Screening for biosurfactant production
- Drop collapse test
- Oil displacement test
- Emulsification activity
- Optimization of biosurfactant production
- Biosurfactant extraction and characterization
- Functional group analyses
- Surface tension measurement
- Biodegradation analysis of crude oil
- Determination of alkane hydroxylase activity
- Statistical analysis
10 µL of cell-free supernatant was added to this oil film and immediately the diameter of the formed clear zone was measured. The diameter of the clear zone is directly proportional to the surface activity of the biosurfactant (Morikawa et al., 1993). Later, the height of the emulsion layer formed in the mixture was measured and E24 was calculated using equation 3.1,.
The nature of the peak (stretch/bend) and its intensity describe the chemical groups present in the.
Results and discussion
- Optimization of culture conditions for biosurfactant production
- Characterization of the produced biosurfactant
- Biodegradation analysis of crude oil
- Relation between biosurfactant production and crude oil degradation
- Determination of alkane hydroxylase activity
- Relationship between AH and oil degradation and biosurfactant production
The overall breakdown of crude oil was obtained as 58 ± 5% in the presence of glucose and 40 ± 4% in the absence. However, the current Agrobacterium strain showed relatively better degradation in the presence of glucose (optimized C source). From Figure 3.6, biosurfactant production was expected to increase significantly (p < 0.05) in the presence of glucose.
The iAH activity showed a similar trend for the degradation of crude oil in the presence and absence of glucose (data not shown).
Conclusions
Biosurfactant mediated crude oil biodegradation by isolated Bacillus subtilis RSL 2
Introduction
In the previous Chapter 3, the need to optimize bacterial growth conditions for maximizing biosurfactant production to achieve effective crude oil biodegradation capability was elaborated. This chapter presents crude biosurfactant as an inherent biostimulatory tool for mediating the bacterial crude oil biodegradation. Several researchers have worked on this line to improve the production of biosurfactants to achieve a higher degradation of spilled crude oil (Patowary et al., 2018).
Furthermore, to reveal the role of biosurfactants in oil utilization, the produced biosurfactant was supplemented with crude oil at different concentrations, and the resulting effects on microbial oil degradation were investigated.
Materials and methods 1. Chemicals and reagents
- Isolation and screening of bacterial isolates
- Identification of selected isolates
- Optimization of nitrogen source
- Optimization of culture conditions
- Estimation of produced biosurfactant
- Estimation of residual crude oil
- Study of biosurfactant production utilizing crude oil under optimized conditions
- Estimation of Alkane Hydroxylase Activity
- Biosurfactant characterization studies
- Biosurfactant assisted biodegradation studies
- Statistical analysis
These enriched bacteria were incubated in BH media with 1 % (v/v) crude oil as the sole carbon source for a week. The cells were harvested upon incubation, and the cell-free broth was analyzed to estimate residual crude oil and COD as mentioned in Chapter 3, section 3.2.5. The selected strain was incubated at thus optimized pH, temperature and crude oil concentration to analyze its degradation and biosurfactant production capacity.
The cell-free supernatant was studied for surface tension measurement, biosurfactant production (Chapter 3, section 3.2.4) and crude oil residual estimation (Chapter 3, section 3.2.5) (Sharma et al., 2019).
Results and discussion
- Isolation, screening and identification studies for selected strains
- Optimization of Nitrogen source
- Optimization of culture conditions
- Estimation of Alkane hydroxylase enzyme activity
- Characterization of produced biosurfactant
The effect of different N sources on the growth and biosurfactant production capacity of Bacillus subtilis RSL 2 was further analyzed. Biosurfactants are surface-active biomolecules that reduce the surface tension of the culture medium (Sharma et al., 2019). Study of crude oil degradation and biosurfactant production under optimized conditions Under optimized conditions (pH 4.0, 25 °C and 1 g/L crude oil), B.
The hydroxylation activity of this enzyme is largely regulated by the availability of substrate (alkanes) and cofactor (NADH) (Ji et al., 2013).
- The implication of biosurfactants in biodegradation behavior
- Role of concentration of biosurfactant on bio-stimulation
- Antibacterial and cell-surface hydrophobicity properties of crude biosurfactant
- Effect of biosurfactant feeding on microbial biodegradation activity
- Conclusions
It was observed that in the case of simultaneous biosurfactant-assisted biodegradation (B+C), there was improved bacterial growth, biosurfactant production and crude oil degradation compared to sequential inoculation (BC). In the case of simultaneous feeding of biosurfactant and oil, the maximum oil degradation of 72% was achieved, 1.6 times higher than the control, i.e. without biosurfactant. However, in the case of sequential feeding (BC) of biosurfactant followed by microbes, the system performance was lower than simultaneous feeding (B+C), i.e., 55% oil degradation and 4.5 g/L biosurfactant.
However, there is no coherent literature on the role of lipopeptide biosurfactants in the biodegradation of crude oil.
Design of microbial consortium and exploring their hydrocarbon biodegradation ability
Introduction
Various studies have reviewed the potential degradation of aliphatic and aromatic hydrocarbons in bioremediation (Chettri and Singh, 2019); however, a few reports have also indicated an inhibitory effect of co-contamination of aliphatic with aromatic hydrocarbons, limiting the overall biodegradability of axenic cultures (Ghorbannezhad et al., 2021). The use of indigenous bacteria eliminates this gap, as they are well acclimatized to such co-contaminated environments (Elumalai et al., 2021; Rabodonirina et al., 2019). In this direction, the formation of a consortium using such an indigenous bacterium has gained much attention in hydrocarbon bioremediation studies due to their various advantages over pure/axenic cultures, such as (a) increased metabolic diversity, (b) a wide spectrum of degradative genes, and (c ) greater tolerance and adaptability to unfavorable physiological conditions (Varjani et al., 2021).
In this chapter, three native bacteria (Bacillus subtilis RSL2, Agrobacterium fabrum SLAJ 731 and Pseudomonas aeruginosa P7815) are investigated based on their previously reported ability to biodegrade carbohydrates and produce biosurfactants (Sharma et al., 2019; Tiwari et al. , 2017; Verma et al., 2020).
Materials and Methods 1. Chemicals
- Microorganism and growth conditions
- Microbial hydrocarbon biodegradation studies
- Estimation of residual hydrocarbon
- Kinetics of hydrocarbon degradation
- Degradative enzymes activity determination
- Statistical analysis
The study aimed to gain insight into hydrocarbon degradation kinetics, bacterial growth and degradative enzyme activities in the aforementioned conditions and investigate its suitability in oil-contaminated sites. The value of 𝐶0 was 50 ppm for the decomposition of individual hydrocarbons and 25 ppm in the case of a binary mixture. 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.
As previously reported in the literature, enzyme activity was expressed as a catechol concentration (mM) oxidized per minute (Elumalai et al., 2021).
- Biodegradation of PHE
- Biodegradation of the binary mixture of substrates
- Insights into hydrocarbon biodegradation
The increased biodegradability of HEX in the microconsortium of Bacillus subtilis and Pseudomonas aeruginosa has been reported elsewhere (Dehghani et al., 2018; Nozari et al., 2018). The highest degradation in the case of the microcosm is associated with the maximum biomass yield (Figure 5.2(A)). Similar to the biodegradation kinetics of the sole PHE, in the presence of HEX as co-substrate.
Thus, this study explored the activity of these key enzymes (AH and C23DO) in the biodegradation of HEX and PHE.
Conclusions
An integrated strategy of hydrophobic surface-induced biosorption and microbial biodegradation for the enhanced crude oil remediation
Introduction
Nevertheless, the harsh environmental conditions slow down their biodegradability, which limits their actual applications at the contaminated site (Sharma et al., 2019; Sharma et al., 2019). This study aimed to mitigate two major drawbacks of bacterial oil bioremediation, viz. the biotoxicity of the oil at higher concentrations and limited bioaffinity of oil. The bagasse surface was modified by forming hydrophobic octyl SAMs, which to the best of our knowledge was explored for the biosorption of oil for the first time.
Bacteria Pseudomonas aeruginosa has been reported elsewhere for its effective biosurfactant production capacity (Sabarinathan et al., 2021; Sharma and Pandey, 2021) and is a potential candidate for the crude oil biodegradation study.
Materials and Methods 1. Materials
- Preparation of biosorbent
Proposed mechanism of the surface modification of bagasse by forming octyl SAMs
- Characterization of the prepared biosorbent (bagasse)
- Crude oil adsorption studies
- Optimization of adsorbate and adsorbent concentrations
- Biosorbent stability/ reusability analysis
- Integration of bio-sorption and biodegradation
- Insights into the integrated process
- Integrated crude oil biodegradation using fed-batch study
- Microbial growth kinetics study
- Oil biodegradation and biosurfactant production estimation
- Results and discussion
- Surface modification of bagasse by forming the octyl SAMs
- Characterization of the modified bagasse 1. X-Ray Diffraction study
- Oil adsorption study using biosorbents
- Optimization of adsorbate and adsorbent concentrations
In the case of a fed batch in the biosorbent study, an initial biosorption of 8 hours using modified bagasse was performed. The change in the surface chemistry of bagasse after acid treatment and chemical modification was analyzed by FTIR spectra. Therefore, such changes in the surface properties suggested that the octyl groups graft onto the surface of the bagasse.
BET analysis supported the surface modification of bagasse with octyl groups, resulting in a change in the adsorption isotherm curve.
