Studies on the Production of Biosurfactant

Abstract

Petroleum hydrocarbons are an integral part of modern developed society as various petroleum fractions provide essential resources for energy, transportation, synthesis of plastics and chemicals, etc. They constitute a large and diverse class of around 250 hydrocarbon compounds consisting of varying constituents and molecular complexity ranging from complex mixture of saturates, aromatics, resins and asphaltenes. The extensive production and use of these hydrocarbons has resulted in widespread environmental contamination. These hydrocarbons reach the environment from leaking underground storage tanks, petroleum refineries and bulk storage facilities, harbour operations, broken oil pipelines, effluent discharges from petroleum industries, spills of petroleum products in chemical plants and transportation processes. Moreover, these hydrocarbons fall into the category of persistent pollutants. When these pollutants are released into the environment; they cause air, water as well as soil pollution. Contamination by petroleum hydrocarbons is a major environmental concern since many of its constituents are highly toxic, carcinogenic and are poorly biodegradable in nature. The contamination of environment by these hydrocarbons can also result in uptake and accumulation of these contaminants in food chains, thereby causing harm to the flora and fauna. Many of these contaminated sites threaten to become sources of contamination to drinking water supplies and thereby, constitute substantial health hazards. Due to the serious and long-term damage caused to the ecosystems, terrestrial life, human health and natural resources; there is a need to remediate the sites which are by petroleum hydrocarbons. The processes leading to the eventual removal of hydrocarbon pollutants from the environment involves various physical, chemical and biological methods or a combination of them. Physical and chemical remedial methods include adsorption, incineration, thermal desorption, solvent extraction, evaporation, etc. These methods are expensive, requires high energy input and also results in significant greenhouse gasemissions. Moreover, they involve the transfer of the contaminant to another medium rather than eliminating the contaminant. Bioremediation has proven to be an efficient, ecofriendly and cost-effective approach to alleviate petroleum hydrocarbon contamination from the environment. The driving force for petroleum biodegradation is the ability of microorganisms to utilize hydrocarbons for their growth and energy needs. One of the widely accepted bioremediation methods of petroleum hydrocarbons is biodegradation. The biodegradation of petroleum hydrocarbons is affected by many factors such as water, oxygen and nutrients. In addition, the lack or reduced bioavailability of petroleum hydrocarbons to the microorganisms affects the biodegradation of these hydrocarbons. One of the options to increase bioavailability of the petroleum hydrocarbons to the microorganism is the use of surfactants. Surfactants emulsify the petroleum hydrocarbons, increase the surface area and thereby, increase the rate of biodegradation of these hydrocarbons. Surfactants used in the remediation of petroleum hydrocarbons earlier were synthetic surfactants which are synthesized from petroleum based products. Since synthetic surfactants are derived from petroleum based products, they are commonly toxic to ecosystems and resistant to complete degradation. Moreover, they act like secondary pollutants in the environment. An increase in the concern about environmental protection has recently caused the consideration of alternatives to synthetic surfactants. Surfactants produced by microorganisms, called biosurfactants, are gaining importance as they exhibit lower toxicity, higher biodegradability, better environmental compatibility and selectivity. They are versatile process chemicals used in various industries such as cosmetic, petroleum, pharmaceutical, etc. While reviewing the literature, it has been observed that there is less data with respect to isolation and identification of potential surfactant producing microorganisms,studies on conditions required for the maximum production of surfactant and utilization of the surfactants in the removal of petroleum hydrocarbons from the affected medium. In this context, the objectives of the present research were formulated. Studies were initiated for the isolation and screening of surfactant potential producing microorganisms, identification of a potential surfactant producing bacteria, studies on the effect of various process parameters on surfactant production by the potential surfactant producer and the usage of surfactant in the biodegradation of crude oil. Reports in the literature suggest that the prior exposure of microbial community in the soil as well as water environments to the petroleum hydrocarbon contaminant increases the incidences of the isolation of surfactant producing microorganisms due to the acclimatization of microorganisms to the contaminated environment. It has been postulated by various researchers that the function of biosurfactant is related to hydrocarbon uptake and therefore, a spontaneous release of biosurfactant occurs in the presence of the hydrocarbon substrate. Hence, in the present study, soil and water samples were collected from various petroleum hydrocarbon contaminated localities in and around Mangalore, Karnataka. The soil and water samples were subjected to enrichment with crude oil in order to increase the chances of isolating microorganisms possessing the ability to produce surfactant. The study resulted in isolation of several microorganisms which were further screened for their ability to produce surfactant. Among several isolates, a bacterial isolate, designated as potential extracellular surfactant producer based on its ability to produce halos on selective Cetyl Trimethyl Ammonium Bromide (CTAB)-methylene blue agar medium, rapid drop collapse reaction and reduction in surface tension from 71.39 mN/m to 29.33 mN/m. Based on microscopic studies, biochemical tests and 16S ribosomal DNA sequencing, the candidate bacterial strain 2B, was identified as a novel Pseudomonas sp.Hence, the 16S ribosomal DNA sequence of the novel isolated bacterium was submitted in the GenBank database with an accession number JF683582. In the present research, we report surfactant production by the novel Pseudomonas sp. 2B. In the present research work, we also have compared the data of Pseudomonas sp. 2B. with that of already reported surfactant producer, Pseudomonas aeruginosa (ATCC 10145), that was procured from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune, Maharashtra, India. The bacterial strain, Pseudomonas aeruginosa (ATCC 10145), was selected based on references cited in the literature. The effect of various process parameters influencing surfactant production by Pseudomonas sp. 2B and Pseudomonas aeruginosa, respectively, was studied. The process parameters assessed for their ability to produce maximum surfactant by the bacterial strains included inoculum size, initial medium pH, incubation temperature, agitation speed, type and concentration of carbon source, type of nitrogen source, inducer, buffer and salinity. Pseudomonas sp. 2B produced maximum surfactant at 2% (v/v) inoculum size, initial production medium pH 7, incubation temperature of 37oC, agitation speed of 150 rpm, 30 g/L (w/v) glucose as carbon source, using a combination of peptone and potassium nitrate, olive oil as inducer, Tris HCl buffer and 1% (w/v) NaCl concentration. Maximum surfactant was produced by Pseudomonas aeruginosa at 3% (v/v) inoculum size, initial production medium pH 7, incubation temperature of 37oC, agitation speed of 150 rpm, 30 g/L (w/v) glucose as carbon source, using a combination of yeast extract and ammonium chloride, n-hexadecane as inducer, Tris HCl buffer and 0.5% (w/v) NaCl concentration. Plackett-Burman method was used to screen process variables affecting surfactant production by the bacterial strains. Glucose as carbon source, potassium nitrate as nitrogen source and olive oil as inducer had significant effect on surfactant production by Pseudomonas sp. 2B whereas glucose as carbon source, ammonium chloride as nitrogensource and n-hexadecane as inducer had significant effect on surfactant production by Pseudomonas aeruginosa. To obtain the optimal concentrations of these process variables leading to maximum surfactant production by the bacterial strains, Response Surface Methodology (RSM) was used. The optimum concentration of factors leading to maximum surfactant production by Pseudomonas sp. 2B was found to be 35.7645 g/L of glucose, 3.5% of olive oil and 5.5425 g/L of potassium nitrate. A maximum of 14.63 g/L of surfactant was produced by Pseudomonas sp. 2B using the RSM studies, the corresponding surface tension of the cell-free broth showed lowest value, i.e. 21.98 mN/m. The optimum concentration of factors leading to maximum surfactant production by Pseudomonas aeruginosa was found to be 35.7645 g/L of glucose, 3.5% of nhexadecane and 5.6274 g/L of ammonium chloride. Using the RSM studies, a maximum of 10.69 g/L of surfactant was produced by Pseudomonas aeruginosa, the corresponding surface tension of the cell-free broth was found to be 25.31 mN/m. During the production of surfactant by the bacterial strains, the quantity of cellular biomass, specific growth rate (μ), maximum growth rate (μmax) and substrate utilization constant (Ks) were determined. In addition, kinetic parameters were evaluated in terms of yield factors-surfactant production to substrate utilization (YP/S), dry cell biomass to substrate utilization (YX/S) and surfactant production to dry cell biomass (YP/X). The study further revealed that the surfactant produced by both the bacterial strains were “primary metabolites” since the production of surfactant coincided with exponential growth phase of the bacterial strains. The surfactant produced by the bacterial strains, Pseudomonas sp. 2B and Pseudomonas aeruginosa, respectively, were subjected to extraction as well as partial purification. Acidification followed by chloroform: methanol mixture (2:1) extraction was effective in the extraction of the extracellular surfactant from the cell-free broth of 2B and Pseudomonas aeruginosa, respectively, as both polar and non-polar components present in the surfactant could be extracted as compared to other extraction methods. Theresults of the column chromatography experiments indicated that the surfactants produced by the bacterial strains were made up of different moieties as suggested by the surface tension values of the different fractions eluted during the experiments. The surfactant produced by Pseudomonas sp. 2B and Pseudomonas aeruginosa, respectively, were characterized using thin layer chromatography, biochemical analysis, fourier transform infrared spectroscopy and liquid-chromatography-mass spectrometric techniques. The results revealed that the surfactant produced by both the bacterial strains were rhamnolipoproteins. The cell-free broth containing the surfactant as well as partially purified surfactant produced by the bacterial strains, Pseudomonas sp. 2B and Pseudomonas aeruginosa, respectively, were found to be stable over a wide range of temperature, pH and salinity. The study also revealed that the cell-free broth could be directly applied without any purification step since the surface tension of the cell-free broth did not vary significantly from that of the partially purified surfactant in varied environmental conditions. The efficiency of surfactant produced by Pseudomonas sp. 2B and Pseudomonas aeruginosa, respectively, was tested in the biodegradation of crude oil by Nocardia hydrocarboxydans NCIM 2386. 95.5% and 93.5% of crude oil degradation was achieved over a span of 42 days in the presence of surfactants produced by Pseudomonas sp. 2B and Pseudomonas aeruginosa, respectively. In the control flask, 65.25% of crude oil biodegradation was observed. This suggests that the surfactant produced by the bacterial strains can be used for the remediation of petroleum hydrocarbon contaminated sites.