Biosurfactant produced by novel Pseudomonas sp. WJ6 with biodegradation of n-alkanes and polycyclic aromatic hydrocarbons

Highlights

• A novel Pseudomonas strain was isolated from heavy oil contaminated soil.

• It can convert n-alkanes and polycyclic aromatic hydrocarbons to lipopeptides surfactant.

• Lipopeptides surfactant have different structures when using different hydrocarbons.

• Lipopetides surfactant showed great performance on heavy oil sand washing.

Abstract

Alkanes and polycyclic aromatic hydrocarbons (PAHs) have threatened the environment due to toxicity and poor bioavailability. Interest in degradation of these hazardous materials by biosurfactant-producing bacteria has been steadily increasing in recent years. In this work, a novel biosurfactant-producing Pseudomonas sp. WJ6 was isolated to degrade a wide range of n-alkanes and polycyclic aromatic hydrocarbons. Production of lipopeptide biosurfactant was observed in all biodegradable studies. These lipopeptides were purified and identified by C18 RP-HPLC system and electrospray ionization-mass spectrometry. Results of structural analysis showed that these lipopeptides generated from different hydrocarbons were classified to be surfactin, fengycin and lichenysin. Heavy-oil sludge washing experiments demonstrated that lipopeptides produced by Pseudomonas sp. WJ6 have 92.46% of heavy-oil washing efficiency. The obtained results indicate that this novel bacterial strain and its lipopeptides have great potentials in the environmental remediation and petroleum recovery.

Introduction

Petroleum is the most important and predominant energy resources and raw materials of chemical industry in modern society. However, spills, leaks, and other releases of petroleum often result in the contamination of soil and groundwater, especially when associated with accidental spills on a large scale [1]. Main sources of petroleum contamination are petrochemical industries, oil field installations, petroleum plants, liquid fuel distribution and storage devices, transportation equipment for petroleum products, airports and illegal drillings in pipe lines [2]. These pollutions have significant environmental impacts, and present substantial hazards to human health and destroy the ecological balance which may take years or even decades to recover.

Numerous researches have been done to diminish environmental threads of petroleum and its risks to human health and ecological system. Biodegradation of petroleum hydrocarbons by microorganisms (such as bacteria, fungi, or yeast, and bacteria) has been recognized as one predominant, effective and environment-friendly way by which crude oil is eliminated from contaminated sites [3]. Bacterial strains able to degrade petroleum hydrocarbons are known to be ubiquitous in nature. For this reason, biodegradation by bacteria has been considered a potentially useful tool in the cleansing of oil spills and the treatment of oil residues [4], [5].

Petroleum is a complex mixture of hydrocarbons and related compounds generally classified into four fractions: aliphatics (alkanes), aromatics, polars or resins and asphaltenes [6]. Alkanes can constitute 50% to 95% of crude oil, depending on the oil source. Alkanes of shorter chain length (C10–C20) are more toxic, while those of larger chain length (C20–40) are hydrophobic solids difficult to degrade [7], [8]. Polycyclic aromatic hydrocarbons (PAHs) are a widespread class of environmental chemical toxic pollutants, and has several possible sources in the environment exist, such as pyrolytic, diagenetic, and petroleum hydrocarbons [9]. PAHs are aromatic compounds containing from two to eight conjugated ring systems, and can have a range of substituents such as alkyl, nitro, and amino groups in their structure. Nitrogen, sulfur, and oxygen atoms can also be incorporated into their ring system [10]. Crude oils contain 0.2 to 7% PAHs, with configurations ranging from two to six rings [11]. And asphaltenes constituted 5% to 20% of crude oil was detected with multiple PAH units containing 2–5 or more rings inside asphaltene molecules and some other heteroatom-containing (N, S and O) aromatic compounds [12]. Therefore, alkanes and PAHs could be treated as typical and predominant fraction of crude oil for biodegradation research.

Many literatures have reported the great abilities of bacteria to utilize/degrade hydrocarbons with different carbon numbers and structures [13], [14], [15], [16], [17]. However, the general low bioavailability of alkanes and PAHs, which are highly recalcitrant molecules that can persist in the environment due to their hydrophobicity and low water solubility, is a hindrance to microbial biodegradation for achieving regulatory end-points. Consequently, there are much more interests in isolating, identifying and understanding potential mechanisms by which alkanes or PAHs degrading microorganisms surmount the challenges imposed by these poorly available substrates.

Production of biosurfactants by bacteria is considered an important microbial strategy that influences the bioavailability of hydrophobic chemicals by changing the surface properties of bacterial cell or by dissolving and emulsifying these non-hydrophilic hydrocarbons, and as well as releasing (or washing) the traped hydrocarbons of porous medium in contaminated soil. Biosurfactants are amphiphilic molecules consisting of hydrophobic and hydrophilic moieties that tend to interact with interfaces of various polarities and reduce the surface and interfacial tension of solutions [18], [19]. These surface active materials generally exhibit low toxicity, high biodegradability and ecological acceptability, which make biosurfactants as potential alternatives of chemically synthesized surfactants in a variety of field such as cosmetics, food, pharmaceutics, agriculture, environmental remediation and petroleum industry [20], [21]. Many types of biosurfactants have been synthesized from bacteria belonging to a wide variety of genera, such as Pseudomonas, Bacillus, Acinetobacter and Mycobacterium [22]. Lipopeptides are a group of biosurfactants with consisting of a hydrophobic fatty acid moiety and a hydrophilic peptide moiety in molecule. It has low critical micelle concentration (CMC), stable emulsification properties, powerful surface activities and excellent foaming characteristics, and shows stable physical–chemical properties at different temperatures and pH levels. Lipopeptides are mostly synthesized by genus Bacillus with hydrophilic substrates (e.g. molasses, glucose, source) as carbon source, and hardly generated with hydrophobic hydrocarbons as sole carbon source, such as alkanes and PAHs [23], [24]. Pseudomonas species are always considered to be ubiquitous hydrocarbon-degrader and have been shown prominent ability of degrading alkanes and PAHs, and could be isolated from diverse environments [14], [23], [25]. It is well known that Pseudomonas sp. strains synthesize the glycolipid biosurfactants (Rhamnolipids) when utilizing the hydrophobic substrates [26]. However, it is rarely reported in literatures that Pseudomonas sp. could convert alkanes or PAHs to lipopeptide surfactant.

The objective of this study is to isolate the promising hydrocarbon-degrading and biosurfactant-producing bacterial strain for bioremediation of petroleum-contaminated soil, and to evaluate the abilities of biodegradation of hazardous hydrocarbons, and to analyze the characteristics and heavy oil-washing potentials of produced biosurfactant. For this purpose, the heavy oil-contaminated soil was collected from Xinjiang Oilfield for screening the high-performed bacterial strains. And we obtained one novel Pseudomonas sp. WJ6 which show great abilities of degrading the n-alkanes and PAHs compounds. And structural diversity of lipopeptides biosurfactant produced during degradation was analyzed. Additionally, characterization of surface activities and heavy oil sand washing of these lipopeptides were evaluated.