Isolation, identification, and crude oil degradation characteristics of a high-temperature, hydrocarbon-degrading strain

https://doi.org/10.1016/j.marpolbul.2015.09.053Get rights and content

Highlights

  • A bacillus licheniformis strain Y-1 was isolated, and the crude oil degradation rate of 60.2% was got in only 5 days.

  • Strain Y-1 showed strong tolerance to high salinity, alkalinity, and temperature.

  • Degradation of both short-chain alkanes and long-chain alkanes were obvious.

  • Emplastic produced by strain Y-1 at high temperature intensified the emulsifying effect.

Abstract

In this work, a hydrocarbon-degrading bacterium Y-1 isolated from petroleum contaminated soil in the Dagang Oilfield was investigated for its potential effect in biodegradation of crude oil. According to the analysis of 16S rRNA sequences, strain Y-1 was identified as Bacillus licheniformis. The growth parameters such as pH, temperature, and salinity were optimised and 60.2% degradation of crude oil removal was observed in 5 days. The strain Y-1 showed strong tolerance to high salinity, alkalinity, and temperature. Emplastic produced by strain Y-1 at high temperatures could be applied as biosurfactant. Gas chromatography analysis demonstrated that the strain Y-1 efficiently degraded different alkanes from crude oil, and the emplastic produced by strain Y-1 promoted the degradation rates of long-chain alkanes when the temperature increased to 55 °C. Therefore, strain Y-1 would play an important role in the area of crude oil contaminant bioremediation even in some extreme conditions.

Introduction

With the increasing demand for oil, and oil products, in various fields, petroleum hydrocarbon pollution (PHP) is becoming more serious. Such pollution damages both marine, and terrestrial ecosystems, it has attracted much attention (Bao et al., 2012, Oren, 1990). PHP results from the exploitation and transport of oil, and the processing of oil products. In the exploitation and processing of oil, large volumes of waste water and residues containing oil are produced, and crude oil spillages happen frequently in the transporting of oil (Deng et al., 2014). Oil spills often result in both immediate and long-term environmental damage (Ricardo et al., 2013, Suneel et al., 1996). Statistics shows that about 1.3 million tonnes (Bao et al., 2012) of oil have been spilled in the oceans: PHP not only significantly damages the ecosystem in ocean waters and coastal soils, but also influences economic development and the lives of residents.

Microbial biodegradation technology has gained increasing acceptance in the remediation of oil pollution. Compared with traditional physico-chemical treatments, biodegradation is more environmentally friendly, cost-effective, and efficient (Das and Mukherjee, 2007, Zhang et al., 2012). Numerous studies have proved that microbial biodegradation technology serves potentially significant applications (Wentzel et al., 2007). In the remediation of xenobiotic pollutants (such as oil), natural microbial populations are considered the most basic and reliable bioremediation mechanism (Cappello et al., 2007). Many microorganisms can degrade short-chain petroleum hydrocarbons. However, microorganisms which can degrade long-chain petroleum hydrocarbons and complex polycyclic aromatic hydrocarbons (PAHs) are key to the remediation of oil pollution (Kenzo et al., 2008).

The effectiveness of microbial biodegradation is often limited by the low bioavailability of hydrocarbons to microorganisms (Nitschke and Pastore, 2006, Van et al., 2003). Biosurfactants are surface active molecules produced by certain strains, which either adhere to the cell surface or are excreted extracellularly in the growth culture medium (Suneel et al., 1996). During the culturing process, certain microorganisms secrete biosurfactants into the medium which can increase the surface area of hydrophobic substrates by reducing surface tension of the culture, and further increase their bioavailability (Chandankere et al., 2014, Ramos et al., 1991, Rufino et al., 2008). So attention, to date, is focused on hydrocarbon degrading microorganisms with biosurfactant-producing capabilities, and several species have been provided, such as Bacillus, Alcaligenes, Pseudomonas, and Corynebacterium to this end (Calvo et al., 2004, Rengathavasi et al., 2011, Toledo and Gonzalez, 2008, Zhang et al., 2010).

Dagang Oilfield in Tianjin, China is next to the Bohai Sea and the soil close to the mined oil fields suffers from heavy oil pollution and salinisation. Therefore, there are high requirements impinging upon the microbial consortium responsible for remediating oil pollution in the area. Here, a Bacillus licheniformis Y-1 was isolated from a soil with heavy oil-contamination in the Dagang Oilfield. As the bacterial strain shows high degradation capacity for oil, salt, a high alkali bearing capacity, and high temperature resistance, it has research and application significance. The gene of the strain was identified, the growth and its oil degradation behaviour were optimised, and the degradation characteristics of the strain for petroleum hydrocarbons were analysed.

Section snippets

Crude oil

The crude oil used was collected from the oil pipeline of Dagang Oilfield (latitude: 38° 49′ N; longitude: 117° 31′ E). After standing for 48 h, the oil was separated from water and the supernatant crude oil was collected and reserved. 0# diesel was obtained from the Sinopec gas station in Huayuan, Tianjin, China.

Media

T-medium was used as the nutrient medium. The original inorganic salt medium contained: 10 g of NaCl, 1 g of NH4NO3, 0.5 g of KH2PO4, 1 g of K2HPO4, 0.5 g of MgSO4, 0.2 g of CaCl2, 1 mL of

Emulsifying effect and hydrocarbon-degradation rate

By streaking and isolating, four strains, named Y-1, Y-2, Y-3, and Y-4, were obtained. In the degradation experiments conducted with crude oil as the single carbon unit, it was observed that the crude oil in the conical flask inoculated with Y-1 was dispersed extensively in the medium. The solubility of crude oil in water was improved and thereby microorganisms and oil came into contact more effectively, which accelerated the degradation. Furthermore, the degradation rate of Y-1 for oil reached

Discussion

A hydrocarbon-degrading strain (Y-1) which applied oil as the single carbon unit in its growth was isolated from heavily polluted soil around the production region of the Dagang Oilfield in Tianjin, China. Results showed that the strain had the closet relative with B. licheniformis and the typical biochemical and physiological characteristics of strain Y-1 also meet the standards of B. licheniformis in Bergey's Manual of Determinative for Bacteriology. Based on this, the strain was judged to

Conclusion

A high-efficiency hydrocarbon-degrading strain (Y-1) was isolated from heavily oil-contaminated soil from around the Dagang Oilfield, Tianjin, China. It was identified as a type of B. licheniformis. The strain showed strong tolerance to high salinity, alkalinity, and temperature. It produced emplastic at high temperatures which could be applied as a surfactant to intensify the emulsifying effect. The strain could not only utilise short-chain alkanes, but also degrade long-chain alkanes with

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