Predominant culturable crude oil-degrading bacteria in the coast of Kuwait

Abstract

Total of 272 crude oil-degrading bacteria were isolated from seven locations along the coast of Kuwait. The analysis of the 16S rDNA sequences of isolated bacteria revealed the predominance of six bacterial genera: Pseudomonas, Bacillus, Staphylococcus, Acinetobacter, Kocuria and Micrococcus. Investigation of the factors associated with bacterial predominance revealed that, dominant culturable crude oil-degrading bacteria were better crude oil utilizers than the less frequently occurring isolates. Bacterial predominance was also influenced by the ability of bacteria to adapt to the level of organic content available. Predominant culturable bacteria constituted 89.7–54.2% of the total crude oil-degrading bacterial communities. Using 16S-RFLP analyses to assess the diversity of the dominant crude oil-degrading bacterial genera, four phylotypes of Pseudomonas sp. and seven phylotypes of Bacillus sp. were determined. This suggested high degree of diversity of crude oil-degrading bacterial population at the strain level, but low diversity at the genus level.

Introduction

Crude oil is considered a major pollutant of Kuwait marine environment. Hence, efforts are directed to investigate factors affecting its bioremediation (Sorkhoh et al., 1990, Radwan et al., 1995, Readman et al., 1996, Banat et al., 1998, Radwan et al., 2002, Obuekwe et al., 2003, AL-Saleh and Obuekwe, 2005). During the Gulf war the coast of Kuwait witnessed the largest oil spill ever known, with more than 1,500,000 tons of crude oil spill covering some 1500 km² of sea surface and polluting more than 500 km of coastline (Reynolds 1993, Sadiq and McCain, 1993). The coastal areas of Kuwait are continuously impacted by crude oil attributed to natural oil seepage, accidental damage to pipelines, accidental spillage from tankers, and oil production from marine wells (Massoud et al., 1996).

Natural removal of crude oil from marine sediments is slow, and oil deposits continue to have long term effects on the benthic community (Blumer 1970, Shriadah 1998). The persistence of a pollutant in the environment is influenced by the nature of the contaminant, the amount of the contaminant present and the interplay between chemical, geological, physical and biological characteristics of the contaminated site. Among the biological factors, the diversity of microbial species and their metabolic capabilities constitute important factors in pollutants removal (Alexander 1999).

Since crude oil is made of a mixture of compounds, and since individual microorganisms metabolize only a limited range of hydrocarbon substrates (Britton 1984), biodegradation of crude oil requires mixture of different bacterial groups or consortia functioning to degrade a wider range of hydrocarbons (Bordenave et al., 2007). Contaminated marine environments are inhabited by a range of selected microorganisms able to tolerate and remediate pollutants that impacted the environment, leading to the dominance of pollutant-tolerant bacteria. Hence, bacterial communities in contaminated sites are typically less diverse than those in non-stressed systems (Harayama et al., 2004). Another characteristic of marine environments is that, the vast majority of bacteria (90–99%) are uncultivable (Rozsak and Colwell, 1987, Amann et al., 1995), hence, the analysis of microbial communities that contribute to in situ hydrocarbon biodegradation activities has been a challenge to microbiologists (Rollins and Colwell, 1986, Wilkinson 1988, Sadiq and McCain, 1993). Therefore, the use of rRNA approaches to study changes in the structure of marine microbial communities during oil spills provides a means of detecting non-culturable organisms (Harayama et al., 2004). For, this purpose, 16S rRNA gene (rDNA) clone libraries, fluorescence in situ hybridization (FISH) analysis with targeted oligonucleotide probes, and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified rDNA have been used in revealing the large diversity of marine bacteria (Wise et al., 1997, Cottrell and Kirchman, 2000). Molecular approaches were also used to fingerprint and investigate changes in bacterial communities subjected to stress factors (Fredrickson and Balkwill, 1998, Röling et al., 2004). For example, DGGE analyses were used to compare bacterial communities in crude oil-treated and untreated sediments. No significant differences were detected and two bacterial genera Alcanivorax borkumensis and Pseudomonas stutzeri dominated the treated and untreated sediments (Röling et al., 2004). Unfortunately, the effectiveness of petroleum pollutants bioremediation remains a controversial issue since the apparent biodegradative potential of bacteria is the result of the interaction of complex physical, chemical and biological factors (Siron et al., 1995, Alexander, 1999).

Identification of major organisms responsible for the biodegradation of pollutants and characterization of their biodegradative potentials is imperative for the comprehension, evaluation, and development of in situ bioremediation strategies (Cohen 2002, Harayama et al., 2004). In the current study, the community structure of the crude oil-degrading bacteria isolated along the cost of Kuwait was characterized. Dominant strains were investigated for their potential to mineralize crude oil, ability to grow in nutrient-rich and poor media, and their genetic affiliation.