Characterization of the bacterial archaeal diversity in hydrocarbon-contaminated soil

Abstract

A polyphasic approach combining culture-based methods with molecular methods is useful to expand knowledge on microbial diversity in contaminated soil.

Microbial diversity was examined in soil samples from a former industrial site in the European Alps (mainly used for aluminum production and heavily contaminated with petroleum hydrocarbons) by culture-dependent and culture-independent methods. The physiologically active eubacterial community, as revealed by fluorescence-in-situ-hybridization (FISH), accounted for 6.7% of the total (DAPI-stained) bacterial community. 4.4% and 2.0% of the DAPI-stained cells could be attributed to culturable, heterotrophic bacteria able to grow at 20 °C and 10 °C, respectively. The majority of culturable bacterial isolates (34/48) belonged to the Proteobacteria (with a predominance of Alphaproteobacteria and Gammaproteobacteria), while the remaining isolates were affiliated with the Actinobacteria, Cytophaga–Flavobacterium–Bacteroides and Firmicutes. A high fraction of the culturable, heterotrophic bacterial population was able to utilize hydrocarbons. Actinobacteria were the most versatile and efficient degraders of diesel oil, n-alkanes, phenol and PAHs. The bacterial 16S rRNA gene clone library contained 390 clones that grouped into 68 phylotypes related to the Proteobacteria, Bacteroidetes, Actinobacteria and Spirochaetes. The archaeal 16S rRNA gene library contained 202 clones and 15 phylotypes belonging to the phylum Euryarchaeota; sequences were closely related to those of methanogenic archaea of the orders Methanomicrobiales, Methanosarcinales, Methanobacteriales and Thermoplasmatales. A number of bacterial and archaeal phylotypes in the clone libraries shared high similarities with strains previously described to be involved in hydrocarbon biodegradation.

Knowledge of the bacterial and archaeal diversity in the studied soil is important in order to get a better insight into the microbial structure of contaminated environments and to better exploit the bioremediation potential by identifying potential hydrocarbon degraders and consequently developing appropriate bioremediation strategies.

Introduction

Petroleum hydrocarbons are the most widespread contaminants in the environment. The contamination of soil with high levels of hydrocarbons results in an increased soil organic carbon content, which – depending on composition and concentration – may be utilized for microbial growth or may be toxic to microorganisms (Bossert and Bartha, 1984, Alexander, 1999, Maier et al., 2000). The impact of low and high doses of environmental pollutants such as hydrocarbons can range from stimulation to total inhibition of microorganisms (Ramakrishnan et al., 2010). The capacity of a broad spectrum of microorganisms to utilize hydrocarbons as the sole source of carbon and energy was the basis for the development of biological remediation methods. The ability to degrade hydrocarbons is widespread among soil microorganisms. They may adapt rapidly to the contamination, as demonstrated by significantly increased numbers of hydrocarbon degraders after a pollution event (Margesin and Schinner, 2001, Greer et al., 2010).

Microbial community structures in hydrocarbon-contaminated soils are influenced by a number of factors, such as soil type, concentration and bioavailability of the contaminants, nutrient contents, temperature, oxygen content and pH (Margesin and Schinner, 2001, Greer et al., 2010). To evaluate soil microbial community composition in contaminated soils, culture-dependent and culture-independent methods have been used (Margesin and Schinner, 2005, Alonso-Gutierrez et al., 2009, Fabiani et al., 2009). Microbial abundance is often based on culture-dependent methods. However, culturable cells may only represent less than 1% of the total microbial community in an environment (Amann et al., 1995, Rappé and Giovannoni, 2003) and numerous bacteria enter a viable but non-culturable (VBNC) state in response to environmental stress (McDougald et al., 2009). Therefore, culture-independent, molecular assays, such as profiling soil DNA, rRNA, or phospholipid fatty acids, are increasingly used in environmental microbiology. Culture-independent approaches have been claimed to be more reliable for diversity analyses given the established cultivation methods favor the isolation of fast-growing microorganisms (Felske et al., 1999). Direct recovery of bacterial 16S rDNA from soil theoretically represents the entire microbial population from environmental samples (Spiegelman et al., 2005). However, molecular methods also have their limitations, such as variable efficiency of lysis and DNA extraction and differential amplification of target genes (Kirk et al., 2004).

Studies on microbial community composition in contaminated alpine soils have focused so far on the bacterial population (Margesin et al., 2003b, Labbé et al., 2007, Margesin et al., 2007), whereas information on the impact of Archaea is missing. In this study, we used a combination of culture-dependent and culture-independent methods (analysis of Bacteria and Archaea 16S rRNA gene clone libraries and fluorescence-in-situ-hybridization (FISH)) to investigate the microbial diversity in soil samples from an Alpine hydrocarbon-contaminated industrial site. Knowledge of the bacterial and archaeal diversity in the studied soil is important in order to get a better insight into the microbial structure of contaminated environments and to better exploit the bioremediation potential by identifying potential hydrocarbon degraders and consequently developing appropriate bioremediation strategies. Since both traditional, culture-based, and molecular methods have their limitations (Kirk et al., 2004), a multi-technique (polyphasic) approach combining these methods is advantageous.