Impact of Petroleum Hydrocarbon Contamination on the Indigenous Soil Microbial Community
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Ann Microbiol (2015) 65:359–369 DOI 10.1007/s13213-014-0868-1 ORIGINAL ARTICLE Impact of petroleum hydrocarbon contamination on the indigenous soil microbial community Simrita Cheema & Meeta Lavania & Banwari Lal Received: 12 July 2013 /Accepted: 9 March 2014 /Published online: 2 May 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Changes in the microbial community structure in importance, especially since the site had high alkaline and agricultural soils and soils contaminated with hydrocarbons saline characteristics. Soils (arid, alpine and polar) which are were evaluated using the culture-independent method of 16S nutrient and moisture limited are typically often dominated by rRNA gene sequence analysis. The bacterial composition was Actinobacteria that are well adapted to low-resource environ- more diverse in the agricultural soil (AS) samples in terms of ments and do not show major changes in community structure number of species and Shannon diversity index [6.6 vs. 1.94 as a result of hydrocarbon contamination. for the hydrocarbon-contaminated soil (HCS)]. Twelve known bacterial groups were identified in the AS: Keywords Diversity . High alkalinity . Proteobacteria (41 % of bacterial community), Ectothiorhodospiraceae . Polyhydroxyalkanoates . Ectoines Actinobacteria (34 %), Acidobacteria (5 %), Firmicutes (4 %), Chloroflexi (4 %), Bacteroidetes (3 %), Gemmatimonadetes, Planctomycetes, Verrucomicrobia, Introduction Armatimonadetes, Cyanobacteria,TM7andArchaea (the lat- ter 7 each accounting for 1–2%) . In comparison, the clonal Microbes are an essential part of all life forms on Earth. They library from the HCS samples included members from only can live in extreme environments and possess adaptive capa- five groups: Proteobacteria (85 %) and Bacteriodetes, bilities that enable them to adjust to changes in living condi- Actinobacteria, Chloroflexi and Verrucomicrobia (the latter tions. Many microorganisms have been used in industrial- four collectively accounting for 15 %). The family scale processes for the production of antibiotics, preparation Ectothiorhodospiraceae was the most dominant family within of food and beverages, leather industry, production of the Proteobacteria isolated from the HCS. These microbes are biofuels, such as ethanol, among others in which they carry known to synthesize a number of biotechnologically useful out various chemical biotranformations. Microbial communi- products, such as polyhydroxyalkanoates and ectoines, and ties have for billions of generations contributed towards their dominance in the sampled area suggests the possibility of transforming the world around them. Their applications are discovering better adapted novel genes of commercial too widespread to describe in detail, and they represent by far the richest repertoire of molecular and chemical diversity in nature (Kapur and Jain 2004). S. Cheema Centre for Bioresources and Biotechnology, TERI University, Plot The diversity of microorganisms has been explored across No.10, Institutional Area, Vasant Kunj, New Delhi 110070, India a wide range of environments, such as water, soils, the human : : body (Achtman and Wagner 2008), hydrocarbon- S. Cheema M. Lavania B. Lal contaminated soils (Liang et al. 2011), the Arctic and deep Microbial Biotechnology Division, TERI, India Habitat Centre, Lodhi Road, New Delhi 110003, India sea vents (Stoeck et al. 2007). Among the various habitats, soil is considered to house the maximum number of microbes, and B. Lal (*) it has been estimated that 1 g of sediment may contain 1010 Environmental and Industrial Biotechnology, TERI University, bacteria, which is the highest for any environment (Torsvik Darbari Seth Block, India Habitat Center, Lodhi Road, New Delhi 110003, India et al. 1996). However, the cultivation efficiency of soil mi- e-mail: [email protected] crobes is only 0.3 % (Torsvik et al. 1996) due to limitations in 360 Ann Microbiol (2015) 65:359–369 traditional cultivation methods. This implies that most soil inputs of composted cow manure; there has also been no bacteria are difficult to isolate on laboratory media due to tillage and no removal of crop residues during this same time unknown culture conditions, insufficient growth nutrients period. An oil spill occurred in April 2008 which resulted in and the lack of cells in cultivable state. This drawback has one of the agricultural fields being contaminated with hydro- been largely overcome by culture-independent approaches carbons. In our study, soil collected from this site was consid- (Handelsman 2004) which are currently being exploited at a ered as the HCS. Within the same area, we also collected soil very fast pace and have allowed researchers and scientists to samples from uncontaminated AS. gain a better understanding of the number of microbial In both the cases (HCS and AS), about 10-g samples of the communities. surface soil were collected at depths ranging from 0 to 10 after Soil microbial communities residing at sites contaminated the top 3 cm of the soil surface was removed, in the rainy with hydrocarbons are one of the most complex and diverse season of July 2008. Ten samples collected from each site assemblages. Their roles and activities in these soils are the were randomly mixed, and subsamples were used for micro- focus of much interest (Liang et al. 2011). It is expected that bial community analysis. Soil samples were kept in polyeth- such information will result in the identification of many ylene whirl bags on ice and transported to the laboratory. The microorganisms which may represent a potential resource samples were stored at −80 °C until further analysis. All for various biotechnological products, bioenergy production efforts were made to avoid surface contamination. and novel biotransformations (Kalia et al. 2003). In addition, such studies will help to determine bacterial communities with Physiochemical analyses of the soil sample a high tolerance for hydrocarbon contamination. Their phys- iological response and stronger biodegradation efficiency can Physiochemical analysis of the soil samples was performed as ultimately be exploited for developing economic bioremedia- described by Marinari et al. (2006). Total organic carbon tion technologies. (TOC) was determined by the dichromate oxidation method In the study reported here, we investigated a site contam- (Nelson and Sommers 1982). Soil total lead (Pb), copper, inated with hydrocarbons using a culture-independent ap- cobalt (Co), arsenic, cadmium, selenium and chromium (Cr) proach in order to gain insight into the microbial community were analysed by digesting 0.1 g soil with HCLO4–HNO3– composition. Microbial diversity from a neighbouring uncon- HF (Nautiyal et al. 2010). The digested solution was washed taminated agricultural soil (AS) was also analysed to better into a flask, and deionized water was added to a fixed volume. understand the effects of environmental conditions and soil The digested solutions were analysed with atomic absorption characteristics on microbial diversity. Our results suggest that spectroscopy (model-AA 300; Perkin Elmer, Waltham, MA) the phylogenetic diversity and distribution in the for heavy metals. Soil pH and electric conductivity were hydrocarbon-contaminated soil (HCS) was quite distinct and measured in 1:10 w/v aqueous solution using a pH meter different from that in the unpolluted AS. They also indicates (Thermo Fisher Scientific, Waltham, MA). that microorganisms differ with habitats, are less diverse in contaminated sites and are dominated by populations that are Extraction of total petroleum hydrocarbon from contaminated well adapted to survive under these conditions. soil Total petroleum hydrocarbon (TPH) was extracted in an equal Materials and methods ratio of hexane and dichloromethane (1:1, each 100 ml) from 10 g of HCS and AS using the Soxhlet method (Soxtherm; Sampling site Gerardt GmbH, Königswinter, Germany). Extracts were dried at room temperature by evaporating the solvents under a The heavy oil belt of Gujarat, India is 30 km long, with a gentle nitrogen stream in the fume hood. After evaporation surface area of about 45 km2. The majority shareholder is the the amount of residual TPH recovered was determined gravi- Oil & Natural Gas Corporation Ltd., and several exploratory metrically (Mishra et al. 2001). oil wells with an extensive pipeline network are located at various sites in this oil belt. The village of Kalol, located Analysis of TPH fractions by gas chromatography 27 km east of Ahmadabad city (73°15′N/ 72°28′60E) also falls within this belt. It is characterized by a mean annual Following gravimetric quantification, the residual TPH was rainfall of 742 mm and maximum and minimum temperatures fractionated into alkane, aromatic, asphaltene and nitrogen– ranging between 45 °C and 4 °C, respectively. The farmers sulphur–oxygen fractions in a silica gel column (Mishra et al. grow mainly Pennisetum glaucum (L), Ricinus communis and 2001). The TPH sample (200 mg) was dissolved in a mixture Brassica juncea in an inter-cropping cycle. For the past 14 of hexane, loaded onto a silica gel column and eluted with years, the crops have been under organic management with solvents of different polarity. The alkane and aromatic Ann Microbiol (2015) 65:359–369 361 fractions were eluted with 100 ml of hexane and dichloro- Sequencing methane, respectively. Aliphatic and aromatic fractions were analysed by gas chromatography (GC-FID; 6890 series N; The template used for