Influence of compost amendments on the diversity of alkane degrading bacteria in hydrocarbon contaminated soils

ORIGINAL RESEARCH ARTICLE published: 13 March 2014 doi: 10.3389/fmicb.2014.00096 Effects of different compost amendments on the abundance and composition of alkB harboring bacterial communities in a soil under industrial use contaminated with hydrocarbons Stefanie Wallisch 1 , Tjasa Gril 1 , Xia Dong 1 , Gerd Welzl 1 , Christian Bruns 2 , Ester Heath 3 , Marion Engel 1 , Marjetka Suhadolc 4 and Michael Schloter 1* 1 Research Unit Environmental Genomics, Helmholtz Zentrum München, Munich, Germany Organic Agricultural Sciences, University of Kassel, Witzenhausen, Germany 3 Jožef Stefan Institute, Ljubljana, Slovenia 4 Biotechnical Faculty,Center for Soil and Environmental Science,University of Ljubljana, Ljubljana, Slovenia 2 Edited by: Paul Bodelier, Netherlands Institute of Ecology (NIOO-KNAW), Netherlands Reviewed by: Paul Bodelier, Netherlands Institute of Ecology (NIOO-KNAW), Netherlands Guy Charles Abell, CSIRO, Australia *Correspondence: Michael Schloter, Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Ingolstädter Landstraße 1, 85764 Neuherberg, Germany e-mail: Alkane degrading microorganisms play an important role for the bioremediation of petrogenic contaminated environments. In this study, we investigated the effects of compost addition on the abundance and diversity of bacteria harboring the alkane monooxygenase gene (alkB) in an oil-contaminated soil originated from an industrial zone in Celje, Slovenia (Technosol). Soil without any amendments (control soil) and soil amended with two composts differing in their maturation stage and nutrient availability, were incubated under controlled conditions in a microcosm experiment and sampled after 0, 6, 12, and 36 weeks of incubation. As expected the addition of compost stimulated the degradation of alkanes in the investigated soil shortly after the addition. By using quantitative real-time PCR higher number of alkB genes were detected in soil samples amended with compost compared to the control soils. To get an insight into the composition of alkB harboring microbial communities, we performed next generation sequencing of amplicons of alkB gene fragment. Richness and diversity of alkB gene harboring prokaryotes was higher in soil mixed with compost compared to control soils with stronger effects of the less maturated, nutrient poor compost. The phylogenetic analysis of communities suggested that the addition of compost stimulated the abundance of alkB harboring Actinobacteria during the experiment independent from the maturation stage of the compost. AlkB harboring γ-proteobacteria like Shewanella or Hydrocarboniphaga as well as α-proteobacteria of the genus Agrobacterium responded also positively to the addition of compost to soil. The amendment of the less maturated, nutrient poor compost resulted in addition in a large increase of alkB harboring bacteria of the Cytophaga group (Microscilla) mainly at the early sampling time points. Our data indicates that compost amendments significantly change abundance and diversity pattern of alkB harboring microbes in Technosol and might be a useful agent to stimulate bioremediation of hydrocarbons in contaminated soils. Keywords: alkane monooxygenase alkB, compost, contaminated soils, bioremediation, next generation sequencing INTRODUCTION Since almost 150 years industry has been using petroleum-based products as major source of energy (OPEC, 2012). Therefore, the number of industrialized as well as remote areas that show a serious degree of contamination with substances which derive from petroleum is continuously increasing, as the risk for accidental spills or leaks during oil exploration, the industrial manufacturing processes and transport is high (SOER, 2010). Linear and branched alkanes, cycloalkanes and other aromatic compounds being part of petroleum are then released to the environment and have contaminated soil ecosystems as well as water bodies (Pinedo et al., 2013). www.frontiersin.org However, alkanes are also naturally produced by many living organisms. Alkane derivatives are for example part of plant waxes (Eglinton et al., 1962), pheromones produced by animals (Mori, 2007) or fungal spores (Oró et al., 1966; Fisher et al., 1972). Thus, many microbes are able to degrade alkanes either as mean of detoxification or as source of energy (Ayala and Torres, 2004). Depending on the length of the carbon chain, different types of alkane hydroxylases have been described so far: Alkanes with one to four carbon residues (C1 –C4 ) are mainly degraded by the methane monooxygenases (Jiang et al., 2010) as well as propane- (Steffan et al., 1997) and butane oxygenases (Dubbels et al., 2007); alkanes with medium chain length (C6 –C11 ) are March 2014 | Volume 5 | Article 96 | 1 Wallisch et al. metabolized by the alkane monooxygenase system (Kloos et al., 2006), which is mainly found amongst bacteria or by the fungal cytochrome P450 enzyme system (Van Beilen and Funhoff, 2005); long chain alkanes (>C20 ) are preferably transformed by LadA and other hydroxylases (Ji et al., 2013). One organism can harbor several alkane degrading enzyme systems that are activated depending on the quality of the alkanes (Rojo, 2009). Microbes play a very important role in the development of remediation strategies of sites contaminated with alkanes. However, mainly in soils with technical origin (Technosols) the potential to degrade alkanes after a contamination with petroleum is low. This might be a result of the overall low microbial biomass and activity as a consequence of missing nutrients (Scalenghe and Ferraris, 2009). Furthermore, the low levels of natural alkanes due to the spare vegetation cover might have prohibited the development of an alkane degrading microflora in Technosols (Séré et al., 2008). Compost material, which is mainly based on plant derived litter material, contains high numbers of microbes that are capable to degrade hydrocarbons (Keeling et al., 1994). Therefore, in the past it has been investigated in several studies, if the addition of compost material may stimulate the degradation of pollutants like hydrocarbons in Technosols (e.g., Beaudin et al., 1999; Van Gestel et al., 2003). However, despite the often reported positive effects of compost amendments on the degradation of hydrocarbons in Technosols, it is not clear if these positive effects of compost addition are related to the introduced microbes and their genetic potential to degrade alkanes or to a general shift of microbial community structure in soil as a result of extra nutrients provided by the compost. Therefore, we investigated in this study the role of composts on the abundance and diversity of alkane degrading microbes in an alkane contaminated Technosol in a laboratory study. We used two contrasting types of composts of different maturation stages, which are characterized by contrasting nutrient levels, to investigate the influence of the compost type (...truncated)