Microb Ecol DOI 10.1007/s00248-017-0973-3
ENVIRONMENTAL MICROBIOLOGY
Distribution of Anaerobic Hydrocarbon-Degrading Bacteria in Soils from King George Island, Maritime Antarctica Dayanna Souza Sampaio 1 & Juliana Rodrigues Barboza Almeida 1 & Hugo E. de Jesus 1 & Alexandre S. Rosado 1 & Lucy Seldin 1 & Diogo Jurelevicius 1,2
Received: 7 December 2016 / Accepted: 24 March 2017 # Springer Science+Business Media New York 2017
Abstract Anaerobic diesel fuel Arctic (DFA) degradation has already been demonstrated in Antarctic soils. However, studies comparing the distribution of anaerobic bacterial groups and of anaerobic hydrocarbon-degrading bacteria in Antarctic soils containing different concentrations of DFA are scarce. In this study, functional genes were used to study the diversity and distribution of anaerobic hydrocarbon-degrading bacteria (bamA, assA, and bssA) and of sulfate-reducing bacteria (SRB—apsR) in highly, intermediate, and non-DFAcontaminated soils collected during the summers of 2009, 2010, and 2011 from King George Island, Antarctica. Signatures of bamA genes were detected in all soils analyzed, whereas bssA and assA were found in only 4 of 10 soils. The concentration of DFA was the main factor influencing the distribution of bamA-containing bacteria and of SRB in the analyzed soils, as shown by PCR-DGGE results. bamA sequences related to genes previously described in Desulfuromonas, Lautropia, Magnetospirillum, Sulfuritalea, Rhodovolum, Rhodomicrobium, Azoarcus, Geobacter, Ramlibacter, and Gemmatimonas genera were dominant in King George Island soils. Although DFA modulated the Electronic supplementary material The online version of this article (doi:10.1007/s00248-017-0973-3) contains supplementary material, which is available to authorized users. * Diogo Jurelevicius
[email protected] 1
Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
2
Laboratório de Genética Microbiana, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPPG), Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco I, Ilha do Fundão, Rio de Janeiro 21941-590, Brazil
distribution of bamA-hosting bacteria, DFA concentration was not related to bamA abundance in the soils studied here. This result suggests that King George Island soils show functional redundancy for aromatic hydrocarbon degradation. The results obtained in this study support the hypothesis that specialized anaerobic hydrocarbon-degrading bacteria have been selected by hydrocarbon concentrations present in King George Island soils. Keywords Anaerobic bacterial community . Anaerobic hydrocarbon-degrading bacterial population . Adenosine 5′-phosphosulphate reductase . Alkyl- and benzyl-succinate synthase . Benzoyl-CoA reductase . Antarctic soils
Introduction Environmental contamination with petroleum hydrocarbons is recurrently reported in polar environments. Filler et al. [1] estimated the presence of 200 contaminated sites in Antarctica. Successful remediation of hydrocarbon-contaminated soils is rare in these sites [2]. On King George Island of the Antarctic Peninsula, the accidental diesel fuel Arctic (DFA) spill around Comandante Ferraz Antarctic Station (EACF, Brazil) that occurred in 1986 is still detectable decades later by the high concentration of total petroleum hydrocarbon (TPH) present in its soils [3]. To date, most studies about Antarctic soil bioremediation have sought aerobic hydrocarbon-degrading bacteria [3–6] and their genes coding for oxygen-dependent alkane monooxygenases [3, 7–10], and for aromatic-ring-hydroxylating dioxygenases [11, 12]. However, Powell et al. [13] showed that hydrocarbon degradation in fertilized hydrocarbon-contaminated Antarctic soils was higher under anaerobiosis than under aerobic conditions. In addition, Powell et al. [13] demonstrated that O2 input in
Sampaio D. S. et al.
anaerobic soils suppressed aliphatic and aromatic hydrocarbon degradation in Antarctic microcosms [13]. Consistent with the results presented by Powell et al. [13], Vázques et al. [14] showed enrichment of genes related to bacterial anaerobic metabolism, such as denitrification, in fertilized microcosms constructed with hydrocarbon-contaminated Antarctic soils. Similarly, Yergeau et al. [15] reported increased expression of denitrification genes in fertilized microcosms containing hydrocarbon-contaminated Arctic soil. Furthermore, the presence of genes coding for benzoyl-CoA reductase (bamA)—a universal biomarker for anaerobic degradation of aromatic compounds [16]—and of genes coding for benzyl- and alkyl-succinate synthase genes (assA and bssA) related to anaerobic aromatic and aliphatic hydrocarbon degradation, respectively [17], has been demonstrated in Antarctic soils [13, 18]. It is important to highlight that Antarctic soils are generally ice-covered, which reduces O2 diffusion and microbial aerobic metabolism. As shown by Teixeira et al. [19], bacterial communities of non-contaminated King George Island soils predominantly comprised representatives of anaerobic bacteria. Furthermore, the annual thaw and the presence of high levels of organic carbons in contaminated soils also contribute to a reduction in oxygen potential, thereby creating anaerobic zones [20]. Thus, anaerobic hydrocarbon degradation is of crucial importance for the natural attenuation of Antarctic soils. Peixoto et al. [21] isolated different anaerobic bacteria with the catabolic potential to degrade xenobiotic compounds from King George Island soils. However, while clearly informative, results obtained to date have not identified the bacterial community capable of anaerobically degrading hydrocarbons in Antarctic soils. Anaerobic hydrocarbon degradation has already been demonstrated in diverse anaerobic bacterial groups, such as in sulfate-, iron-, nitrate-, and nitritereducing bacteria [22, 23]. To encompass this diversity, molecular analyses based on catabolic gene markers have been used to study anaerobic hydrocarbon-degrading bacteria in different environments [17, 23–26]. Here, we determined the presence and distribution of anaerobic bacteria in DFAcontaminated and non-contaminated soils collected from King George Island. Specifically, we examined whether contamination influenced the distributions of sulfate-reducing bacteria (SRB—used here as a model of the anaerobic bacterial community) and anaerobic hydrocarbon-degrading bacteria (using signatures of the genes bamA, assA, and bssA). We used genetic fingerprinting, quantitative PCR, and clone libraries to study the anaerobic bacterial communities in 30 soil samples (representing 10 different sampling locations). We then analyzed these data with non-metric multidimensional scaling (NMDS) to determine the principles structuring anaerobic bacterial communities among DFA-contaminated and non-contaminated soils from King George Island, Antarctica.
Methods Site Description The samples used in this study were collected from around Comandante Ferraz Antarctic Station (EACF) in the summers of 2009 (samples sA, sB, and sC), 2010 (s1, s2, s3, s4, and s5), and 2011 (Ctr and SN), as described in detail by Jurelevicius et al. [12], Cury et al. [18], and Jesus et al. [3], respectively. All necessary permits were obtained for the field studies described here. In total, 30 samples were used, representing triplicates of each collecting site (9 samples collected in 2009, 15 samples from 2010, and 6 samples from 2011). Physical and chemical properties of the soil samples, including total petroleum hydrocarbon (TPH) content, were previously characterized [3, 12, 18]. We used these established TPH values to categorize the soil samples as highly (>1000 μg/g of THP in soil), intermediate (from 110 to 1000 μg/g of THP), and noncontaminated (
