Characterization of the microbial community from the marine sediment of the Venice lagoon capable of reductive dechlorination of coplanar polychlorinated biphenyls (PCBs)

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Abstract

The native microbial community of a contaminated sediment from Brentella Canal (Venice Lagoon, Italy) was enriched in slurry microcosms consisting of sterile sediment suspended in sterile site water in the presence of 3,3′,4,4′-tetrachlorobiphenyl, 3,3′,4,4′,5- and 2,3′,4,4′,5-pentachlorobiphenyls, 3,3′,4,4′,5,5′- and 2,3,3′,4,4′,5-hexachlorobiphenyls. The enrichment cultures were characterized at each subculturing step by 16S rRNA gene Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE) analysis. About 90% of spiked polychlorinated biphenyls (PCBs) were stoichiometrically converted into di- and tri-chlorinated congeners by each enriched culture via dechlorination of flanked para chlorines and ortho-flanked meta chlorines. A 2-fold increase in PCB-dechlorination rate, the disappearance of lag phase, as well as a remarkable increase of sulfate consumption and a decline of methanogenic activity, were observed throughout subculturing. A reduction of complexity of the archaeal community, which was composed by Methanomicrobiales and Methanosarcinales, was also observed as a result of culture enrichment. The bacterial community included members of the Alpha, Gamma, Delta and Epsilon divisions of Proteobacteria, Firmicutes and Chloroflexi. Two sequence phylotypes related to the genus Sulforovum and the species Desulfococcus multivorans and two Chloroflexi enriched throughout subculturing, thus suggesting that these bacteria were involved in PCB dechlorination in the marine sediments of Brentella canal.

Introduction

Polychlorinated biphenyls (PCBs) are priority pollutants that extensively accumulate in the anoxic reduced subsurface sediments of aquatic ecosystems because of their very low water solubility and high chemical stability [1]. Due to their strong hydrophobicity, PCBs persisting in such sediments enter the food chain through sediment dwelling organisms, thus accumulating in the fatty tissues of animals and humans, where they can exert multiple adverse health effects [2], [3].

Despite their recalcitrance to biodegradation, the microbial mediated anaerobic reductive dechlorination of PCBs was reported in a variety of anaerobic sediments [4], [5], [6]. The process consists in the sequential reduction of highly chlorinated, toxic and bioaccumulable PCBs into lesser chlorinated congeners commonly having lower toxicity, lower bioaccumulation potential and higher susceptibility to undergo mineralization by aerobic bacteria [6]. If occurring in situ, it might therefore mediate a remarkable detoxification of the site and/or reduce of the volume of sediments that need to be removed by expensive and highly impacting dredging operations [7].

PCB dechlorination and PCB-dechlorinating microbial communities have been mainly studied in slurry or sediment-free cultures of a number of freshwater habitats developed in synthetic mineral media supplemented with target PCBs [4], [5], [6], where reductive dechlorination has been ascribed to the activity of different indigenous dehalogenating bacteria [5], [6].

During the last decade, a number of studies investigated the structure and composition of PCB-dechlorinating microbial communities from freshwater habitats. Several species of the delta subgroup of Proteobacteria, low-G + C Gram positive bacteria and Thermotogales, along with a single species with low sequence similarity to Dehalococcoides ethenogenes, were found to be predominant during ortho-dechlorination of 2,3,5,6-tetrachlorobiphenyl in a methanogenic enrichment culture of an estuarine sediment collected from the Baltimore Harbor suspended in a salts medium [8]. A Dehalococcoides-like bacterium, namely bacterium o-17, was later identified as the 2,3,5,6-tetrachlorobiphenyl ortho-dechlorinating microbe in sediment-free enrichments obtained from the same culture [9]. Similar results were described for a 2,3,4,5-tetrachlorobiphenyl para-dechlorinating sediment-free culture enriched from Charleston Harbor estuarine sediments, where Deltaproteobacteria, low-G + C Gram positive bacteria, Thermotogales and Green non-sulfur bacteria were detected [10], along with a Dehalococcoides-like microorganism, namely bacterium DF-1, later identified as the dechlorinator [11]. Several phylotypes closely related to the o-17/DF-1 group as well as sequences highly similar to Dehalococcoides spp. were detected in other sediment-free cultures enriched from estuarine sediments of Baltimore Harbor capable of dechlorinating different single PCB congeners [12], [13] as well as Aroclor 1260 PCBs [14]. PCB-dechlorinating communities enriched from the Woods Pond section of Housatonic river and Hudson river in sediment-free mineral medium amended with single PCB congeners or Aroclor 1260 were found to contain several different phylotypes, such as Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Clostridiales, Bacteroidetes and Dehalococcoides spp. [15], [16], [17], the latter of which were indicated as the dechlorinators. On the contrary, no Dehalococcoides spp. or o-17/DF-1-like microorganisms were detected in sediment slurry cultures that were inoculated with microorganisms eluted from St. Lawrence river, where the enrichment of sequences highly homologous to Clostridium pascui and Dehalobacter restrictus was correlated to the dechlorination of spiked Aroclor 1248 PCBs [18].

Much less is known about PCB reductive dechlorination processes and the dehalorespiring microbial communities potentially active in marine sediments [1], [19], [20], [21], [22], [23], [24], where the occurrence of different biogeochemical conditions, such as higher salinity, availability of sulfate and occurrence of a wide variety of halogenated organic compounds naturally produced by indigenous organisms, might favor the enrichment of dehalogenating microorganisms different from those occurring in freshwater habitats. Furthermore, knowledge on the activity and diversity of the native sediment microbial communities enriched in the presence of water from the site, i.e., under biogeochemical conditions very close to those occurring in situ, might provide new insights on PCB dehalogenating strains in sediment and information of special relevance to design a tailored in situ biostimulation [7].

PCB dechlorination was recently documented in five contaminated marine sediments of the Brentella Canal (Porto Marghera, Venice Lagoon) when suspended either in a synthetic marine medium or in water coming from the site, i.e., under laboratory geochemical conditions very close to those occurring in situ. Interestingly, the dechlorination was more extensive in the presence of the site water than in mineral medium [22], thus suggesting the ability of the PCB-dechlorinating microbial community of the sediment to actively work under the conditions occurring in situ. In addition, the process was selective towards meta and para positions of PCB molecules and was apparently mediated by sulfate-reducing spore forming bacteria [23]. Finally, it was not primed by the addition of exogenous PCBs, including coplanar very toxic PCBs, which however were extensively dechlorinated with rate, extent and selectivity higher than those described so far in the literature [24]. Therefore, it was of special interest to enrich and to characterize the sediment native microbial communities in charge of the dechlorination process. Culturing conditions miming the biogeochemical features of the site were adopted in order to enrich the microflora better representing that occurring at the site. This might provide information on the actual potential of sediments to undergo in situ natural restoration.

Section snippets

PCB-dechlorinating cultures

The microbial community from a coplanar PCBs dechlorinating primary slurry culture [24] consisting of a PCB contaminated sediment of the Brentella canal (first industrial area of Porto Marghera, Venice Lagoon, Italy) suspended at 25% (v/v) in its own site water, hereinafter designated M1C, was subcultured in sterile slurry microcosms consisting of autoclave-sterilized sediment suspended in filter-sterilized water coming from the same site, in order to simulate the biogeochemical conditions

PCB dechlorination and other microbial activities

Spiked 3,3′,4,4′,5,5′- and 2,3,3′,4,4′,5-hexachlorobiphenyl, 3,3′,4,4′,5- and 2,3′,4,4′,5-pentachlorobiphenyl and 3,3′,4,4′-tetrachlorobiphenyl were initially detected in the slurry culture M2C (i.e., the first subculturing step inoculated with M1C culture) at the concentration of 406 ± 21, 369 ± 19, 405 ± 28, 439 ± 37 and 382 ± 40 μmol kg−1 of dry sediment, respectively. Some lower chlorinated PCBs not occurring in the spiked mixture were also detected in the microcosm at the beginning of incubation (Fig.

Conclusions

The PCB dehalogenating microbial community of a marine sediment from the Brentella canal (Venice lagoon), was enriched in laboratory slurry microcosms consisting of sediment suspended in the water of the site, i.e., under biogeochemical conditions that mimic those occurring in situ. The enriched culture dechlorinated five of the most toxic, dioxin-like coplanar PCB congeners with rate and extent remarkably higher than those reported so far in the literature [31]. Similarly to other reports [8],

Acknowledgments

The authors sincerely thank Dr. J.R. Peréz-Jimenéz and Prof. L.J. Kerkhof (Rutgers University, NJ) for their support in the T-RFLP analysis, and Dr. E. Zanotto (Venice Port Authority, Italy) for having supplied the sediment and water samples employed in the study. This research was funded by the Italian MIUR and the ENVIREN laboratories cluster of the Emilia Romagna Region (Italy).

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