Chemical-physical parameters and microbial community changes induced by electrodes polarization inhibit PCB dechlorination in a marine sediment.

Microbial reductive dechlorination of organohalogenated pollutants is often limited by the scarcity of electron donors, that can be overcome with microbial electrochemical technologies (METs). In this study, polarized electrodes buried in marine sediment microcosms were investigated to stimulate PCB reductive dechlorination under potentiostatic (-0.7 V vs Ag/AgCl) and galvanostatic conditions (0.025 mA·cm-2-0.05 mA·cm-2), using graphite rod as cathode and iron plate as sacrificial anode. A single circuit and a novel two antiparallel circuits configuration (2AP) were investigated. Single circuit polarization impacted the sediment pH and redox potential (ORP) proportionally to the intensity of the electrical input and inhibited PCB reductive dechlorination. The effects on the sediment's pH and ORP, along with the inhibition of PCB reductive dechlorination, were mitigated in the 2AP system. Electrodes polarization stimulated sulfate-reduction and promoted the enrichment of bacterial clades potentially involved in sulfate-reduction as well as in sulfur oxidation. This suggested the electrons provided were consumed by competitors of organohalide respiring bacteria and specifically sequestered by sulfur cycling, which may represent the main factor limiting the applicability of METs for stimulating PCB reductive dechlorination in marine sediments.

Site-specific response of sediment microbial community to supplementation of polyhydroxyalkanoates as biostimulants for PCB reductive dechlorination.

The use of biodegradable plastics is constantly raising, increasing the likeliness for these polymers to end up in the environment. Environmental applications foreseeing the intentional release of biodegradable plastics have been also recently proposed, e.g., for polyhydroxyalkanoates (PHAs) acting as slow hydrogen releasing compounds to stimulate microbial reductive dehalogenation processes. However, the effects of their release into the environment on the ecosystems still need to be thoroughly explored. In this work, the use of PHAs to enhance the microbial reductive dechlorination of polychlorobiphenyls (PCBs) and their impact on the metabolic and compositional features of the resident microbial community have been investigated in laboratory microcosms of a polluted marine sediment from Mar Piccolo (Taranto, Italy), and compared with recent findings on a different contaminated marine sediment from Pialassa della Baiona (Ravenna, Italy). A decreased biostimulation efficiency of PHAs on PCBs reductive dechlorination was observed in the sediment from Mar Piccolo, with respect to the sediment from Pialassa della Baiona, suggesting that the sediments' physical-chemical characteristics and/or the biodiversity and composition of its microbial community might play a key role in determining the outcome of this biostimulation strategy. Regardless of the sediment origin, PHAs were found to have a specific and pervasive effect on the sediment microbial community, reducing its biodiversity, defining a newly arranged microbial core of primary degraders and consequently affecting, in a site-specific way, the abundance of subdominant bacteria, possibly cross-feeders. Such potential to dramatically change the structure of autochthonous microbial communities should be carefully considered, since it might have secondary effects, e.g., on the natural biogeochemical cycles.

Bacterial colonization dynamics of different microplastic types in an anoxic salt marsh sediment and impact of adsorbed polychlorinated biphenyls on the plastisphere.

Plastic debris dispersed into the environment provide a substrate for microbial colonization, constituting a new human-made ecosystem called "plastisphere", and altering the microbial species distribution in aquatic, coastal and benthic ecosystems. The study aims at exploring the interaction among microplastics (MPs) made of different polymers, a persistent organic contaminant (polychlorinated biphenyls, PCBs), and the environmental microbial communities, in an anoxic marine sediment. Plastic pellets were incubated in the field in a salt marsh anoxic sediment, to observe the stages of plastisphere formation, by quantitative PCR and 16S rRNA gene sequencing, and PCB dechlorination activity on the MPs surface. Microbes from the sediment rapidly colonized the different microplastics types, with PVC recruiting a peculiar community enriched in sulfate-reducing bacteria. The composition of the plastisphere varied along the 1-year incubation possibly in response either to warmer temperatures in spring-summer or to microhabitat's changes due to the progressive plastic surface weathering. Even if PCB contaminated MPs were able to recruit potentially dehalogenating taxa, actual dechlorination was not detectable after 1 year. This suggests that the concentration of potentially dehalorespiring bacteria in the natural environment could be too low for the onset of the dechlorination process on MP-sorbed contaminants. Our study, which is among very few available longitudinally exploring the plastisphere composition in an anoxic sediment context, is the first exploring the fate and possible biodegradation of persistent organic pollutants sorbed on MPs reaching the seafloor.

Unraveling the Metabolic Potential of Asgardarchaeota in a Sediment from the Mediterranean Hydrocarbon-Contaminated Water Basin Mar Piccolo (Taranto, Italy).

Increasing number of metagenome sequencing studies have proposed a central metabolic role of still understudied Archaeal members in natural and artificial ecosystems. However, their role in hydrocarbon cycling, particularly in the anaerobic biodegradation of aliphatic and aromatic hydrocarbons, is still mostly unknown in both marine and terrestrial environments. In this work, we focused our study on the metagenomic characterization of the archaeal community inhabiting the Mar Piccolo (Taranto, Italy, central Mediterranean) sediments heavily contaminated by petroleum hydrocarbons and polychlorinated biphenyls (PCB). Among metagenomic bins reconstructed from Mar Piccolo microbial community, we have identified members of the Asgardarchaeota superphylum that has been recently proposed to play a central role in hydrocarbon cycling in natural ecosystems under anoxic conditions. In particular, we found members affiliated with Thorarchaeota, Heimdallarchaeota, and Lokiarchaeota phyla and analyzed their genomic potential involved in central metabolism and hydrocarbon biodegradation. Metabolic prediction based on metagenomic analysis identified the malonyl-CoA and benzoyl-CoA routes as the pathways involved in aliphatic and aromatic biodegradation in these Asgardarchaeota members. This is the first study to give insight into the archaeal community functionality and connection to hydrocarbon degradation in marine sediment historically contaminated by hydrocarbons.

Microbial colonization of different microplastic types and biotransformation of sorbed PCBs by a marine anaerobic bacterial community.

We investigated the colonization dynamics of different microplastic (MP) pellets, namely, polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), polypropylene (PP) and polyvinyl chloride (PVC), either pristine or contaminated with polychlorinated biphenyls (PCBs), by an organohalide respiring marine microbial community and its biotransformation activity towards PCBs sorbed on MPs, in anaerobic laboratory microcosms of a marine sediment. All MPs were rapidly colonized by the microbial community within 2 weeks of incubation, when approximately 1010 16S rRNA gene copies cm-2 were detected on PVC, 109 copies cm-2 on PE, and 108 copies cm-2 on PET, PP and PS. A greater biofilm growth on PVC pellets than other MPs was confirmed by quantification of the reducing sugars of the EPS and biofilm staining with crystal violet. Illumina sequencing of the 16S rRNA genes and Principal Coordinate Analysis (PCoA) revealed that the biofilm community on MPs significantly differed from the sediment community, being enriched of chemoorganotrophic fermenting species, and was significantly affected by the type of polymer. The presence of sorbed PCBs did not significantly affect the overall community composition, and mainly resulted in the enrichment of Dehalococcoidia, i.e., of the organohalide respiring members of the community. Reductive dechlorination of PCBs sorbed to MPs was observed after 2 weeks of incubation, when the average number of chlorines per biphenyl molecule was reduced from 5.2 to 4.8-4.3, and was faster (35.2 ± 1.9 to 61.2 ± 5.8 µmol of Cl removed kgMP-1 week-1) than that of sediment-sorbed ones (33.9 ± 9.1 µmol of Cl removed kgsediment-1 week-1), which started only after 10 weeks of incubation. These data suggest that microbial colonization of contaminated MPs might change the composition of sorbed PCB mixtures and therefore the toxicity associated to PCB-polluted MPs.

Identification of two organohalide-respiring Dehalococcoidia associated to different dechlorination activities in PCB-impacted marine sediments.

BACKGROUND: Microbial reductive dechlorination of polychlorinated biphenyls (PCBs) plays a major role in detoxifying anoxic contaminated freshwater and marine sediments from PCBs. Known members of the phylum Chloroflexi are typically responsible for this activity in freshwater sediments, whereas less is known about the microorganisms responsible for this activity in marine sediments. PCB-respiring activities were detected in PCB-impacted marine sediments of the Venice Lagoon. The aim of this work was to identify the indigenous organohalide-respiring microorganisms in such environments and assess their dechlorination specificity against spiked Aroclor™ 1254 PCBs under laboratory conditions resembling the in situ biogeochemistry. RESULTS: High PCB dechlorination activities (from 150 ± 7 to 380 ± 44 µmol of chlorine removed kg-1 week-1) were detected in three out of six sediments sampled from different locations of the lagoon. An uncultured non-Dehalococcoides phylotype of the class Dehalococcoidia closely related to Dehalobium chlorocoercia DF-1, namely phylotype VLD-1, was detected and enriched up to 109 16S rRNA gene copies per gram of sediment where dechlorination activities were higher and 25-4/24-4 and 25-2/24-2/4-4 chlorobiphenyls (CB) accumulated as the main tri-/dichlorinated products. Conversely, a different phylotype closely related to the SF1/m-1 clade, namely VLD-2, also enriched highly where lower dechlorination activity and the accumulation of 25-3 CB as main tri-chlorinated product occurred, albeit in the simultaneous presence of VLD-1. Both phylotypes showed growth yields higher or comparable to known organohalide respirers and neither phylotypes enriched in sediment cultures not exhibiting dechlorination. CONCLUSIONS: These findings confirm the presence of different PCB-respiring microorganisms in the indigenous microbial communities of Venice Lagoon sediments and relate two non-Dehalococcoides phylotypes of the class Dehalococcoidia to different PCB dechlorination rates and specificities.

Genomic and phenotypic characterization of the species Acinetobacter venetianus.

Crude oil is a complex mixture of hydrocarbons and other organic compounds that can produce serious environmental problems and whose removal is highly demanding in terms of human and technological resources. The potential use of microbes as bioremediation agents is one of the most promising fields in this area. Members of the species Acinetobacter venetianus have been previously characterized for their capability to degrade n-alkanes and thus may represent interesting model systems to implement this process. Although a preliminary experimental characterization of the overall hydrocarbon degradation capability has been performed for five of them, to date, the genetic/genomic features underlying such molecular processes have not been identified. Here we have integrated genomic and phenotypic information for six A. venetianus strains, i.e. VE-C3, RAG-1(T), LUH 13518, LUH 7437, LUH 5627 and LUH 8758. Besides providing a thorough description of the A. venetianus species, these data were exploited to infer the genetic features (presence/absence patterns of genes) and the short-term evolutionary events possibly responsible for the variability in n-alkane degradation efficiency of these strains, including the mechanisms of interaction with the fuel droplet and the subsequent catabolism of this pollutant.

Microbial dehalogenation of organohalides in marine and estuarine environments.

Marine sediments are the ultimate sink and a major entry way into the food chain for many highly halogenated and strongly hydrophobic organic pollutants, such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polybrominated diphenylethers (PBDEs) and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). Microbial reductive dehalogenation in anaerobic sediments can transform these contaminants into less toxic and more easily biodegradable products. Although little is still known about the diversity of respiratory dehalogenating bacteria and their catabolic genes in marine habitats, the occurrence of dehalogenation under actual site conditions has been reported. This suggests that the activity of dehalogenating microbes may contribute, if properly stimulated, to the in situ bioremediation of marine and estuarine contaminated sediments.

Application of a molecular based approach for the early detection of short term 3-chloroaniline shock loads on activated sludge bacterial community and functionality.

Microbial processes are central elements in wastewater treatment plants (WWTPs) to mineralize the organic matter, to degrade pollutants and to decrease the amount of suspended solids. This activity can be disrupted by organic and inorganic pollutants present in wastewater streams. Hence, it is of primary importance to investigate and monitor the structure and functionality of the sludge-resident microbial communities. We simulated a 3-chloroaniline (3-CA) shock load in 3-CA adapted and non-adapted semi-continuous activated-sludge (SCAS) reactors to selectively stress the Ammonia Oxidizing Bacteria (AOB). Recently developed setting-independent theoretical interpretation of molecular DNA and RNA fingerprinting patterns were used to evaluate the responses of the microbial populations. Ammonium accumulation in treated reactors upon 3-CA addition confirmed the disruption of the functionality under stress conditions. Molecular analyses coupled to their interpretation highlighted that shock loaded reactors clustered separately from non-treated ones, especially when AOBs community was specifically targeted. Furthermore, the interpretation of RNA-based analyses, as compared to DNA-based ones, allowed to more promptly depicting shifts in a stressed community. We showed that the use of RNA-based molecular tools in combination with a new set of parameters is a powerful tool to link functional failures with microbial structure modifications in WWTPs, providing a potential tool for a rational optimization of the processes (Microbial Resource Management - MRM).

Environmental conditions and community evenness determine the outcome of biological invasion.

Biological invasion is widely studied, however, conclusions on the outcome of this process mainly originate from observations in systems that leave a large number of experimental variables uncontrolled. Here using a fully controlled system consisting of assembled bacterial communities, we evaluate the degree of invasion and the effect on the community functionality in relation to the initial community evenness under specific environmental stressors. We show that evenness influences the level of invasion and that the introduced species can promote functionality under stress. The evenness-invasibility relationship is negative in the absence and neutral in the presence of stress. Under these conditions, the introduced species is able to maintain the functionality of uneven communities. These results indicate that communities, initially having the same genetic background, in the presence of the same invader, react in a different way with respect to invasibility and functionality depending on specific environmental conditions and community evenness.

Bioaugmentation of a historically contaminated soil by polychlorinated biphenyls with Lentinus tigrinus.

BACKGROUND: Several species belonging to the ecological group of white-rot basidiomycetes are able to bring about the remediation of matrices contaminated by a large variety of anthropic organic pollutants. Among them, polychlorobiphenyls (PCBs) are characterized by a high recalcitrance due to both their low bioavailability and the inability of natural microbial communities to degrade them at significant rates and extents. Objective of this study was to assess the impact of a maize stalk-immobilized Lentinus tigrinus CBS 577.79 inoculant combined with soybean oil (SO), as a possible PCB-mobilizing agent, on the bioremediation and resident microbiota of an actual Aroclor 1260 historically contaminated soil under unsaturated solid-phase conditions. RESULTS: Best overall PCB depletions (33.6 ± 0.3%) and dechlorination (23.2 ± 1.3%) were found after 60 d incubation in the absence of SO where, however, the fungus appeared to exert adverse effects on both the growth of biphenyl- and chlorobenzoate-degrading bacteria and the abundance of genes coding for both biphenyl dioxygenase (bph) and catechol-2,3-dioxygenase. A significant (P < 0.001) linear inverse relationship between depletion yields and degree of chlorination was observed in both augmented and control microcosms in the absence of SO; conversely, this negative correlation was not evident in SO-amended microcosms where the additive inhibited the biodegradation of low chlorinated congeners. The presence of SO, in fact, resulted in lower abundances of both biphenyl-degrading bacteria and bph. CONCLUSIONS: The PCB depletion extents obtained in the presence of L. tigrinus are by far higher than those reported in other remediation studies conducted under unsaturated solid phase conditions on actual site soils historically contaminated by Aroclor 1260. These results suggest that the bioaugmentation strategy with the maize stalk-immobilized mycelium of this species might be promising in the reclamation of PCB-contaminated soils. The addition of SO to matrices contaminated by technical PCB mixtures, such as Aroclor 1242 and Delor 103 and characterized by a large preponderance of low chlorinated congeners, might not be advisable.

A Chloroflexi bacterium dechlorinates polychlorinated biphenyls in marine sediments under in situ-like biogeochemical conditions.

We investigated the reductive dechlorination of Aroclor 1254 PCBs by a coplanar PCB-dechlorinating microbial community enriched from an actual site contaminated marine sediment of the Venice lagoon in sterile slurry microcosms of the same sediment suspended in its site water, i.e., under biogeochemical conditions that closely mime those occurring in situ. The culture dechlorinated more than 75% of the penta- through hepta-chlorinated biphenyls to tri- and tetra-chlorinated congeners in 30 weeks. The dechlorination rate was reduced by the addition of H(2) and short chain fatty acids, which stimulated sulfate-reduction and methane production, and markedly increased by the presence of vancomycin or ampicillin. DGGE analysis of 16S rRNA genes on PCB-spiked and PCB-free cultures ruled out sulfate-reducing and methanogenic bacteria and revealed the presence of a single Chloroflexi phylotype closely related to the uncultured bacteria m-1 and SF1 associated to PCB dechlorination. These findings suggest that a single dechlorinator is responsible for the observed extensive dechlorination of Aroclor 1254 and that a Chloroflexi species similar to those already detected in freshwater and estuarine contaminated sediments mediates PCB dechlorination in the marine sediment adopted in this study under biogeochemical conditions resembling those occurring in situ in the Brentella Canal of Venice Lagoon.

Selection of commercial hydrolytic enzymes with potential antifouling activity in marine environments.

In this work, the marine antifouling potential of some commercially available hydrolytic enzymes acting on the main constituents of extracellular polymeric substances (EPS) involved in bacterial biofilm formation was determined. The selected protease (i.e., alpha-chymotrypsin from bovine pancreas), carbohydrase (i.e., alpha-amylase from porcine pancreas) and lipase (from porcine pancreas) exhibited remarkable hydrolytic activities towards target macromolecules typically composing EPS under a wide range of pHs (6.5-9.0 for alpha-chymotrysin and alpha-amylase; 7.0-8.5 for the lipase) and temperatures (from 10 °C to 30 °C), as well as relevant half-lives (from about 2 weeks to about 2 months), in a marine synthetic water. The activity displayed by each enzyme was poorly affected by the co-presence of the other enzymes, thus indicating their suitability to be employed in combination. None of the enzymes was able to inhibit the formation of biofilm by an actual site marine microbial community when applied singly. However, a mixture of the same enzymes reduced biofilm formation by about 90% without affecting planktonic growth of the same microbial community. This indicates that multiple hydrolytic activities are required to efficiently prevent biofilm formation by complex microbial communities, and that the mixture of enzymes selected in this study has the potential to be employed as an environmental friendly antifouling agent in marine antifouling coatings.

Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid.

Vanillin is one of the most important flavors in the food industry and there is great interest in its production through biotechnological processes starting from natural substrates such as ferulic acid. Among bacteria, recombinant Escherichia coli strains are the most efficient vanillin producers, whereas Pseudomonas spp. strains, although possessing a broader metabolic versatility, rapidly metabolize various phenolic compounds including vanillin. In order to develop a robust Pseudomonas strain that can produce vanillin in high yields and at high productivity, the vanillin dehydrogenase (vdh)-encoding gene of Pseudomonas fluorescens BF13 strain was inactivated via targeted mutagenesis. The results demonstrated that engineered derivatives of strain BF13 accumulate vanillin if inactivation of vdh is associated with concurrent expression of structural genes for feruloyl-CoA synthetase (fcs) and hydratase/aldolase (ech) from a low-copy plasmid. The conversion of ferulic acid to vanillin was enhanced by optimization of growth conditions, growth phase and parameters of the bioconversion process. The developed strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin produced by a Pseudomonas strain to date and opens new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes which contain ferulic acid.

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

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.