Andrea Negroni

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.

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.

Influence of chemical and architectural modifications on the enzymatic hydrolysis of poly(butylene succinate)

Copolymers of poly(butylene succinate) (PBS) containing diethylene succinate sequences (PBSPDGS) with different molecular architectures were prepared via reactive blending in the presence of a Ti-based catalyst. In particular, a block copolymer with long sequences and a random one with very short sequences were synthesized, characterized and investigated in terms of enzymatic biodegradability. For comparison, the parent homopolymer PBS has been also prepared by the usual two-stage melt polycondensation. Preliminary biodegradation tests based on the highly sensitive film opacity assay indicated that lipase from Candida cylindracea was the most effective among four different commercially available lipases (e.g., those from Candida rugosa, Candida cylindracea, Aspergillus niveus and hog pancreas) and a serine protease (α-chymotrypsin from bovine pancreas), and that optimal test conditions were 50 enzyme U mL−1, 30 °C and pH 7.0. Under such conditions, copolymers degraded to a much higher extent as compared to PBS. Moreover, the random copolymer degraded 100 times faster than the block one. ATRIR analysis and DSC measurements indicated that the enzyme attacked the amorphous phase first. Further, NMR analysis indicated that enzyme hydrolysis involved preferentially ester groups of DGS sequences, more hydrophilic than the others. These findings confirm previous evidence on the correlation between polymers biodegradation rate and their hydrophilic and amorphous degree. More importantly, they indicate (i) that dramatic increases in polyesters biodegradability can be obtained by introducing ether-oxygen atoms into the polymer chain and (ii) that biodegradability of oxygen etheroatom-containing copolyesters might be tuned within a wide range of rates through the modification of their molecular architecture.

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.

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.

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.

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

BackgroundSeveral 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.ResultsBest 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.ConclusionsThe 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.

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.

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-1T, 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.

Biological fate of Diuron and Sea-nine® 211 and their effect on primary microbial activities in slurries of a contaminated sediment from Venice Lagoon

The individual fate of the industrial booster biocides Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and Sea-nine® 211 (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one) in a marine sediment of Porto Marghera (Venice Lagoon, Italy) was investigated, under anaerobic and aerobic conditions, in slurry-phase microcosms consisting of sediment suspended in its own overlaying water, i.e. under biogeochemical conditions which closely mimic those occurring in situ. Biocides were singly supplied as methanol solutions to a final concentration of 50xa0mg/kg of dry sediment. No Diuron degradation occurred throughout 3xa0months of incubation under any of the conditions tested. Conversely, Sea-nine®211 was promptly and completely degraded, regardless of the presence of oxygen. Under anoxic conditions, neither sulphate reduction nor methanogenesis occurred significantly in the non-amended microcosms, probably because of the very low availability of substrates in the actual site sediment and water employed. In contrast, methanogenesis occurred in the biocide amended microcosms where methanol was supplied as the carrier for biocides. No sulphate consumption was observed in the same microcosms. The large methane production observed in the parallel biocide-free control microcosms supplemented with methanol suggested that methylotrophic methanogens were the main compounds responsible for the detected activities. Diuron was found to intensify methane production, probably because it exerted an inhibitory effect on some competitors of indigenous methylotrophic methanogens.