Stefano Fedi

Microbial degradation of chloroform

Chloroform (CF) is largely produced by both anthropogenic and natural sources. It is detected in ground and surface water sources and it represents the most abundant halocarbon in the atmosphere. Microbial CF degradation occurs under both aerobic and anaerobic conditions. Apart from a few reports describing the utilization of CF as a terminal electron acceptor during growth, CF degradation was mainly reported as a cometabolic process. CF aerobic cometabolism is supported by growth on short-chain alkanes (i.e., methane, propane, butane, and hexane), aromatic hydrocarbons (i.e., toluene and phenol), and ammonia via the activity of monooxygenases (MOs) operatively divided into different families. The main factors affecting CF cometabolism are (1) the inhibition of CF degradation exerted by the growth substrate, (2) the need for reductant supply to maintain MO activity, and (3) the toxicity of CF degradation products. Under anaerobic conditions, CF degradation was mainly associated to the activity of methanogens, although some examples of CF-degrading sulfate-reducing, fermenting, and acetogenic bacteria are reported in the literature. Higher CF toxicity levels and lower degradation rates were shown by anaerobic systems in comparison to the aerobic ones. Applied physiological and genetic aspects of microbial cometabolism of CF will be presented along with bioremediation perspectives.

The membrane-bound respiratory chain of Pseudomonas pseudoalcaligenes KF707 cells grown in the presence or absence of potassium tellurite

The respiratory chain of Pseudomonas pseudoalcaligenes KF707 in membranes isolated from cells grown in the presence or absence of the toxic oxyanion tellurite (TeO3(2-)) was examined. Aerobic growth in the absence of tellurite shows an NADH-dependent respiration which is 80% catalysed by the cytochrome (cyt) bc1-containing pathway leading to two terminal membrane-bound cyt c oxidases inhibited by different concentrations of KCN (IC50 0.2 and 1 microM). A third oxidase, catalysing the remaining 20% of the cyanide-resistant respiration and fully inhibited by 2-3 mM KCN, is also present; this latter pathway accounts for 60-70% of the total NADH-dependent respiration in membranes from cells grown in LB medium supplemented with potassium tellurite (35 microg x ml(-1)). Two high-potential b-type haems (E(m,7) +395 and 318 mV) are redox centres of a membrane-bound cyt c oxidase (possibly of the cbb3 type) which shows a 50% decrease of its activity in parallel with a similar decrease of the c-type haem content (mostly soluble cyt c) in membranes from tellurite-grown cells; the latter type of cells specifically contain a cyt b type at +203 mV (pH 9.0) which is likely to be involved in cyanide-resistant respiration. Comparison of the growth curve of KF707 cells in parallel with tellurite uptake showed that intracellular accumulation of tellurium (Te(0)) crystallites starts from the mid-exponential growth phase, whereas tellurite-induced changes of the respiratory chain are already evident during the early stages of growth. These data were interpreted as showing that reduction of tellurite to tellurium and tellurite-dependent modifications of the respiratory chain are unrelated processes in P. pseudoalcaligenes KF707.

Polychlorinated biphenyl degradation activities and hybridization analyses of fifteen aerobic strains isolated from a PCB-contaminated site.

Fifteen bacterial strains using biphenyl as sole carbon and energy source, obtained from different positions and depths of a polychlorinated biphenyl (PCB)-contaminated area, were analyzed for their basic metabolic phenotypes and subjected to genomic DNA hybridization screening for the presence of well characterized bph operons such as those of Pseudomonas pseudoalcaligenes KF707 and Rhodococcus globerulus P6. Most of the isolates belonged to the gamma-subdivision (Pseudomonas stutzeri, P. plutida, P. fluorescens and Vibrio logei species) and to the beta-subdivision (genera Alcaligenes, Comamonas, Ralstonia) of the Proteobacteria. All the isolates were able to cometabolize different low chlorinated PCB congeners. Among the dichlorinated biphenyls tested, a lower degradation capacity was observed for the di-ortho substituted congeners, whereas high levels of degradation were observed for the di-meta and di-para isomers, whether they were chlorinated on one or on both rings. The PCB congeners nonsubstituted in the 2,3 or 2,3 and 3,4 positions were also degraded by most of the isolated strains, which were, however, unable to significantly metabolize PCBs with more than 3 chlorine atoms. Five of the isolated strains were also able to degrade some of the tri- and tetrachlorobiphenyls tested. Southern hybridization analysis showed a strong homology between four of the fifteen isolated strains and the bph operon obtained from P. pseudoalcaligenes strain KF707. Conversely, none of the isolates here examined showed homology with the bph operon of R. globerulus strain P6. In line with this, the KF707 bph probe strongly hybridized with DNA of a significant number of bacterial colonies obtained from selected locations in the contaminated area using biphenyl-supplemented minimal medium agar plates.

Tolerance of Pseudomonas pseudoalcaligenes KF707 to metals, polychlorobiphenyls and chlorobenzoates: effects on chemotaxis-, biofilm- and planktonic-grown cells

Pseudomonas pseudoalcaligenes KF707 is a polychlorinated biphenyls (PCBs) degrader, also tolerant to several toxic metals and metalloids. The work presented here examines for the first time the chemotactic response of P. pseudoalcaligenes KF707 to biphenyl and intermediates of the PCB biodegradation pathway in the presence and absence of metals. Chemotaxis analyses showed that biphenyl, benzoic acid and chlorobenzoic acids acted as chemoattractants for KF707 cells and that metal cations such as Ni(2+) and Cu(2+) strongly affected the chemotactic response. Toxicity profiles of various metals on KF707 cells grown on succinate or biphenyl as planktonic and biofilm were determined both in the presence and in the absence of PCBs. Notably, KF707 cells from both biofilms and planktonic cultures were tolerant to high amounts (up to 0.5 g L(-1)) of Aroclor 1242, a commercial mixture of PCBs. Together, the data show that KF707 cells are chemotactic and can form a biofilm in the presence of Aroclor 1242 and specific metals. These findings provide new perspectives on the effectiveness of using PCB-degrading bacterial strains in bioremediation strategies of metal-co-contaminated sites.

Analyses of both the alkB gene transcriptional start site and alkB promoter-inducing properties of Rhodococcus sp. strain BCP1 grown on n-alkanes.

ABSTRACT Rhodococcus sp. strain BCP1, known for its capacity to grow on short-chain n-alkanes (C2 to C7) and to cometabolize chlorinated solvents, was found to also utilize medium- and long-chain n-alkanes (C12 to C24) as energy and carbon sources. To examine this feature in detail, a chromosomal region which includes the alkB gene cluster encoding a non-heme di-iron monooxygenase (alkB), two rubredoxins, and one rubredoxin reductase was cloned from the BCP1 genome. Furthermore, the activity of the alkB gene promoter (P alkB ) was examined in the presence of gaseous, liquid, and solid n-alkanes along with intermediates of the putative n-alkane degradation pathway. A recombinant plasmid, pTPalkBLacZ, was constructed by inserting the lacZ gene downstream of P alkB , and it was used to transform Rhodococcus sp. strain BCP1. Measurements of β-galactosidase activity showed that P alkB is induced by C6 to C22 n-alkanes. Conversely, C2 to C5 and >C22 n-alkanes and alkenes, such as hexene, were not inducers of alkB expression. The effects on P alkB expression induced by alternative carbon sources along with putative products of n-hexane metabolism were also evaluated. This report highlights the great versatility of Rhodococcus sp. strain BCP1 and defines for the first time the alkB gene transcriptional start site and the alkB promoter-inducing capacities for substrates different from n-alkanes in a Rhodococcus strain.

A kinetic study of biohydrogen production from glucose, molasses and cheese whey by suspended and attached cells of Thermotoga neapolitana.

Batch tests of H2 production from glucose, molasses and cheese whey by suspended and immobilized cells of Thermotoga neapolitana were conducted to develop a kinetic model of the process. H2 production was inhibited by neither H2 (up to 0.7 mg L(-1)) nor O2 (up to 0.2 mg L(-1)). The H2 specific rates obtained at different substrate concentrations were successfully interpolated with Andrews inhibition model. With glucose and molasses, biofilms performed better than suspended cells. The suspended-cell process was successfully scaled-up to a 19-L bioreactor. Assays co-fed with molasses and cheese whey led to higher H2 productivities and H2/substrate yields than the single-substrate tests. The simulation of the suspended-cell continuous-flow process indicated the potential attainment of H2 productivities higher than those of the batch tests (up to 3.6 mmol H2 h(-1) L(-1) for molasses and 0.67 mmol H2 h(-1) L(-1) for cheese whey) and allowed the identification of the optimal dilution rate.

Genome Sequence of the Polychlorinated-Biphenyl Degrader Pseudomonas pseudoalcaligenes KF707

Pseudomonas pseudoalcaligenes KF707 is a soil polychlorinated biphenyl (PCB) degrader, able to grow both planktonically and as a biofilm in the presence of various toxic metals and metalloids. Here we report the genome sequence (5,957,359 bp) of P. pseudoalcaligenes KF707, which provides insights into metabolic degradation pathways, flagellar motility, and chemotaxis.

Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.

Use of potassium tellurite for testing the survival and viability of Pseudomonas pseudoalcaligenes KF707 in soil microcosms contaminated with polychlorinated biphenyls

This study shows that the oxyanion tellurite TeO3(2-) can be used as a tool to detect and quantify the release in soil microcosms of Pseudomonas pseudoalcaligenes KF707, a strain spontaneously resistant to tellurite with a minimal inhibitory concentration (MIC) of 150 microg ml(-1). KF707 cells which carry the genes for degradation of a wide range of polychlorinated biphenyl congeners (PCBs) were used for inoculation of laboratory microcosms prepared with two different PCB-contaminated soils (Ci/s and Di/s) in the presence or absence of biphenyl as carbon source. In all microcosms supplemented with biphenyl, significant survival of strain KF707 was noted over a time period of 35 days; conversely, in microcosms containing Ci/s soil without biphenyl addition a rapid decrease in KF707 inoculated cells was observed. By comparing the number of inoculated KF707 cells with the number of indigenous bacteria growing on biphenyl (IBGB) of both Ci/s and Di/s microcosms, it could be concluded that the KF707/IBGB ratio is a relevant parameter in determining the fate of the added strain. The efficacy of potassium tellurite as a selective marker to monitor strain KF707 in laboratory microcosms was confirmed by ARDRA analyses of the 16S rDNA, while the isolated indigenous bacteria growing on biphenyl were identified as members of three different species of the genus Pseudomonas. We also report that in microcosms inoculated with KF707 cells in the absence of biphenyl, only low chlorinated biphenyls were degraded.

Biotransformation of a highly chlorinated PCB mixture in an activated sludge collected from a Membrane Biological Reactor (MBR) subjected to anaerobic digestion

The role of anaerobic digestion (AD) on the decontamination and biomethanization of a PCB-spiked sludge obtained from a Membrane Biological Reactor (MBR) pilot plant was investigated throughout a 10-month batch experiment. The study was carried out under mesophilic (35°C) and thermophilic (55°C) conditions and was monitored by means of an integrated chemical, microbiological and molecular biology strategy. Remarkable PCB depletions (higher than 50% of the overall spiked PCBs) and dechlorinations were achieved under methanogenic conditions. The process was not affected by yeast extract addition. Both acetoclastic and hydrogenotrophic methanogens, together with some fermentative eubacteria, were found to persist in all PCB biodegrading microcosms. This finding, together with those obtained from parallel microcosms where specific populations were selectively inhibited, suggested that native methanogens played a key role in the biodegradation and dechlorination of the spiked PCBs. Taken together, the results of this study indicate that AD is a feasible option for the decontamination and the efficient disposal (with the production of a CH(4)-rich biogas) of contaminated MBR sludge, which can be then employed as a fertilizer for agricultural purposes.