Lucia Cavalca

Intrinsic bioremediability of an aromatic hydrocarbon-polluted groundwater: diversity of bacterial population and toluene monoxygenase genes.

The functional and phylogenetic biodiversity of bacterial communities in a benzene, toluene, ethylbenzene and xylene (BTEX)-polluted groundwater was analysed. To evaluate the feasibility of using an air sparging treatment to enhance bacterial degradative capabilities, the presence of degrading microorganisms was monitored. The amplification of gene fragments corresponding to toluene monooxygenase (tmo), catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and toluene dioxygenase genes in DNA extracted directly from the groundwater samples was associated with the presence of indigenous degrading bacteria. Five months of air injection reduced species diversity in the cultivable community (as calculated by the Shannon-Weaver index), while little change was noted in the degree of biodiversity in the total bacterial community, as characterised by denaturing gradient gel electrophoresis (DGGE) analysis. BTEX-degrading strains belonged to the genera Pseudomonas, Microbacterium, Azoarcus, Mycobacterium and Bradyrhizobium. The degrading capacities of three strains in batch liquid cultures were also studied. In some of these microorganisms different pathways for toluene degradation seemed to operate simultaneously. Pseudomonas strains of the P24 operational taxonomic unit, able to grow only on catechol and not on BTEX, were the most abundant, and were present in the groundwater community at all stages of treatment, as evidenced both by cultivation approaches and by DGGE profiles. The presence of different tmo-like genes in phylogenetically distant strains of Pseudomonas, Mycobacterium and Bradyrhizobium suggested recent horizontal gene transfer in the groundwater.

Arsenic-resistant bacteria associated with roots of the wild Cirsium arvense (L.) plant from an arsenic polluted soil, and screening of potential plant growth-promoting characteristics

A rhizobacterial community, associated with the roots of wild thistle Cirsium arvense (L.) growing in an arsenic polluted soil, was studied by fluorescence in situ hybridization (FISH) analysis in conjunction with cultivation-based methods. In the bulk, rhizosphere, and rhizoplane fractions of the soil, the qualitative picture obtained by FISH analysis of the main phylogenetic bacterial groups was similar and was predominantly comprised of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The arsenic-resistant isolates belonged to 13 genera, the most abundant being those of Bacillus, Achromobacter, Brevundimonas, Microbacterium, and Ochrobactrum. Most bacteria grew in the presence of high arsenic concentrations (over 100mM arsenate and 10mM arsenite). Most strains possessed the ArsC, ArsB and ACR3 genes homologous to arsenate reductase and to the two classes of arsenite efflux pumps, respectively, peculiar to the ars operon of the arsenic detoxification system. ArsB and ACR3 were present simultaneously in highly resistant strains. An inconsistency between 16S rRNA phylogenetic affiliations and the arsenate reductase sequences of the strains was observed, indicating possible horizontal transfer of arsenic resistance genes in the soil bacterial community. Several isolates were able to reduce arsenate and to oxidise arsenite. In particular, Ancylobacter dichloromethanicum strain As3-1b possessed both characteristics, and arsenite oxidation occurred in the strain also under chemoautotrophic conditions. Some rhizobacteria produced siderophores, indole acetic acid and 1-amino-cyclopropane-1-carboxylic acid deaminase, thus possessing potential plant growth-promoting traits.

Arsenic-resistant bacteria isolated from agricultural soils of Bangladesh and characterization of arsenate-reducing strains

Aims:  To analyse the arsenic‐resistant bacterial communities of two agricultural soils of Bangladesh, to isolate arsenic‐resistant bacteria, to study their potential role in arsenic transformation and to investigate the genetic determinants for arsenic resistance among the isolates.

Microbial transformations of arsenic: perspectives for biological removal of arsenic from water

Arsenic is present in many environments and is released by various natural processes and anthropogenic actions. Although arsenic is recognized to cause a wide range of adverse health effects in humans, diverse bacteria can metabolize it by detoxification and energy conservation reactions. This review highlights the current understanding of the ecology, biochemistry and genomics of these bacteria, and their potential application in the treatment of arsenic-polluted water.

Distribution of catabolic pathways in some hydrocarbon-degrading bacteria from a subsurface polluted soil

Enrichment cultures on naphtha solvent were used to select aromatic hydrocarbon-degrading bacteria from a BTEX (benzene, toluene, ethylbenzene, xylene)-contaminated subsoil obtained from beneath a paint factory located in Milan, Italy. Fifteen isolated strains were studied for their different biodegradative capacities. Among these, 13 were able to grow on naphtha solvent. Ten were identified as Pseudomonas putida and three as Pseudomonas aureofaciens. Two other degraders were identified as Pseudomonas aeruginosa and Alcaligenes xylosoxidans subsp. denitrificans. Further molecular characterization of the isolates was carried out by randomly amplified polymorphic DNA analysis to ascertain that all the studied strains belonged to different haplotypes. The isolates were characterized for the presence of genes encoding for toluene dioxygenase, xylene monooxygenase and catechol 2,3-dioxygenase by polymerase chain reaction analysis and by Southern analysis. P. putida strain CM23, which showed homology with xylA,M, xylE and todC1C2BA genes, possessed multiple pathways which enabled the strain to grow on benzene, toluene and m-xylene.

Diversity of tfdC genes: distribution and polymorphism among 2,4-dichlorophenoxyacetic acid degrading soil bacteria

The aim of the present work was to study the occurrence, distribution and diversity of 1,2-dichlorocatechol dioxygenase genes among 2,4-dichlorophenoxyacetic acid degrading bacteria. Phylogenetic relationships between the 31 strains or isolates were evaluated by amplified ribosomal DNA restriction analysis of the 16S rDNA gene. All the strains could be assigned to the β or γ subdivisions of the Proteobacteria. tfdC genes were detected by PCR amplification using degenerated primers. Two specific probes were produced from Ralstonia eutropha strain JMP134 and from a soil isolate strain PLAE6 which was grouped with Variovorax paradoxus. Sequence analysis of the probes revealed that they were homologous to the tfdC genes of JMP134 located on plasmid pJP4 and to the tfdC gene of Pseudomonas putida strain PaW85 located on plasmid pEST4011. The localization and the copy number of tfdC genes were determined by hybridization of plasmid profiles and genomic DNA restriction fragment length polymorphism profiles with the two probes. Most of the strains were found to bear tfdC genes on plasmids ranging from 78 to 532 kb; two strains without any plasmids were also found to hybridize with the probes, revealing a chromosomal localization of catabolic genes. Sequence analysis of the PCR products from different strains confirmed that four different classes of chlorocatechol 1,2-dioxygenase genes were present in the strains and isolates studied.

Arsenic transforming abilities of groundwater bacteria and the combined use of Aliihoeflea sp. strain 2WW and goethite in metalloid removal

Several technologies have been developed for lowering arsenic in drinking waters below the World Health Organization limit of 10 μg/L. When in the presence of the reduced form of inorganic arsenic, i.e. arsenite, one options is pre-oxidation of arsenite to arsenate and adsorption on iron-based materials. Microbial oxidation of arsenite is considered a sustainable alternative to the chemical oxidants. In this contest, the present study investigates arsenic redox transformation abilities of bacterial strains in reductive groundwater from Lombardia (Italy), where arsenite was the main arsenic species. Twenty isolates were able to reduce 75 mg/L arsenate to arsenite, and they were affiliated to the genera Pseudomonas, Achromobacter and Rhodococcus and genes of the ars operon were detected. Three arsenite oxidizing strains were isolated: they belonged to Rhodococcus sp., Achromobacter sp. and Aliihoeflea sp., and aioA genes for arsenite oxidase were detected in Aliihoeflea sp. strain 2WW and in Achromobacter sp. strain 1L. Uninduced resting cells of strain 2WW were used in combination with goethite for arsenic removal in a model system, in order to test the feasibility of an arsenic removal process. In the presence of 200 μg/L arsenite, the combined 2WW-goethite system removed 95% of arsenic, thus lowering it to 8 μg/L. These results indicate that arsenite oxidation by strain 2WW combined to goethite adsorption is a promising approach for arsenic removal from contaminated groundwater.

Influences of dissolved oxygen concentration on biocathodic microbial communities in microbial fuel cells

Dissolved oxygen (DO) at cathodic interface is a critical factor influencing microbial fuel cells (MFC) performance. In this work, three MFCs were operated with cathode under different DO conditions: i) air-breathing (A-MFC); ii) water-submerged (W-MFC) and iii) assisted by photosynthetic microorganisms (P-MFC). A plateau of maximum current was reached at 1.06±0.03mA, 1.48±0.06mA and 1.66±0.04mA, increasing respectively for W-MFC, P-MFC and A-MFC. Electrochemical and microbiological tools (Illumina sequencing, confocal microscopy and biofilm cryosectioning) were used to explore anodic and cathodic biofilm in each MFC type. In all cases, biocathodes improved oxygen reduction reaction (ORR) as compared to abiotic condition and A-MFC was the best performing system. Photosynthetic cultures in the cathodic chamber supplied high DO level, up to 16mgO2L-1, which sustained aerobic microbial community in P-MFC biocathode. Halomonas, Pseudomonas and other microaerophilic genera reached >50% of the total OTUs. The presence of sulfur reducing bacteria (Desulfuromonas) and purple non-sulfur bacteria in A-MFC biocathode suggested that the recirculation of sulfur compounds could shuttle electrons to sustain the reduction of oxygen as final electron acceptor. The low DO concentration limited the cathode in W-MFC. A model of two different possible microbial mechanisms is proposed which can drive predominantly cathodic ORR.

Chlorophenol removal from soil suspensions: effects of a specialised microbial inoculum and a degradable analogue

Two soils of different contamination history were tested in slurry for their self-remediability towards mono-, di- and trisubstituted chlorophenols. The landfill soil showed poor ability in removing the compounds. Instead, the soil from the golf course, treated for many years with a 2,4,6-trichlorophenol derivative (Prochloraz), remediated different concentrations of the same 2,4,6TCP,2,4-dichlorophenol and monochlorophenol isomers, singly and in mixtures, at varying degradation rates. Ralstonia eutropha TCP, a specialised microorganism capable of degrading 2,4,6TCP, proved highly efficient in removing the compound from both tested soils. The same microbial inoculum allowed total removal of the ternary mixture of monochlorophenol isomers from the golf course soil, but it did not accelerate the removal of the same compounds when singly supplied. The addition of phenol as a degradable analogue was more effective in co-metabolically removing not only the single monochlorophenols, but also their mixtures, the removal occurring faster and independently of the presence of the microbial inoculum. From the golf course soil, a microorganism, phenotypically and genetically identical to R. eutropha TCP, was isolated and classified as R. eutropha TCP II.

Enzymatic and genetic profiles in environmental strains grown on polycyclic aromatic hydrocarbons

The possible generation of oxidative stress induced by aromatic hydrocarbon degradation suggests that ancillary enzyme activities could facilitate the utilization of polycyclic aromatic hydrocarbons as sole carbon source. To investigate the metabolic profiles of low molecular weight polycyclic aromatic hydrocarbon-degrading strains of Sphingobium chlorophenolicum, Rhodococcusaetherovorans, Rhodococcus opacus and Mycobacterium smegmatis, the determination of the activity of putative detoxifying enzymes (rhodanese-like and glutathione S-transferase proteins) was combined with genetic analyses. All the studied strains were able to utilize phenanthrene or naphthalene. Glutathione S-transferase activity was found in S. chlorophenolicum strains grown on phenanthrene and it was related to the presence of the bphK gene, since modulation of glutathione S-transferase activity by phenanthrene paralleled the induction of glutathione S-transferase transcript in the S. chlorophenolicum strains. No glutathione S-transferase activity was detectable in R.aetherovorans, R. opacus and in M. smegmatis strains. All strains showed 3-mercaptopyruvate:cyanide sulfurtransferase activity. A rhodanese-like SseA protein was immunodetected in R.aetherovorans, R. opacus and in M. smegmatis strains, where increase of 3-mercaptopyruvate:cyanide sulfurtransferase activity was significantly induced by growth on phenanthrene.