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.

Draft Genome Sequence of the Arsenite-Oxidizing Strain Aliihoeflea sp. 2WW, Isolated from Arsenic-Contaminated Groundwater.

Here, we report the draft genome sequence of the arsenite-oxidizing bacterium Aliihoeflea sp. strain 2WW, which consists of a 4.15-Mb chromosome and contains different genes that are involved in arsenic transformations.

Rhizosphere colonization and arsenic translocation in sunflower (Helianthus annuus L.) by arsenate reducing Alcaligenes sp. strain Dhal-L.

In the present study, six arsenic-resistant strains previously isolated were tested for their plant growth promoting characteristics and heavy metal resistance, in order to choose one model strain as an inoculum for sunflower plants in pot experiments. The aim was to investigate the effect of arsenic-resistant strain on sunflower growth and on arsenic uptake from arsenic contaminated soil. Based on plant growth promoting characteristics and heavy metal resistance, Alcaligenes sp. strain Dhal-L was chosen as an inoculum. Beside the ability to reduce arsenate to arsenite via an Ars operon, the strain exhibited 1-amino-cyclopropane-1-carboxylic acid deaminase activity and it was also able to produce siderophore and indole acetic acid. Pot experiments were conducted with an agricultural soil contaminated with arsenic (214 mg kg⁻¹). A real time PCR method was set up based on the quantification of ACR3(2) type of arsenite efflux pump carried by Alcaligenes sp. strain Dhal-L, in order to monitor presence and colonisation of the strain in the bulk and rhizospheric soil. As a result of strain inoculation, arsenic uptake by plants was increased by 53 %, whereas ACR3(2) gene copy number in rhizospheric soil was 100 times higher in inoculated than in control pots, indicating the colonisation of strain. The results indicated that the presence of arsenate reducing strains in the rhizosphere of sunflower influences arsenic mobilization and promotes arsenic uptake by plant.

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.

Aerobic biodegradation of propylene glycol by soil bacteria.

Propylene glycol (PG) is a main component of aircraft deicing fluids and its extensive use in Northern airports is a source of soil and groundwater contamination. Bacterial consortia able to grow on PG as sole carbon and energy source were selected from soil samples taken along the runways of Oslo Airport Gardermoen site (Norway). DGGE analysis of enrichment cultures showed that PG-degrading populations were mainly composed by Pseudomonas species, although Bacteroidetes were found, as well. Nineteen bacterial strains, able to grow on PG as sole carbon and energy source, were isolated and identified as different Pseudomonas species. Maximum specific growth rate of mixed cultures in the absence of nutrient limitation was 0.014 h(-1) at 4 °C. Substrate C:N:P molar ratios calculated on the basis of measured growth yields are in good agreement with the suggested values for biostimulation reported in literature. Therefore, the addition of nutrients is suggested as a suitable technique to sustain PG aerobic degradation at the maximum rate by autochthonous microorganisms of unsaturated soil profile.

Arsenite oxidation in Ancylobacter dichloromethanicus As3-1b strain: detection of genes involved in arsenite oxidation and CO2 fixation.

The aim of this study was to characterize a facultative chemolithotrophic arsenite-oxidizing bacterium by evaluating the growth and the rate of arsenite oxidation and to investigate the genetic determinants for arsenic resistance and CO(2) fixation. The strain under study, Ancylobacter dichloromethanicus As3-1b, in a minimal medium containing 3 mM of arsenite as electron donor and 6 mM of CO(2)-bicarbonate as the C source, has a doubling time (t(d)) of 8.1 h. Growth and arsenite oxidation were significantly enhanced by the presence of 0.01 % yeast extract, decreasing the t(d) to 4.3 h. The strain carried arsenite oxidase (aioA) gene highly similar to those of previously reported arsenite-oxidizing Alpha-proteobacteria. The RuBisCO Type-I (cbbL) gene was amplified and sequenced too, underscoring the ability of As3-1b to carry out autotrophic As(III) oxidation. The results suggest that A. dichloromethanicus As3-1b can be a good candidate for the oxidation of arsenite in polluted waters or groundwaters.

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.

Assessment of bacterial community structure in a long-term copper-polluted ex-vineyard soil.

The influence of long-term copper contamination on the diversity of bacterial communities was investigated in an ex-vineyard soil. Two sites of the same area but exhibiting different 3-fold exchangeable copper (Ex-Cu) concentrations were analysed. Culturable bacterial community structure was assessed using a variety of approaches: determination of culturable bacteria number, analyses of 132 isolates, and denaturing gradient gel lectrophoresis (DGGE) patterns of bacterial biomass grown on agar plates and of soil DNA. There was no significant difference in the number of total heterotrophs at the two sites, whereas the percentage of fast-growing bacteria growing in 1 day, was lower at the site with the higher Ex-Cu content. A high percentage of Cu-tolerant bacteria was found in both sites (63-70%) and it was relatively independent of the Cu content. Shifts in species composition of the culturable bacterial community were detected by analysing isolates from the two soils, Gram-positive bacteria prevailed in the less-polluted soil while Gram-negative bacteria in the more-polluted soil. Each sample site had a community with a different metal resistance pattern. Our study seems to indicate that in this soil ecosystem, copper influenced the culturable bacterial communities, affecting the structural diversity and altering some of the metal resistance of the microorganisms. The Sorensen similarity index calculated on DGGE profiles of 16S rDNA of total and culturable bacterial communities indicated a different species composition at the two sites, although both sites had the same biodiversity degree and different dominance.

Biodegradation of phenanthrene and analysis of degrading cultures in the presence of a model organo-mineral matrix and of a simulated NAPL phase.

Two mixed bacterial cultures (C(B-BT) and C(I-AT)) degraded phenanthrene when it was: (i) in the presence of either hexadecane as a non aqueous phase liquid or a montmorillonite-Al(OH)x-humic acid complex as a model organo-mineral matrix; (ii) sorbed to the complex, either alone or in the presence of hexadecane. The cultures had different kinetic behaviours towards phenanthrene with or without hexadecane. The degradation of Phe alone as well as that of Phe in hexadecane ended in 8 and 15 days with C(B-BT) and C(I-AT) cultures, respectively. Hexadecane increased Phe bioavailability for C(I-AT) bacteria which degraded Phe according to first-order kinetics. The same effect was observed for C(B-BT) bacteria, but with an initial 2 days lag phase and in accordance with zero-order kinetics. The presence of hexadecane did not affect the degradation of phenanthrene sorbed and aged on the complex by C(I-AT )culture. This capability was exhibited also after experimental aging of 30 days. The dynamics of the bacterial community composition was investigated through PCR-DGGE (denaturing gradient gel electrophoresis) of 16S rRNA gene fragments. Individual bands changed their intensity during the incubation time, implying that particular microbe's relative abundance changed according to the culture conditions. Isolation of phenanthrene and/or hexadecane degraders was in accord with cultivation-independent data. Growth-dependent changes in the cell surface hydrophobicity of the two cultures and of the isolates suggested that modulation of cell surface hydrophobicity probably played an important role for an efficient phenanthrene assimilation/uptake.

Bioremediation and monitoring of aromatic-polluted habitats.

Bioremediation may restore contaminated soils through the broad biodegradative capabilities evolved by microorganisms towards undesirable organic compounds. Understanding bioremediation and its effectiveness is rapidly advancing, bringing available molecular approaches for examining the presence and expression of the key genes involved in microbial processes. These methods are continuously improving and require further development and validation of primer- and probe-based analyses and expansion of databases for alternative microbial markers. Phylogenetic marker approaches provide tools to determine which organisms are present or generally active in a community; functional gene markers provide only information concerning the distribution or transcript levels (deoxyribonucleic acid [DNA]- or messenger ribonucleic acid [mRNA]-based approaches) of specific gene populations across environmental gradients. Stable isotope probing methods offer great potential to identify microorganisms that metabolize and assimilate specific substrates in environmental samples, incorporating usually a rare isotope (i.e., (13)C) into their DNA and RNA. DNA and RNA in situ characterization allows the determination of the species actually involved in the processes being measured. DNA microarrays may analyze the expression of thousands of genes in a soil simultaneously. A global analysis of which genes are being expressed under various conditions in contaminated soils will reveal the metabolic status of microorganisms and indicate environmental modifications accelerating bioremediation.

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, Rhodococcus aetherovorans, 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.

Analysis of rhizobacterial communities in perennial Graminaceae from polluted water meadow soil, and screening of metal-resistant, potentially plant growth-promoting bacteria.

This study investigates the impact of long-term heavy metal contamination on the culturable, heterotrophic, functional and genetic diversity of rhizobacterial communities of perennial grasses in water meadow soil. The culturable heterotrophic diversity was investigated by colony appearance on solid LB medium. Genetic diversity was measured as bands in denaturing gradient gel electrophoresis (DGGE) obtained directly from rhizosphere soil and rhizoplane DNA extracts, and from the corresponding culturable communities. In the two rhizospheric fractions the DGGE profiles of the direct DNA extracts were similar and stable among replicates, whereas in the enriched cultures the profiles of the fractions differed, but among the replicates they were similar. One hundred isolates were collected into 33 different operational taxonomic units by use of amplified internal transcribed spacers and into 19 heavy metal-resistant phenotypes. The phylogenetic position of strains belonging to 18 operational taxonomic units, representing more than 80% of the isolates, was determined by 16S rRNA gene sequencing. Several heavy metal-resistant strains were isolated from rhizoplane. Finally, metal-resistant rhizobacteria were tested for plant growth-promoting characteristics; some were found to contain 1-aminocyclopropane-1-carboxylic acid deaminase and/or to produce indole acetic acid and siderophores. Two strains resistant to cadmium and zinc, Pseudomonas tolaasii RP23 and Pseudomonas fluorescens RS9, had all three plant growth-promoting characteristics. Our findings suggest that bacteria can respond to soil metal contamination, and the described methodological approach appears promising for targeting potential plant growth-promoting rhizobacteria.