New clusters of arsenite oxidase and unusual bacterial groups in enrichments from arsenic-contaminated soil

In the present study cultivation-dependent and molecular methods were applied in combination to investigate the arsenite-oxidizing communities in enrichment cultures from arsenic and lead smelter-impacted soils with respect to both 16S rRNA and arsenite oxidase gene diversity. Enrichments with arsenite as the only electron donor resulted in completely different communities than enrichments with yeast extract and the simultaneous presence of arsenite. The lithoautotrophic community appeared to be dominated by Ferrimicrobium-related Actinobacteria, unusual Acidobacteria, Myxobacteria, and α-Proteobacteria but the heterotrophic community comprised many Dokdonella-related γ-Proteobacteria. Gene sequences of clones encoding arsenite oxidase from the enrichment for lithoautotrophs belonged to three major clusters with sequences from non-cultivated microorganisms. So, primers used to detect arsenite oxidase genes could amplify the genes from many α-, β- and γ-Proteobacteria, but not from various strains of the other phyla present in the enrichment for lithotrophs. This was also observed for the isolates where arsenite oxidase genes from new proteobacterial isolates of the genera Burkholderia, Bosea, Alcaligenes, Bradyrhizobium and Methylobacterium could be amplified but the genes of the new Rhodococcus isolate S43 could not. The results indicate that the ability to oxidize arsenite is widespread in various unusual taxa, and molecular methods for their detection require further improvement.     

Redox cycling of arsenic by the hydrothermal marine bacterium Marinobacter santoriniensis

Marinobacter santoriniensis NKSG1^T is a mesophilic, dissimilatory arsenate-reducing and arsenite-oxidizing bacterium isolated from an arsenate-reducing enrichment culture. The inoculum was obtained from arsenic-rich shallow marine hydrothermal sediment from Santorini, Greece, with evidence of arsenic redox cycling. Growth studies demonstrated M. santoriniensis NKSG1^T is capable of conserving energy from the reduction of arsenate [As(V)] with acetate or lactate as the electron donor, and of oxidizing arsenite [As(III)] heterotrophically with oxygen as the electron acceptor. The oxidation of As(III) coincided with the expression of the aoxB gene encoding for the catalytic molybdopterin subunit of the heterodimeric arsenite oxidase operon, indicating the reaction is enzymatically controlled, and M. santoriniensis NKSG1^T is a heterotrophic As(III)-oxidizing bacterium. Although it is clear that this organism also performs dissimilatory As(V) reduction, no amplification of the arrA arsenate reductase gene was attained using a range of primers and PCR conditions. Marinobacter santoriniensis NKSG1^T belongs to a genus of bacteria widely occurring in marine environments, including hydrothermal sediments, and is among the first marine bacteria shown to be capable of either anaerobic As(V) respiration or aerobic As(III) oxidation.     

Distribution of Arsenite-Oxidizing Bacteria and its Correlation with Temperature in Hot Springs of the Tibetan-Yunnan Geothermal Zone in Western China

The distribution of arsenite-oxidizing bacteria in response to temperature is of great importance to an understanding of biogeochemical cycling of arsenic in geothermal features. The abundance and diversity of arsenite-oxidizing bacteria were investigated in the geothermal features of Tengchong County of Yunnan Province, Dachaidan County of Qinghai Province, and Tibet. The abundance of aioA genes, which encode the large subunit of arsenite oxidase, was determined by quantitative polymerase chain reactions. The diversity of aioA genes was studied by PCR-cloning-based phylogenetic analyses. The results showed that the aioA gene abundance increased as temperature decreased, whereas its diversity at the OTU level (97% cutoff) increased with increasing temperature. This suggests that temperature played an important role in affecting aioA gene distribution and thus arsenic speciation. The aioA gene population (at OTU level) differed among the studied regions, indicating geographic isolation may be an important factor controlling aioA gene distribution in hot springs.     

Effects of the high pressure of homogenization on some spoiling micro-organisms, representative of fruit juice microflora, inoculated in saline solution

Aims:  This study was aimed to investigate the effects of a high-pressure homogenization (HPH) treatment on some micro-organisms, involved in the spoilage of fruit juices.
Methods and Results:  Lactobacillus plantarum , Lactobacillus brevis , Bacillus coagulans cells, Saccharomyces bayanus , Pichia membranaefaciens and Rhodotorula bacarum were separately inoculated in a saline solution (0·9% NaCl); the initial inoculum was ca. 5 log CFU ml⁻¹. Then, the samples were processed through a homogenizer at 10–150 MPa for 1, 2 or 3 times. Yeasts were completely inactivated at 50–110 MPa with a single pass treatment, while lactic acid bacteria counts were reduced to approximately 1 log CFU ml⁻¹ after a three-steps HPH processing.
Conclusions:  Yeasts were the most sensitive micro-organisms, followed by B. coagulans . On the other hand, lactic acid bacteria appeared resistant to HPH.
Significance and Impact of the Study:  The results of this study provided some useful information on the susceptibility of microflora of juices to homogenization; moreover, they suggested that HPH could be used successfully to inactivate yeasts.     

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.
Methods and Results:  Enrichment cultures were performed in a minimal medium in the presence of As(III) and As(V) to isolate resistant bacteria. Twenty-one arsenic-resistant bacteria belonging to different genera of Gram-positive and Gram-negative bacteria were isolated. The isolates, with the exception of Oceanimonas doudoroffii Dhal Rw, reduced 2 mmol l⁻¹ As(V) completely to As(III) in aerobic conditions. Putative gene fragments for arsenite efflux pumps were amplified in isolates from Dhal soil and a putative arsenate reductase gene fragment was amplified from a Bacillus sp. from Rice soil.
Conclusions:  Phylogenetically diverse arsenic-resistant bacteria present in agricultural soils of Bangladesh are capable of reducing arsenate to arsenite under aerobic conditions apparently for detoxification purpose.
Significance and Impact of the Study:  This study provides results on identification, levels of arsenic resistance and reduction of arsenate by the bacterial isolates which could play an important role in arsenic cycling in the two arsenic-contaminated soils in Bangladesh.     

Enumeration and characterization of culturable arsenate resistant bacteria in a large estuary

Arsenic is a toxic element that exists in two major inorganic forms, arsenate and arsenite. A number of bacteria have been shown to resist arsenic exposure, and even more bacteria appear to possess the genes for arsenic resistance. In this study, the numbers of culturable arsenate-resistant bacteria present in water at three coastal sites in the Lake Pontchartrain estuary, Louisiana, was determined. Despite insignificant (less than 1.33 μM) levels of arsenic in this system, 20–50% of the viable count of bacteria showed appreciable arsenate resistance, suggesting that arsenic-resistant bacteria are common and widespread. A diverse array of arsenate-resistant isolates was obtained, with 16S rRNA sequence analysis indicating 37 different bacterial strains, representing six major bacterial groups. Many of these isolates were affiliated with groups of bacteria that have been poorly characterized in terms of arsenic resistance, such as the Betaproteobacteria or Flavobacteria. Some isolates were capable of tolerating very high (>100 mM) levels of arsenate, although arsenite resistance was generally much lower. The results suggest that arsenic-resistant bacteria are common, even in environments with insignificant arsenic contamination, and that many different groups of aquatic bacteria show appreciable arsenic resistance.     

Molecular Characterization of the Total Bacteria and Dissimilatory Arsenate-Reducing Bacteria in Core Sediments of the Jianghan Plain,Central China

Arsenic contamination in groundwater has been reported in the Jianghan Plain of China since 2005, yet little is known about the microbial communities involved in As mobilization in this area, especially the dissimilatory arsenate-reducing bacteria (DARB) communities. Here, we conducted a cultivation-independent investigation on core sediments collected from a region with arsenic-contaminated groundwater in the Jianghan Plain to reveal the total bacteria and DARB community structures. Highly diverse As-resistant bacteria communities were found from sediment samples via high-throughput sequencing of 16S rRNA genes. Notably, we identified 27 unique arrA gene (encoding the alpha subunit of dissimilatory arsenate reductase) phylotypes, none of which was related to any previously described arrA gene sequence. This suggests a novel and unique DARB community in the sediments of the Jianghan Plain and expands our knowledge about the distribution and diversity of this group of bacteria in natural environments. Moreover, RDA and CCA demonstrated that total bacterial communities and specific functional groups are controlled by different environmental factors. Specifically, sediment pH, NH₄⁺, total nitrogen, total Fe, total organic carbon and total phosphorus were the key factors driving total bacterial community compositions, while As significantly shaped DARB community structures. This report is the first to describe DARB communities and their correlation with environmental factors in Jianghan Plain sediments, which could give us clues about the origin of the arsenic contamination of groundwater in this region.     

A new aerobic chemolithoautotrophic arsenic oxidizing microorganism isolated from a high Andean watershed

Biological arsenic oxidation has been suggested as a key biogeochemical process that controls the mobilization and fate of this metalloid in aqueous environments. To the best of our knowledge, only four aerobic chemolithoautotrophic arsenite-oxidizing (CAO) bacteria have been shown to grow via direct arsenic oxidation and to have the essential genes for chemolithoautotrophic arsenite oxidation. In this study, a new CAO bacterium was isolated from a high Andean watershed evidencing natural dissolved arsenic attenuation. The bacterial isolate, designated TS-1, is closely related to the Ancylobacter genus, in the Alphaproteobacteria class. Results showed that TS-1 has genes for arsenite oxidation and carbon fixation. The dependence of bacterial growth from arsenite oxidation was demonstrated. In addition, a mathematical model was suggested and the kinetic parameters were obtained by simultaneously fitting the biomass growth, arsenite depletion curves, and arsenate production. This research increases the knowledge of chemolithoautotrophic arsenic oxidizing microorganisms and its potential role as a driver for natural arsenic attenuation.     

The culturable bacteria isolated from organic-rich black shale potentially useful in biometallurgical procedures

Aims:  The aim of this study was the isolation and characterization of micro-organisms from Lubin copper mine potentially useful in biotechnology of metal recovery from copper bearing black shale.
Methods and Results:  Eight bacterial strains were isolated from black shale ore. Phylogenetic analysis based on 16S rRNA gene homology showed that five strains belonged to the γ-Proteobacteria, one to the Firmicutes and two to the Actinobacteria. The ability of the isolates to transform bituminous shale and use them as carbon and energy sources, as well as high resistance to metals and metalloids, esterase and lipase activities, assimilation of organic acids, degradation of phenanthrene and siderophores production were shown.
Conclusions:  The indigenous bacteria exhibited a broad range of physiological properties related to geochemical parameters of the examined environment and potentially useful in biometallurgical procedures.
Significance and Impact of the Study:  The results have yielded new insights into the microbiology of black shale. It can be suggested that isolated micro-organisms might play a role in the geochemical cycle of carbon and metals occurring in the organic fraction of black shale ore and might be of potential use in biotechnological procedures for the copper recovery and other valuable metals from tailings containing black shale as well as organic rich ore.     

Aromatic hydrocarbon degradation genes from chronically polluted Subantarctic marine sediments

Aim:  The goal of this study was to identify functional targets to detect polycyclic aromatic hydrocarbon (PAH)-degrading bacterial populations in cold marine ecosystems.
Methods and Results:  We designed a degenerate primer set targeting genes encoding the α subunit of PAH-dioxygenases from Gram-positive bacteria. This primer set was used to amplify gene fragments from metagenomic DNA isolated from Subantarctic marine sediments (Ushuaia Bay, Argentina). These gene fragments were cloned and sequenced. We identified 14 distinct groups of genes, most of them showing significant relatedness with dioxygenases from Gram-positive bacteria of the genera Rhodococcus , Mycobacterium , Nocardioides , Terrabacter and Bacillus . The level of identity with these genes, however, was low to moderate (33–62% at the amino acid level).
Conclusion:  These results indicate the presence of a high diversity of hitherto unidentified dioxygenase genes in this cold polluted environment.
Significance and Impact of the Study:  Subantarctic marine ecosystems are particularly vulnerable to hydrocarbon pollution, and the development of environmental restoration strategies for these environments is pressing. The information obtained in this work will be the starting point for the design of quantitative molecular tools to analyse the abundance and dynamics of these aromatic hydrocarbon-degrading bacterial populations in the marine environment.     

Feather micro-organisms and uropygial antimicrobial defences in a colonial passerine bird

1.  The relationship between the composition of communities of micro-organisms and their hosts remains poorly understood. We conducted extensive field studies of feather-degrading bacteria, other cultivable bacteria, and fungi on the plumage of a migratory bird, the barn swallow Hirundo rustica Linnaeus, to understand the association between micro-organisms, host sociality and host antimicrobial defences, as reflected by the size of the uropygial gland.
2.  The abundance of feather-degrading bacteria, but not other cultivable bacteria or fungi, decreased with increasing size of the uropygial gland.
3.  Females had more feather-degrading bacteria than males.
4.  Barn swallows living in larger colonies had more feather-degrading bacteria than less social conspecifics.
5.  These findings suggest that the uropygial gland plays a specific role in regulating the abundance of feather-degrading bacteria that furthermore depends on the social environment of the host.     

Detection, diversity and expression of aerobic bacterial arsenite oxidase genes

The arsenic (As) drinking water crisis in south and south-east Asia has stimulated intense study of the microbial processes controlling the redox cycling of As in soil-water systems. Microbial oxidation of arsenite is a critical link in the global As cycle, and phylogenetically diverse arsenite-oxidizing microorganisms have been isolated from various aquatic and soil environments. However, despite progress characterizing the metabolism of As in various pure cultures, no functional gene approaches have been developed to determine the importance and distribution of arsenite-oxidizing genes in soil-water-sediment systems. Here we report for the first time the successful amplification of arsenite oxidase-like genes (aroA/asoA/aoxB) from a variety of soil-sediment and geothermal environments where arsenite is known to be oxidized. Prior to the current work, only 16 aroA/asoA/aoxB-like gene sequences were available in GenBank, most of these being putative assignments from homology searches of whole genomes. Although aroA/asoA/aoxB gene sequences are not highly conserved across disparate phyla, degenerate primers were used successfully to characterize over 160 diverse aroA-like sequences from 10 geographically isolated, arsenic-contaminated sites and from 13 arsenite-oxidizing organisms. The primer sets were also useful for confirming the expression of aroA-like genes in an arsenite-oxidizing organism and in geothermal environments where arsenite is oxidized to arsenate. The phylogenetic and ecological diversity of aroA-like sequences obtained from this study suggests that genes for aerobic arsenite oxidation are widely distributed in the bacterial domain, are widespread in soil-water systems containing As, and play a critical role in the biogeochemical cycling of As.     

Isolation, characterization and identification of lactic acid bacteria and yeasts from sour Mifen, a traditional fermented rice noodle from China

Aims:  Considering the effect of natural fermentation on the textural improvement of fermented rice noodles in China and South Asia, and given the lack of reports concerning microbial populations and structure in the fermentation process, this study aims to determine the number of viable micro-organisms and identify the species isolated from the local factories, and to assess their potential use as a starter culture from their enzymatic profiles.
Methods and Results:  Fourteen samples from three local factories were analysed for the presence of micro-organisms. A total of 170 lactic acid bacteria (LAB) and 96 yeasts were isolated from the factories. The isolates were phenotypically characterized by using API 50 CHL kits, API 20 Strep kits, API ID 32 C kits and by performing additional biochemical tests. The enzymatic profiles of isolates were assessed by using API ZYM kits. Lactobacillus plantarum and Saccharomyces cerevisiae were identified as predominant species in the fermented supernatants. A majority of the isolates of LAB and yeasts displayed activities of α-glucosidase, β-glucosidase, lipase and trypsin.
Conclusions:  The microbial composition and strain characteristics present in the fermentation supernatant demonstrate that a majority of micro-organisms have the ability to digest starch, sugar, protein or lipid. It supports our previous work in which the rice starch was modified and purified by fermentation and thus improves the texture of rice noodles.
Significance and Impact of the Study:  The dominant strains would be important in developing a starter culture. The results can form the basis for the improvement of product quality and consistency.     

The <Emphasis Type="Italic">ars</Emphasis> genotype characterization of arsenic-resistant bacteria from arsenic-contaminated gold–silver mines in the Republic of Korea

The isolates were identified on the basis of ars genotype characteristics as well as arsenic oxidation/reduction analysis based on the molecular detection characterization. Diversity, pH range (4.0 to 7.0), location, and ars features were assessed for four arsenic-contaminated pond sites and six arsenic tailings located in the Duck-um mine and Myoung-bong mine areas. The presence of ars genes in the genomes of each bacterial strain was evaluated using polymerase chain reaction. Batch experiment results showed that Pseudomonas putida strains OS-3 and -18 completely oxidized 1 mM of arsenite(III) to arsenate(V) within 35-40 h. In contrast, two arsenate-reducing bacteria isolated from mines, P. putida RS-4 and RS-5, were capable of growing aerobically in growth medium supplemented with up to 66.7 mM arsenate(V), which are significantly higher concentration than those tolerated by other arsenic-resistant bacteria. These results suggest that newly isolated indigenous arsenic-resistant bacteria may provide a better understanding of the molecular geomicrobiology and may be applied to the bioremediation of arsenic-contaminated mines in Korea. Ecologically, the redox potential plays an important role in arsenic toxicity and mobility in As-contaminated mine areas, as it facilitates the biogeochemical cycling activity of Pseudomonas sp. groups.     

Oxidation of Arsenite by a Soil Isolate of Alcaligenes

A strain of Alcaligenes , isolated from soil and grown in nutrient broth in the presence of arsenite, possessed the ability to oxidize arsenite to arsenate. Washed cell suspensions consumed one-half mol of oxygen/mol of arsenite and produced arsenate. The optimum pH for arsenite oxidation was 7.0. The K_m for arsenite was 1.5 × 10^-4 M and V _max was 6.7 μl of oxygen/min. The arsenite-oxidizing enzyme system was induced by growth in arsenite. Response of the arsenite-oxidizing enzyme system to respiratory inhibitors suggested that electrons resulting from arsenite oxidation by an oxido-reductase with a bound flavin are transferred via cytochrome c and cytochrome oxidase to oxygen. The presence of the cytochromes in crude extract was confirmed by spectral measurements.     

Identification of yeast and bacteria involved in the mezcal fermentation of Agave salmiana

Aims:  To identify the yeast and bacteria present in the mezcal fermentation from Agave salmiana .
Methods and Results:  The restriction and sequence analysis of the amplified region, between 18S and 28S rDNA and 16S rDNA genes, were used for the identification of yeast and bacteria, respectively. Eleven different micro-organisms were identified in the mezcal fermentation. Three of them were the following yeast: Clavispora lusitaniae , Pichia fermentans and Kluyveromyces marxianus. The bacteria found were Zymomonas mobilis subsp. mobilis and Zymomonas mobilis subsp. pomaceae, Weissella cibaria , Weissella paramesenteroides , Lactobacillus pontis , Lactobacillus kefiri , Lactobacillus plantarum and Lactobacillus farraginis .
Conclusions:  The phylogenetic analysis of 16S rDNA and ITS sequences showed that microbial diversity present in mezcal is dominated by bacteria, mainly lactic acid bacteria species and Zymomonas mobilis . Pichia fermentans and K. marxianus could be micro-organisms with high potential for the production of some volatile compounds in mezcal.
Significance and Impact of the Study:  We identified the community of bacteria and yeast present in mezcal fermentation from Agave salmiana.     

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.     

Anhydrobiosis quotient: a novel approach to evaluate stability in desiccated bacterial cells

Aim:  The major objective of this study was the development of a methodology to quantify the anhydrobiotic ability of bacteria and its application to evaluate the stability of desiccated bacterial cells using the biocontrol agent Tsukamurella paurometabola C-924 as a model of anhydrobiote.
Methods and Results:  Tsukamurella paurometabola C-924 was desiccated by spray-drying. Samples of desiccated cells were stored at several temperatures and viability and residual moisture were measured at different intervals of time. The term anhydrobiosis quotient (ε) was defined, and a scale of anhydrobiotic ability for classifying micro-organisms in terms of tolerance to desiccation was established (1 ≤  ε  ≤ 15). The anhydrobiosis quotient was used to evaluate the stability of the anhydrobiotic cells. As a main result, changes in the anhydrobiosis quotient at several temperatures were fitted using a reparameterized Weibull model, which was found to be robust for the prediction of the stability at 4°C.
Conclusions:  A novel methodology was developed to evaluate the desiccated state in bacteria. The anhydrobiosis quotient allows the quantitative estimation of the anhydrobiotic ability, and the mathematical model developed allows the prediction of the desiccated state of bacterial populations.
Significance and Impact of the Study:  The new methodology could be applied in studying the anhydrobiosis state of bacterial populations as a predictive tool for industrial and environmental microbiology.     

Genome sequence and characterisation of a freshwater photoarsenotroph,Cereibacter azotoformans strain ORIO,isolated from sediments capable of cyclic light–dark arsenic oxidation and reduction

A freshwater photosynthetic arsenite-oxidizing bacterium, Cereibacter azotoformans strain ORIO, was isolated from Owens River, CA, USA. The waters from Owens River are elevated in arsenic and serve as the headwaters to the Los Angeles Aqueduct. The complete genome sequence of strain ORIO is 4.8 Mb genome (68% G + C content) and comprises two chromosomes and six plasmids. Taxonomic analysis placed ORIO within the Cereibacter genus (formerly Rhodobacter). The ORIO genome contains arxB₂AB₁CD (encoding an arsenite oxidase), arxXSR (regulators) and several ars arsenic resistance genes all co-localised on a 136 kb plasmid, named pORIO3. Phylogenetic analysis of ArxA, the molybdenum-containing arsenite oxidase catalytic subunit, demonstrated photoarsenotrophy is likely to occur within members of the Alphaproteobacteria. ORIO is a mixotroph, oxidises arsenite to arsenate (As(V)) photoheterotrophically, and expresses arxA in cultures grown with arsenite. Further ecophysiology studies with Owens River sediment demonstrated the interconversion of arsenite and As(V) was dependent on light–dark cycling. arxA and arrA (As(V) respiratory reductase) genes were detected in the light–dark cycled sediment metagenomes suggesting syntrophic interactions among arsenotrophs. This work establishes C. azotoformans str. ORIO as a new model organism for studying photoarsenotrophy and light–dark arsenic biogeochemical cycling.     

Genome sequence of the arsenite-oxidizing strain Agrobacterium tumefaciens 5A

Microbial transformations of arsenic influence its mobility and toxicity. We report the draft genome sequence of the arsenite-oxidizing strain Agrobacterium tumefaciens 5A isolated from an As-contaminated soil in the Madison River Valley, MT. A large number of metal (or metalloid) resistance genes, especially contributing to arsenite oxidation, were identified.