Introduction to Bacterial Anhydrobiosis: A General Perspective and the Mechanisms of Desiccation-Associated Damage.

Anhydrobiosis is the ability of selected organisms to lose almost all water and enter a state of reversible ametabolism. Such an organism dries up to a state of equilibrium with dry air. Unless special protective mechanisms exist, desiccation leads to damage, mainly to proteins, nucleic acids, and membrane lipids. A short historical outline of research on extreme dehydration of living organisms and the current state of research are presented. Terminological issues are outlined. The role of water in the cell and the mechanisms of damage occurring in the cell under the desiccation stress are briefly discussed. Particular attention was paid to damage to proteins, nucleic acids, and membrane lipids. Understanding the nature of the changes and damage associated with desiccation is essential for the study of desiccation-tolerance mechanisms and application research. Difficulties related to the definition of life and the limits of life in the scientific discussion, caused by the phenomenon of anhydrobiosis, were also indicated.

Microbiological Sulfide Removal-From Microorganism Isolation to Treatment of Industrial Effluent.

Management of excessive aqueous sulfide is one of the most significant challenges of treating effluent after biological sulfate reduction for metal recovery from hydrometallurgical leachate. The main objective of this study was to characterize and verify the effectiveness of a sulfide-oxidizing bacterial (SOB) consortium isolated from post-mining wastes for sulfide removal from industrial leachate through elemental sulfur production. The isolated SOB has a complete sulfur-oxidizing metabolic system encoded by sox genes and is dominated by the Arcobacter genus. XRD analysis confirmed the presence of elemental sulfur in the collected sediment during cultivation of the SOB in synthetic medium under controlled physicochemical conditions. The growth yield after three days of cultivation reached ~2.34 gprotein/molsulfid, while approximately 84% of sulfide was transformed into elemental sulfur after 5 days of incubation. Verification of isolated SOB on the industrial effluent confirmed that it can be used for effective sulfide concentration reduction (~100% reduced from the initial 75.3 mg/L), but for complete leachate treatment (acceptable for discharged limits), bioaugmentation with other bacteria is required to ensure adequate reduction of chemical oxygen demand (COD).

Bacterial and Fungal Diversity Inside the Medieval Building Constructed with Sandstone Plates and Lime Mortar as an Example of the Microbial Colonization of a Nutrient-Limited Extreme Environment (Wawel Royal Castle, Krakow, Poland).

Biodeterioration is a serious threat to cultural heritage objects and buildings. The deterioration of a given material often incurs irreparable losses in terms of uniqueness and historical value. Hence preventive actions should be taken. One important challenge is to identify microbes involved in the biodeterioration process. In this study, we analyzed the microbial diversity of an ancient architectonical structure of the Rotunda of Sts. Felix and Adauctus, which is a part of the Wawel Royal Castle located in Krakow, Poland. The Rotunda is unavailable to tourists and could be treated as an extreme habitat due to the low content of nutrients coming either from sandstone plates bound with lime mortar or air movement. Microbial diversity was analyzed with the use of the high-throughput sequencing of marker genes corresponding to fragments of 16S rDNA (for Bacteria) and ITS2 (internal transcribed spacer 2) (for Fungi). The results showed that the microbial community adhered to wall surfaces is, to a large extent, endemic. Furthermore, alongside many microorganisms that could be destructive to masonry and mortar (e.g., Pseudomonas, Aspergillus), there were also bacteria, such as species of genera Bacillus, Paenisporosarcina, and Amycolatopsis, that can positively affect wall surface properties by reducing the damage caused by the presence of other microorganisms. We also showed that airborne microorganisms probably have little impact on the biodeterioration process as their abundance in the microbial community adhered to the ancient walls was very low.

Granulated Bog Iron Ores as Sorbents in Passive (Bio)Remediation Systems for Arsenic Removal.

The main element of PbRS (passive (bio)remediation systems) are sorbents, which act as natural filters retaining heavy metals and carriers of microorganisms involved in water treatment. Thus, the effectiveness of PbRS is determined by the quality of the (ad)sorbents, which should be stable under various environmental conditions, have a wide range of applications and be non-toxic to (micro)organisms used in these systems. Our previous studies showed that bog iron ores (BIOs) meet these requirements. However, further investigation of the physical and chemical parameters of BIOs under environmental conditions is required before their large-scale application in PbRS. The aim of this study was (i) to investigate the ability of granulated BIOs (gBIOs) to remove arsenic from various types of contaminated waters, and (ii) to estimate the application potential of gBIOs in technologies dedicated to water treatment. These studies were conducted on synthetic solutions of arsenic and environmental samples of arsenic contaminated water using a set of adsorption columns filled with gBIOs. The experiments performed in a static system revealed that gBIOs are appropriate arsenic and zinc adsorbent. Dynamic adsorption studies confirmed these results and showed, that the actual sorption efficiency of gBIOs depends on the adsorbate concentration and is directly proportional to them. Desorption analysis showed that As-loaded gBIOs are characterized by high chemical stability and they may be reused for the (ad)sorption of other elements, i.e., zinc. It was also shown that gBIOs may be used for remediation of both highly oxygenated waters and groundwater or settling ponds, where the oxygen level is low, as both forms of inorganic arsenic (arsenate and arsenite) were effectively removed. Arsenic concentration after treatment was <100 µg/L, which is below the limit for industrial water.

Raoultella sp. SM1, a novel iron-reducing and uranium-precipitating strain.

The main aim of this study was the characterisation of novel Raoutella isolate, an iron-reducing and uranium-precipitating strain, originating from microbial mats occurring in the sediments of a closed down uranium mine in Kowary (SW Poland). Characterisation was done in the context of its potential role in the functioning of these mats and the possibility to use them in uranium removal/recovery processes. In our experiment, we observed the biological precipitation of iron and uranium's secondary minerals containing oxygen, potassium, sodium and phosphor, which were identified as ningyoite-like minerals. The isolated strain, Raoultella sp. SM1, was also able to dissimilatory reduce iron (III) and uranium (VI) in the presence of citrate as an electron donor. Our studies allowed us to characterise a new strain which may be used as a model microorganism in the study of Fe and U respiratory processes and which may be useful in the bioremediation of uranium-contaminated waters and sediments. During this process, uranium may be immobilised in ningyoite-like minerals and can then be recovered in nano/micro-particle form, which may be easily transformed to uraninite.

Adaptation of Methanogenic Inocula to Anaerobic Digestion of Maize Silage.

A well-balanced microbial consortium is crucial for efficient biogas production. In turn, one of a major factor that influence on the structure of anaerobic digestion (AD) consortium is a source of microorganisms which are used as an inoculum. This study evaluated the influence of inoculum sources (with various origin) on adaptation of a biogas community and the efficiency of the biomethanization of maize silage. As initial inocula for AD of maize silage the samples from: (i) an agricultural biogas plant (ABP) which utilizes maize silage as a main substrate, (ii) cattle slurry (CS), which contain elevated levels of lignocelluloses materials, and (iii) raw sewage sludge (RSS) with low content of plant origin materials were used. The adaptation of methanogenic consortia was monitored during a series of passages, and the functionality of the adapted consortia was verified through start-up operation of AD in two-stage reactors. During the first stages of the adaptation phase, methanogenic consortia occurred very slowly, and only after several passages did the microbial community adapts to allow production of biogas with high methane content. The ABP consortium revealed highest biogas production in the adaptation and in the start-up process. The biodiversity dynamics monitored during adaptation and start-up process showed that community profile changed in a similar direction in three studied consortia. Native communities were very distinct to each other, while at the end of the Phase II of the start-up process microbial diversity profile was similar in all consortia. All adopted bacterial communities were dominated by representatives of Porphyromonadaceae, Rikenellaceae, Ruminococcaceae, and Synergistaceae. A shift from low acetate-preferring acetoclastic Methanosaetaceae (ABP and RSS) and/or hydrogenotrophic Archaea, e.g., Methanomicrobiaceae (CS) prevailing in the inoculum samples to larger populations of high acetate-preferring acetoclastic Methanosarcinaceae was observed by the end of the experiment. As a result, three independent, functional communities that syntrophically produced methane from acetate (primarily) and H2/CO2, methanol and methylamines were adapted. This study provides new insights into the specific process by which different inocula sampled from typical methanogenic environments that are commonly used to initiate industrial installations gradually adapted to allow biogas production from maize silage.

The influence of thermal treatment on bioweathering and arsenic sorption capacity of a natural iron (oxyhydr)oxide-based adsorbent.

Adsorption plays a significant role in remediation of waters contaminated with arsenic, but the efficiency of the process varies depending on the sorbent properties. Bog iron ores (BIOs), characterized by high sorption capacity and widespread availability, seem to be an optimal sorbent of arsenic. However, the use of BIOs for arsenic removal from waters may be limited by the high amount of organic matter, which may stimulate microbial activity, and thus decomposition of the sorbent. The aim of this study was to determine the effect of organic matter removal by thermal transformation (roasting) on the bioavailability of BIOs and their arsenic sorption capacity. For this purpose, the influence of bacterial growth and activity on untreated and treated BIOs, unloaded and loaded with arsenic, was studied. Moreover, the chemical and physical properties (including FTIR and desorption of arsenic) of BIOs were investigated as well. The results show that the removal of organic matter increases the stability of BIOs, and thus reduces the bioavailability of the immobilized arsenic.

Bacterial weathering of fossil organic matter and organic carbon mobilization from subterrestrial Kupferschiefer black shale: long-term laboratory studies.

A large part of the organic carbon present in the lithosphere is trapped in fossil organic matter deposited in sedimentary rocks. Only specialized microorganisms are able to degrade it contributing to the return of the carbon to the global cycle. The role of bacteria in this process is not yet completely understood. In the present laboratory studies, subterrestrial organic-rich ∼256-million-year-old Kupferschiefer black shale was exposed to the activity of an indigenous consortium of lithobiontic bacteria for 365 days under aerobic conditions. An interdisciplinary research approach was applied, consisting of a detailed comparison of the chemical composition of extractable bitumens as well as resistant to extraction kerogen of the unweathered black shale to that of the bioweathered and chemically weathered, identification of mobilized organic compounds and spectrometry-based determination of proteomic composition of the bacterial biofilm. The oxidative bioweathering of bitumens and kerogen was confirmed. The mobilization of organic carbon in the form of oxidized organic compounds, such as monohydroxy and dihydroxy alcohols, aldehydes, monocarboxylic and dicarboxylic acids and esters due to the microbial activity, was documented. The enzymes crucial for the aerobic metabolism of aliphatic and aromatic hydrocarbons such as monooxygenases and dehydrogenases were identified in the epilithic biofilm inhabiting the black shale.

The role of dissimilatory arsenate reducing bacteria in the biogeochemical cycle of arsenic based on the physiological and functional analysis of Aeromonas sp. O23A.

Dissimilatory arsenate reducing bacteria (DARB) are known to contribute to the mobilization of arsenic and other elements from minerals. Despite this, metabolic capabilities of only a few DARB strains have been thoroughly investigated so far, and the influence of these bacteria on the bioavailability of arsenic in the environment is still a topic for discussion. In this study, Aeromonas sp. O23A, one of the dominant DARB strains found in the Zloty Stok mine (SW Poland), was subjected to a detailed physiological and functional analysis aimed to identify the actual environmental impact of this strain. Physiological analyses revealed that O23A is a facultative anaerobe, capable of utilizing arsenate as a respiratory substrate and acetate, citrate and lactate as electron donors. Arsenate reduction was observed within the first 24h of culturing. The strain shows high resistance to arsenic and several other heavy metals (i.a. Cu, Fe, Ni and Zn) as well tolerance to a broad range of physico-chemical conditions. Metabolic preferences of O23A were thoroughly investigated using Biolog™ MicroArray assay. The strain was found to produce hydroxamate siderophores, potentially involved in the mobilization of iron and co-occurring heavy metals from minerals. On the other hand, O23A showed high adherence abilities, and its involvement in biofilm formation may lead to the entrapment of dissolved arsenic species and other toxic ions. The results shed light on the importance of arsenic respiration and resistance in the overall metabolism of Aeromonas sp. O23A and confirmed its key role in the biogeochemical cycle of arsenic, also in the context of self-purification of heavy-metal-contaminated waters.

The effect of the source of microorganisms on adaptation of hydrolytic consortia dedicated to anaerobic digestion of maize silage.

The main aim of this study was to evaluate the effect of the source of microorganisms on the selection of hydrolytic consortia dedicated to anaerobic digestion of maize silage. The selection process was investigated based on the analysis of changes in the hydrolytic activity and the diversity of microbial communities derived from (i) a hydrolyzer of a commercial agricultural biogas plant, (ii) cattle slurry and (iii) raw sewage sludge, during a series of 10 passages. Following the selection process, the adapted consortia were thoroughly analyzed for their ability to utilize maize silage and augmentation of anaerobic digestion communities. The results of selection of the consortia showed that every subsequent passage of each consortium leads to their adaptation to degradation of maize silage, which was manifested by the increased hydrolytic activity of the adapted consortia. Biodiversity analysis (based on the 16S rDNA amplicon sequencing) confirmed the changes microbial community of each consortium, and showed that after the last (10th) passage all microbial communities were dominated by the representatives of Lactobacillaceae, Prevotellaceae, Veillonellaceae. The results of the functional analyses showed that the adapted consortia improved the efficiency of maize silage degradation, as indicated by the increase in the concentration of glucose and volatile fatty acids (VFAs), as well as the soluble chemical oxygen demand (sCOD). Moreover, bioaugmentation of anaerobic digestion communities by the adapted hydrolytic consortia increased biogas yield by 10-29%, depending on the origin of the community. The obtained results also indicate that substrate input (not community origin) was the driving force responsible for the changes in the community structure of hydrolytic consortia dedicated to anaerobic digestion.

Solubilization of Pb-bearing apatite Pb5(PO4)3Cl by bacteria isolated from polluted environment.

The main purpose of this study was to test if microorganisms isolated from heavily polluted environments can enhance dissolution of Pb-apatite (pyromorphite) resulting in remobilization of lead. Three bacterial strains belonging to the genus Pseudomonas isolated from underground mines in SW Poland were used in batch experiments of pyromorphite solubilization carried out in phosphate reach and phosphate poor media. Bacteria growth and evolution of Pb and phosphate concentrations as well as pH were determined. Additionally the concentration of bacterial siderophores in leaching solution was assayed. All bacterial strains were able to grow in both media in the presence of pyromorphite. The number of bacterial cells was from one to two orders of magnitude higher in the phosphate rich media. In the phosphate poor media the only source of P was the dissolving lead apatite. Bacteria enhanced the solubility of pyromorphite resulting in elevated Pb concentrations, up to 853 µg L-1 in phosphate-rich medium and 6112 µg L-1 in phosphate-poor medium, compared to less than 100 µg L-1 in an abiotic control sample. Production of siderophores was characteristic for each culture and was much lower (10-1000 fold) in the phosphate-poor medium. This study demonstrates for the first time that indigenous bacteria can directly and indirectly promote the mobilization of lead from pyromorphite. This phenomenon should be considered in long term risk assessment of Pb contaminated soils after reclamation processes because bacteria can play a significant role in the efficiency of clean-up efforts and overall geochemical cycling of Pb.

Physiological and Metagenomic Analyses of Microbial Mats Involved in Self-Purification of Mine Waters Contaminated with Heavy Metals.

Two microbial mats found inside two old (gold and uranium) mines in Zloty Stok and Kowary located in SW Poland seem to form a natural barrier that traps heavy metals leaking from dewatering systems. We performed complex physiological and metagenomic analyses to determine which microorganisms are the main driving agents responsible for self-purification of the mine waters and identify metabolic processes responsible for the observed features. SEM and energy dispersive X-ray microanalysis showed accumulation of heavy metals on the mat surface, whereas, sorption experiments showed that neither microbial mats were completely saturated with heavy metals present in the mine waters, indicating that they have a large potential to absorb significant quantities of metal. The metagenomic analysis revealed that Methylococcaceae and Methylophilaceae families were the most abundant in both communities, moreover, it strongly suggest that backbones of both mats were formed by filamentous bacteria, such as Leptothrix, Thiothrix, and Beggiatoa. The Kowary bacterial community was enriched with the Helicobacteraceae family, whereas the Zloty Stok community consist mainly of Sphingomonadaceae, Rhodobacteraceae, and Caulobacteraceae families. Functional (culture-based) and metagenome (sequence-based) analyses showed that bacteria involved in immobilization of heavy metals, rather than those engaged in mobilization, were the main driving force within the analyzed communities. In turn, a comparison of functional genes revealed that the biofilm formation and heavy metal resistance (HMR) functions are more desirable in microorganisms engaged in water purification than the ability to utilize heavy metals in the respiratory process (oxidation-reduction). These findings provide insight on the activity of bacteria leading, from biofilm formation to self-purification, of mine waters contaminated with heavy metals.

Arsenic Mobilization from Historically Contaminated Mining Soils in a Continuously Operated Bioreactor: Implications for Risk Assessment.

Concentrations of soil arsenic (As) in the vicinity of the former Zloty Stok gold mine (Lower Silesia, southwest Poland) exceed 1000 µg g(-1) in the area, posing an inherent threat to neighboring bodies of water. This study investigated continuous As mobilization under reducing conditions for more than 3 months. In particular, the capacity of autochthonic microflora that live on natural organic matter as the sole carbon/electron source for mobilizing As was assessed. A biphasic mobilization of As was observed. In the first two months, As mobilization was mainly conferred by Mn dissolution despite the prevalence of Fe (0.1 wt % vs 5.4 for Mn and Fe, respectively) as indicated by multiple regression analysis. Thereafter, the sudden increase in aqueous As[III] (up to 2400 µg L(-1)) was attributed to an almost quintupling of the autochthonic dissimilatory As-reducing community (quantitative polymerase chain reaction). The aqueous speciation influenced by microbial activity led to a reduction of solid phase As species (X-ray absorption fine structure spectroscopy) and a change in the elemental composition of As hotspots (micro X-ray fluorescence mapping). The depletion of most natural dissolved organic matter and the fact that an extensive mobilization of As[III] occurred after two months raises concerns about the long-term stability of historically As-contaminated sites.

Microbial Consortium with High Cellulolytic Activity (MCHCA) for Enhanced Biogas Production.

The use of lignocellulosic biomass as a substrate in agricultural biogas plants is very popular and yields good results. However, the efficiency of anaerobic digestion, and thus biogas production, is not always satisfactory due to the slow or incomplete degradation (hydrolysis) of plant matter. To enhance the solubilization of the lignocellulosic biomass various physical, chemical and biological pretreatment methods are used. The aim of this study was to select and characterize cellulose-degrading bacteria, and to construct a microbial consortium, dedicated for degradation of maize silage and enhancing biogas production from this substrate. Over 100 strains of cellulose-degrading bacteria were isolated from: sewage sludge, hydrolyzer from an agricultural biogas plant, cattle slurry and manure. After physiological characterization of the isolates, 16 strains (representatives of Bacillus, Providencia, and Ochrobactrum genera) were chosen for the construction of a Microbial Consortium with High Cellulolytic Activity, called MCHCA. The selected strains had a high endoglucanase activity (exceeding 0.21 IU/mL CMCase activity) and a wide range of tolerance to various physical and chemical conditions. Lab-scale simulation of biogas production using the selected strains for degradation of maize silage was carried out in a two-bioreactor system, similar to those used in agricultural biogas plants. The obtained results showed that the constructed MCHCA consortium is capable of efficient hydrolysis of maize silage, and increases biogas production by even 38%, depending on the inoculum used for methane fermentation. The results in this work indicate that the mesophilic MCHCA has a great potential for application on industrial scale in agricultural biogas plants.

Determination of factors responsible for the bioweathering of copper minerals from organic-rich copper-bearing Kupferschiefer black shale.

The aim of this study was to investigate the bioweathering of copper minerals present in the alkaline, copper-bearing and organic-rich Kupferschiefer black shale through the action of a consortium of indigenous lithobiontic, heterotrophic, neutrophilic bacteria isolated from this sedimentary rock. The involvement of microorganisms in the direct/enzymatic bioweathering of fossil organic matter of the rock was confirmed. As a result of bacterial activity, a spectrum of various organic compounds such as urea and phosphoric acid tributyl ester were released from the rock. These compounds indirectly act on the copper minerals occurring in the rock and cause them to weather. This process was reflected in the mobilization of copper, iron and sulfur and in changes in the appearance of copper minerals observed under reflected light. The potential role of identified enzymes in biodegradation of fossil organic matter and role of organic compounds released from black shale as a result of this process in copper minerals weathering was discussed. The presented results provide a new insight into the role of chemical compounds released by bacteria during fossil organic matter bioweathering potentially important in the cycling of copper and iron deposited in the sedimentary rock. The originality of the described phenomenon lies in the fact that the bioweathering of fossil organic matter and, consequently, of copper minerals occur simultaneously in the same environment, without any additional sources of energy, electrons and carbon.

Shewanella sp. O23S as a Driving Agent of a System Utilizing Dissimilatory Arsenate-Reducing Bacteria Responsible for Self-Cleaning of Water Contaminated with Arsenic.

The purpose of this study was a detailed characterization of Shewanella sp. O23S, a strain involved in arsenic transformation in ancient gold mine waters contaminated with arsenic and other heavy metals. Physiological analysis of Shewanella sp. O23S showed that it is a facultative anaerobe, capable of growth using arsenate, thiosulfate, nitrate, iron or manganite as a terminal electron acceptor, and lactate or citrate as an electron donor. The strain can grow under anaerobic conditions and utilize arsenate in the respiratory process in a broad range of temperatures (10-37 °C), pH (4-8), salinity (0%-2%), and the presence of heavy metals (Cd, Co, Cr, Cu, Mn, Mo, Se, V and Zn). Under reductive conditions this strain can simultaneously use arsenate and thiosulfate as electron acceptors and produce yellow arsenic (III) sulfide (As2S3) precipitate. Simulation of As-removal from water containing arsenate (2.5 mM) and thiosulfate (5 mM) showed 82.5% efficiency after 21 days of incubation at room temperature. Based on the obtained results, we have proposed a model of a microbially mediated system for self-cleaning of mine waters contaminated with arsenic, in which Shewanella sp. O23S is the main driving agent.

Construction of the recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidation ability.

The plasmid pSinA of Sinorhizobium sp. M14 was used as a source of functional phenotypic modules, encoding proteins involved in arsenite oxidation and arsenic resistance, to obtain recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidative ability. An arsenite oxidation module was cloned into pBBR1MCS-2 vector yielding plasmid vector pAIO1, while an arsenic resistance module was cloned into pCM62 vector yielding plasmid pARS1. Both plasmid constructs were introduced (separately and together) into the cells of phylogenetically distant (representing Alpha-, Beta-, and Gammaproteobacteria) and physiologically diversified (unable to oxidize arsenite and susceptible/resistant to arsenite and arsenate) bacteria. Functional analysis of the modified strains showed that: (i) the plasmid pARS1 can be used for the construction of strains with an increased resistance to arsenite [up to 20mM of As(III), (ii) the presence of the plasmid pAIO1 in bacteria previously unable to oxidize As(III) to As(V), contributes to the acquisition of arsenite oxidation abilities by these cells, (iii) the highest arsenite utilization rate are observed in the culture of strains harbouring both the plasmids pAIO1 and pARS1, (iv) the strains harbouring the plasmid pAIO1 were able to grow on arsenic-contaminated mine waters (∼ 3.0 mg As L(-1)) without any supplementation.

Extracellular compounds produced by bacterial consortium promoting elements mobilization from polymetallic Kupferschiefer black shale (Fore-Sudetic Monocline, Poland).

Culture experiments employing Fe-deficient medium showed that a consortium of indigenous microorganisms isolated from Kupferschiefer black shale produced a mixture of extracellular compounds containing siderophores which could form complexes with a wide range of elements and were able to mediate element mobilization from polymetallic black shale. The mobilization of a diverse array of elements including a number of essential trace elements (Co, Cu, Mn, Mo, Zn) and toxic species (As) was shown. Since the bacteria used in this study were originally obtained from a subsurface copper deposit, these results highlight the potential importance of extracellular compounds in biogeochemical cycles of elements in underground environment and their ecological significance in promoting the uptake of essential trace metals and resistance to toxic elements.

Bacteria diversity and arsenic mobilization in rock biofilm from an ancient gold and arsenic mine.

In this paper we characterize the biofilm community from an ancient Zloty Stok gold and arsenic mine. Bacterial diversity was examined using a culture-independent technique based on 16S rRNA gene amplification, cloning and sequencing. We show that unexpectedly the microbial diversity of this community was extremely high (more than 190 OTUs detected), with the most numerous members from Rhizobiales (α-Proteobacteria). Although the level of rock biofilm diversity was similar to the microbial mat community we have previously characterized in the same adit, its taxonomic composition was completely different. Detailed analysis of functional arrA and aioA genes, chemical properties of siderophores found in pore water as well as the biofilm chemical composition suggest that the biofilm community contributes to arsenic pollution of surrounding water in a biogeochemical cycle similar to the one observed in bacterial mats. To interpret our results concerning the biological arsenic cycle, we applied the theory of ecological pyramids of Charles Elton.

Structural and functional genomics of plasmid pSinA of Sinorhizobium sp. M14 encoding genes for the arsenite oxidation and arsenic resistance.

Plasmid pSinA of Sinorhizobium sp. M14 (Alphaproteobacteria) is the first described, natural, self-transferable plasmid harboring a complete set of genes for oxidation of arsenite. Removal of this plasmid from cells of the host strain caused the loss of resistance to arsenic and heavy metals (Cd, Co, Zn and Hg) and abolished the ability to grow on minimal salt medium supplemented with sodium arsenite as the sole energy source. Plasmid pSinA was introduced into other representatives of Alphaproteobacteria which resulted in acquisition of new abilities concerning arsenic resistance and oxidation, as well as heavy metals resistance. Microcosm experiments revealed that plasmid pSinA can also be transferred via conjugation into other indigenous bacteria from microbial community of As-contaminated soils, including representatives of Alpha- and Gammaproteobacteria. Analysis of "natural" transconjugants showed that pSinA is functional (expresses arsenite oxidase) and is stably maintained in their cells after approximately 60 generations of growth under nonselective conditions. This work clearly demonstrates that pSinA is a self-transferable, broad-host-range plasmid, which plays an important role in horizontal transfer of arsenic metabolism genes.