Isolation, characterization, and genome insights into an anaerobic sulfidogenic Tissierella bacterium from Cu-bearing coins.

Recent reports on antimicrobial effects of metallic Cu prompted this study of anaerobic microbial communities on copper surfaces. Widely circulating copper-containing coinage was used as a potential source for microorganisms that had had human contact and were tolerant to copper. This study reports on the isolation, characterization, and genome of an anaerobic sulfidogenic Tissierella sp. P1from copper-containing brass coinage. Dissimilatory (bi)sulfite reductase dsrAB present in strain P1 genome and the visible absorbance around 630 nm in the cells suggested the presence of a desulfoviridin-type protein. However, the sulfate reduction rate measurements with 35SO42- did not confirm the dissimilatory sulfate reduction by the strain. The P1 genome lacks APS reductase, sulfate adenylyltransferase, DsrC, and DsrMK necessary for dissimilatory sulfate reduction. The isolate produced up to 0.79 mM H2S during growth, possibly due to cysteine synthase (CysK) and/or cysteine desulfhydrase (CdsH) activities, encoded in the genome. The strain can tolerate up to 2.4 mM Cu2+(150 mg/l) in liquid medium, shows affinity to metallic copper, and can survive on copper-containing coins up to three days under ambient air and dry conditions. The genome sequence of strain P1 contained cutC, encoding a copper resistance protein, which distinguishes it from all other Tissierella strains with published genomes.

Isolation, Characterization, and Metal Response of Novel, Acid-Tolerant Penicillium spp. from Extremely Metal-Rich Waters at a Mining Site in Transbaikal (Siberia, Russia).

The role of fungi in metal cycling in acidic environments has been little explored to date. In this study, two acid-tolerant and metal-resistant Penicillium isolates, strains ShG4B and ShG4C, were isolated from a mine site in the Transbaikal area of Siberia (Russia). Waters at the mine site were characterized by extremely high metal concentrations: up to 18 g l-1 Fe and > 2 g l-1 each of Cu, Zn, Al, and As. Both isolates were identified as Penicillium spp. by phylogenetic analyses and they grew well in Czapek medium acidified to pH 2.5. Resistance to Cu, Cd, Ni, Co, and arsenate was in the range of 1-10 g l-1. Further experiments with Penicillium strain ShG4C demonstrated that growth in Cu-containing media was accompanied by the precipitation of Cu-oxalate (moolooite) and the formation of extracellular vesicles enriched in Cu on the mycelia. Vesicles were greatly reduced in size in Cd-containing media and were not formed in the presence of Ni or Co. Cd-oxalate was detected as a crystalline solid phase in Cd-exposed mycelia. Hydrated Ni-sulfate (retgersite) and Co-sulfate (bieberite) were detected in mycelia grown in the presence of Ni and Co, respectively. The results demonstrated that acid-tolerant and metal-resistant Penicillium constitute a component in extremophilic microbiomes, contributing to organic matter breakdown and formation of secondary solid phases at pH ranges found in acid rock drainage.

Selection for novel, acid-tolerant Desulfovibrio spp. from a closed Transbaikal mine site in a temporal pH-gradient bioreactor.

Almost all the known isolates of acidophilic or acid-tolerant sulphate-reducing bacteria (SRB) belong to the spore-forming genus Desulfosporosinus in the Firmicutes. The objective of this study was to isolate acidophilic/acid-tolerant members of the genus Desulfovibrio belonging to deltaproteobacterial SRB. The sample material originated from microbial mat biomass submerged in mine water and was enriched for sulphate reducers by cultivation in anaerobic medium with lactate as an electron donor. A stirred tank bioreactor with the same medium composition was inoculated with the sulphidogenic enrichment. The bioreactor was operated with a temporal pH gradient, changing daily, from an initial pH of 7.3 to a final pH of 3.7. Among the bacteria in the bioreactor culture, Desulfovibrio was the only SRB group retrieved from the bioreactor consortium as observed by 16S rRNA-targeted denaturing gradient gel electrophoresis. Moderately acidophilic/acid-tolerant isolates belonged to Desulfovibrio aerotolerans-Desulfovibrio carbinophilus-Desulfovibrio magneticus and Desulfovibrio idahonensis-Desulfovibrio mexicanus clades within the genus Desulfovibrio. A moderately acidophilic strain, Desulfovibrio sp. VK (pH optimum 5.7) and acid-tolerant Desulfovibrio sp. ED (pH optimum 6.6) dominated in the bioreactor consortium at different time points and were isolated in pure culture.

Kinetics of aerobic and anaerobic biomineralization of atrazine in surface and subsurface agricultural soils in Ohio.

The purpose of this study was to assess atrazine mineralization in surface and subsurface samples retrieved from vertical cores of agricultural soils from two farm sites in Ohio. The Defiance site (NW-Ohio) was on soybean-corn rotation and Piketon (S-Ohio) was on continuous corn cultivation. Both sites had a history of atrazine application for at least a couple of decades. The clay fraction increased at the Defiance site and the organic matter and total N content decreased with depth at both sites. Mineralization of atrazine was assessed by measurement of (14)CO2 during incubation of soil samples with [U-ring-(14)C]-atrazine. Abiotic mineralization was negligible in all soil samples. Aerobic mineralization rate constants declined and the corresponding half-lives increased with depth at the Defiance site. Anaerobic mineralization (supplemented with nitrate) was mostly below the detection at the Defiance site. In Piketon samples, the kinetic parameters of aerobic and anaerobic biomineralization of atrazine displayed considerable scatter among replicate cores and duplicate biometers. In general, this study concludes that data especially for anaerobic biomineralization of atrazine can be more variable as compared to aerobic conditions and cannot be extrapolated from one agricultural site to another.

Characterization of precipitates formed by H(2)S-producing, Cu-resistant Firmicute isolates of Tissierella from human gut and Desulfosporosinus from mine waste.

The purpose of this study was to characterize precipitates formed in anaerobic, H2S-producing cultures of two Tissierella isolates and Desulfosporosinus strain DB. The cultures were grown in Cu-containing media as part of a larger study of Cu resistance in anaerobic sulfidogens. The Tissierella strains produced H2S from peptone. Desulfosporosinus formed H2S from peptone or through dissimilatory sulfate reduction with lactate. Tissierella cultures precipitated iron phosphate, vivianite, but no crystalline phases or Cu sulfides were detected. Multiple Cu sulfides, including chalcopyrite and covellite, were detected in Desulfosporosinus cultures but vivianite was not formed. Ion microprobe spectra and electron microscopic examination showed major variation in the elemental composition and morphological differences depending on incubation conditions. Extended incubation time for at least 1-2 months increased the crystallinity of the precipitates. The results highlight biogeochemical differences in sulfide and phosphate precipitates between the two major groups of Firmicutes although they may share the same habitat including the human intestinal tract.

Nitrate determines the bacterial habitat specialization and impacts microbial functions in a subsurface karst cave.

Karst caves are usually considered as natural laboratories to study pristine microbiomes in subsurface biosphere. However, effects of the increasingly detected nitrate in underground karst ecosystem due to the acid rain impact on microbiota and their functions in subsurface karst caves have remained largely unknown. In this study, samples of weathered rocks and sediments were collected from the Chang Cave, Hubei province and subjected to high-throughput sequencing of 16S rRNA genes. The results showed that nitrate significantly impacted bacterial compositions, interactions, and functions in different habitats. Bacterial communities clustered according to their habitats with distinguished indicator groups identified for each individual habitat. Nitrate shaped the overall bacterial communities across two habitats with a contribution of 27.2%, whereas the pH and TOC, respectively, structured bacterial communities in weathered rocks and sediments. Alpha and beta diversities of bacterial communities increased with nitrate concentration in both habitats, with nitrate directly affecting alpha diversity in sediments, but indirectly on weathered rocks by lowering pH. Nitrate impacted more on bacterial communities in weathered rocks at the genus level than in sediments because more genera significantly correlated with nitrate concentration in weathered rocks. Diverse keystone taxa involved in nitrogen cycling were identified in the co-occurrence networks such as nitrate reducers, ammonium-oxidizers, and N2-fixers. Tax4Fun2 analysis further confirmed the dominance of genes involved in nitrogen cycling. Genes of methane metabolism and carbon fixation were also dominant. The dominance of dissimilatory and assimilatory nitrate reduction in nitrogen cycling substantiated nitrate impact on bacterial functions. Our results for the first time revealed the impact of nitrate on subsurface karst ecosystem in terms of bacterial compositions, interactions, and functions, providing an important reference for further deciphering the disturbance of human activities on the subsurface biosphere.

Methanotrophs dominate methanogens and act as a methane sink in a subterranean karst cave.

Karst caves are potential sinks of atmospheric methane due to microbial consumption. However, knowledge gaps on methanogens (methane producing microorganisms) and their interaction with methane-oxidizing bacteria (MOB) hinder our further understanding about methane dynamics in karst caves. Here we reported methanogenic community composition and their interaction with MOBs in the Heshang Cave to comprehensively understand methane cycling in subsurface biosphere. MOBs in karst cave were dominated by high-affinity MOB, upland soil cluster (USC), with USCγ pmoA gene abundance within the range of 1.34 × 104 to 1.8 × 107 copies·g-1 DW. In contrast, methanogens were dominated by Methanoregula and cluster ZC-I. The mcrA numbers were 7.21 × 103 to 8.31 × 104 copies·g-1 DW, 1-3 orders of magnitude lower than those of MOB. The inter-domain network analysis indicated that MOBs and methanogens cooperated more in the interior of the cave. Despite of the higher number of methanogenic nodes in the network, MOB dominated the keystone taxa, suggesting a leading functional role of MOB. MOB in caves showed a comparable with or higher potential methane oxidizing rate (PMOR, 0.63 ng CH4·g-1 DW·h-1 in sediment versus 11.02 ng CH4·g-1 DW·h-1 in weathered rock) than those in soils, whereas methane produced by methanogens was undetected. Collectively, high absolute abundances of MOB, high PMORs, the dominance of methanotrophic keystone taxa in the inter-domain network confirmed the superiority of MOBs over methanogens in the oligotrophic karst cave, mounting new evidence on caves as an important methane sink in terms of the interaction between methanogens and MOBs.

Elevated antimony concentration stimulates rare taxa of potential autotrophic bacteria in the Xikuangshan groundwater.

Microbial communities composed of few abundant and many rare species are widely involved in the biogeochemical cycles of elements. Yet little is known about the ecological roles of rare taxa in antimony (Sb) contaminated groundwater. Groundwater samples were collected along an Sb concentration gradient in the Xikuangshan antimony mine area and subjected to high through-put sequencing of 16S rRNA genes to investigate the bacterial communities. Results suggested that both abundant and rare sub-communities were dominated by Betaproteobacteria, Gammaproteobacteria, and Alphaproteobacteria, whereas rare sub-communities showed higher alpha-diversities. Multivariate analysis showed that both the abundant and rare taxa were under the stress of Sb, but the impact on rare taxa was greater. Nitrate explained a large part for the variation of the abundant sub-communities, indicating the critical role of nitrate for their activities under anoxic conditions. In contrast, bicarbonate significantly impacted rare sub-communities, suggesting their potential autotrophic characteristics. To further explore the role of rare taxa in the communities and the mechanism of affecting the community composition, a network was constructed to display the co-occurrence pattern of bacterial communities. The rare taxa contributed most of the network nodes and served as keystone species to maintain the stability of community. Abiotic factors (mainly Sb and pH) and bacterial interspecific interactions (interactions between keystone species and other bacterial groups) jointly affect the community dynamics. Functional prediction was performed to further reveal the ecological function of rare taxa in the Sb-disturbed groundwater environment. The results indicated that the rare taxa harbored much more diverse functions than their abundant counterparts. Notably, elevated Sb concentration promoted some potential autotrophic functions in rare taxa such as the oxidation of S-, N-, and Fe(II)-compounds. These results offer new insights into the roles of rare species in elemental cycles in the Sb-impacted groundwater.

Water table level controls methanogenic and methanotrophic communities and methane emissions in a Sphagnum-dominated peatland.

Peatlands are important sources of the greenhouse gas methane emissions equipoised by methanogens and methanotrophs. However, knowledge about how microbial functional groups associated with methane production and oxidation respond to water table fluctuations has been limited to date. Here, methane-related microbial communities and the potentials of methane production and oxidation were determined along sectioned peat layers in a subalpine peatland across four Sphagnum-dominated sites with different water table levels. Methane fluxes were also monitored at these sites. The results showed that mcrA gene copies for methanogens were the highest in the 10- to 15-cm peat layer, which was also characterized by the maximum potential methane production (24.53 ± 1.83 nmol/g/h). Copy numbers of the pmoA gene for type Ia and Ib methanotrophs were enriched in the 0-5 cm peat layer with the highest potential methane oxidation (43.09 ± 3.44 nmol/g/h). For the type II methanotrophs, the pmoA gene copies were higher in the 10- to 15-cm peat layer. Hydrogenotrophic methanogens and type II methanotrophs dominated the methane functional groups. Deterministic process contributed more to methanogenic and methanotrophic community assemblages in comparison with stochastic process. The level of water table significantly shaped methanogenic and methanotrophic community structures and regulated methane fluxes. Compared with vascular plants, Sphagnum mosses significantly reduced the methane emissions in peatlands. Collectively, these findings enhance a comprehensive understanding of the effect of the water table level on methane functional groups, with consequential implications for reducing methane emissions within peatland ecosystems.IMPORTANCEThe water table level is recognized as a critical factor in regulating methane emissions, which are largely dependent on the balance of methanogens and methanotrophs. Previous studies on peat methane emissions have been mostly focused on spatial-temporal variations and the relationship with meteorological conditions. However, the role of the water table level in methane emissions remains unknown. In this work, four representative microhabitats along a water table gradient in a Sphagnum-dominated peatland were sampled to gain an insight into methane functional communities and methane emissions as affected by the water table level. The changes in methane-related microbial community structure and assembly were used to characterize the response to the water table level. This study improves the understanding of the changes in methane-related microbial communities and methane emissions with water table levels in peatlands.

Archaea are better adapted to antimony stress than their bacterial counterparts in Xikuangshan groundwater.

Archaea are important ecological components of microbial communities in various environments, but are currently poorly investigated in antimony (Sb) contaminated groundwater particularly on their ecological differences in comparison with bacteria. To address this issue, groundwater samples were collected from Xikuangshan aquifer along an Sb gradient and subjected to 16S rRNA gene amplicon sequencing and bioinformatic analysis. The results demonstrated that bacterial communities were more susceptibly affected by elevated Sb concentration than their archaeal counterparts, and the positive stimulation of Sb concentration on bacterial diversity coincided with the intermediate disturbance hypothesis. Overall, the balance of environmental variables (Sb, pH, and EC), competitive interactions, and stochastic events jointly regulated bacterial and archaeal communities. Linear fitting analysis revealed that Sb significantly drove the deterministic process (heterogeneous selection) of bacterial communities, whereas stochastic process (dispersal limitation) contributed more to archaeal community assembly. In contract, the assembly of Sb-resistant bacteria and archaea was dominated by the stochastic process (undominated), which implied the important role of diversification and drift instead of selection. Compared with Sb-resistant microorganisms, bacterial and archaeal communities showed lower niche width, which may result from the constraints of Sb concentration and competitive interaction. Moreover, Sb-resistant archaea had a higher niche than that of Sb-resistant bacteria via investing on flexible metabolic pathways such as organic metabolism, ammonia oxidation; and carbon fixation to enhance their competitiveness. Our results offered new insights into the ecological adaptation mechanisms of bacteria and archaea in Sb-contaminated groundwater.

Growth of Dunaliella tertiolecta and associated bacteria in photobioreactors.

The aim of this study was to test three flat-plate photobioreactor configurations for cultivation of marine green alga Dunaliella tertiolecta under non-axenic growth conditions and to characterize and quantify the associated bacteria. The photobioreactor cultivations were conducted using tap water-based media. Static mixers intended to enhance mixing and light utilization did not generally increase algal growth at the low light intensities used. The maximum biomass concentration (measured as volatile suspended solids) and maximum specific growth rate achieved in the flat plate with no mixer were 2.9 g l⁻¹ and 1.3 day⁻¹, respectively. Based on quantitative polymerase chain reaction, bacterial growth followed the growth of D. tertiolecta. Based on 16S rDNA amplification and denaturing gradient gel electrophoresis profiling, heterotrophic bacteria in the D. tertiolecta cultures mainly originated from the non-axenic algal inocula, and tap water heterotrophs were not enriched in high chloride media (3 % salinity). Bacterial communities were relatively stable and reproducible in all flat-plate cultivations and were dominated by Gammaproteobacteria, Flavobacteria, and Alphaproteobacteria.

The Characterization of Microbiome and Interactions on Weathered Rocks in a Subsurface Karst Cave, Central China.

Karst caves are a natural oligotrophic subsurface biosphere widely distributed in southern China. Despite the progress in bacterial and fungal diversity, the knowledge about interactions between bacteria, fungi, and minerals is still limited in caves. Hence, for the first time, we investigated the interaction between bacteria and fungi living on weathered rocks in the Heshang Cave via high-throughput sequencing of 16S rRNA and ITS1 genes, and co-occurrence analysis. The mineral compositions of weathered rocks were analyzed by X-ray diffraction. Bacterial communities were dominated by Actinobacteria (33.68%), followed by Alphaproteobacteria (8.78%), and Planctomycetia (8.73%). In contrast, fungal communities were dominated by Sordariomycetes (21.08%) and Dothideomycetes (14.06%). Mineral substrata, particularly phosphorus-bearing minerals, significantly impacted bacterial (hydroxyapatite) and fungal (fluorapatite) communities as indicated by the redundancy analysis. In comparison with fungi, the development of bacterial communities was more controlled by the environmental selection indicated by the overwhelming contribution of deterministic processes. Co-occurrence network analysis showed that all nodes were positively linked, indicating ubiquitous cooperation within bacterial groups and fungal groups, as well as between bacteria and fungi under oligotrophic conditions in the subsurface biosphere. In total, 19 bacterial ASVs and 34 fungal OTUs were identified as keystone taxa, suggesting the fundamental role of fungi in maintaining the microbial ecosystem on weathered rocks. Ascomycota was most dominant in keystone taxa, accounting for 26.42%, followed by Actinobacteria in bacteria (24.53%). Collectively, our results confirmed the highly diverse bacterial and fungal communities on weathered rocks, and their close cooperation to sustain the subsurface ecosystem. Phosphorus-bearing minerals were of significance in shaping epipetreous bacterial and fungal communities. These observations provide new knowledge about microbial interactions between bacteria, fungi, and minerals in the subterranean biosphere.

Different responses of bacteria and fungi to environmental variables and corresponding community assembly in Sb-contaminated soil.

Bacterial communities in antimony (Sb) polluted soils have been well addressed, whereas the important players fungal communities are far less studied to date. Here, we report different responses of bacterial and fungal communities to Sb contamination and the ecological processes controlling their community assembly. Soil samples in the Xikuangshan mining area were collected and subjected to high through-put sequencing of 16S rRNA and ITS1 to investigate bacterial and fungal communities, respectively, along an Sb gradient. Sb speciation in the soil samples and other physicochemical parameters were analyzed as well. Bacterial communities were dominated by Deltaproteobacteria in the soil with highest Sb concentration, whereas Chloroflexi were dominant in the soil with lowest Sb concentration. Fungal communities in high-Sb soils were predominated by unclassified Fungi, whilst Leotiomycetes were dominant in low-Sb soil samples. Multivariate analysis indicated that Sb, pH and soil texture were the main drivers to strongly impact microbial communities. We further identified Sb-resistant microbial groups via correlation analysis. In total, 18 bacterial amplicon sequence variants (ASVs) were found to potentially involve in biogeochemical cycles such as Sb oxidation, sulfur oxidation or nitrate reduction, whereas 12 fungal ASVs were singled out for potential heavy metal resistance and plant growth promotion. Community assembly analysis revealed that variable selection contributed 100% to bacterial community assembly under acidic or high Sb concentration conditions, whereas homogeneous selection dominated fungal community assembly with a contribution over 78.9%. The community assembly of Sb-resistant microorganisms was mainly controlled by stochastic process. The results offer new insights into microbial ecology in Sb-contaminated soils, especially on the different responses of microbial communities under identical environmental stress and the different ecological processes underlining bacterial and fungal community assembly.

Thermophilic, anaerobic co-digestion of microalgal biomass and cellulose for H2 production.

Microalgal biomass has been a focus in the sustainable energy field, especially biodiesel production. The purpose of this study was to assess the feasibility of treating microalgal biomass and cellulose by anaerobic digestion for H2 production. A microbial consortium, TC60, known to degrade cellulose and other plant polymers, was enriched on a mixture of cellulose and green microalgal biomass of Dunaliella tertiolecta, a marine species, or Chlorella vulgaris, a freshwater species. After five enrichment steps at 60°C, hydrogen yields increased at least 10% under all conditions. Anaerobic digestion of D. tertiolecta and cellulose by TC60 produced 7.7 mmol H2/g volatile solids (VS) which were higher than the levels (2.9-4.2 mmol/g VS) obtained with cellulose and C. vulgaris biomass. Both microalgal slurries contained satellite prokaryotes. The C. vulgaris slurry, without TC60 inoculation, generated H2 levels on par with that of TC60 on cellulose alone. The biomass-fed anaerobic digestion resulted in large shifts in short chain fatty acid concentrations and increased ammonium levels. Growth and H2 production increased when TC60 was grown on a combination of D. tertiolecta and cellulose due to nutrients released from algal cells via lysis. The results indicated that satellite heterotrophs from C. vulgaris produced H2 but the Chlorella biomass was not substantially degraded by TC60. To date, this is the first study to examine H2 production by anaerobic digestion of microalgal biomass. The results indicate that H2 production is feasible but higher yields could be achieved by optimization of the bioprocess conditions including biomass pretreatment.

A potential sanitary sewer overflow treatment technology: fixed-media bioreactors.

Under certain conditions, sanitary sewer overflows (SSOs) containing raw wastewater may be discharged to public land and can contribute to environmental and public health issues. Although this problem has attracted the attention of local, state, and federal government and regulators, relatively little SSO abatement research has been published. This study used fixed-media bioreactors, a proven onsite technology in rural areas, to treat wet weather SSO wastewater and reduce its effects on the receiving water environment. The results of this 32-month laboratory study showed that fixed-media bioreactors, especially sand bioreactors, efficiently removed organic matter, solids, and nutrients during six-hour simulated SSO peak flows. Five-day biochemical oxygen demand (BODs) of the simulated SSO varied between 40 and 125 mg/L. The average effluent concentration of BOD5 was 13 mg/L in sand bioreactors at a hydraulic loading rate of 20.4 cm/h. In addition to having high hydraulic loadings, SSO events occur infrequently. This irregularity requires that treatment systems quickly start up and effectively treat wastewater after a period of no flow. This research found that an interval up to six months between two SSO peak flows did not affect the bioreactor performance. Based on this work, fixed-media bioreactors have the potential to reduce the effects of SSOs on the water environment by following proper design parameters and operation strategies. The pollution loading of approximately 18 g BODs/m2 x h is recommended for the efficient performance of sand bioreactors in the SSO treatment.

Pathobionts: mechanisms of survival, expansion, and interaction with host with a focus on Clostridioides difficile.

Pathobionts are opportunistic microbes that emerge as a result of perturbations in the healthy microbiome due to complex interactions of various genetic, exposomal, microbial, and host factors that lead to their selection and expansion. Their proliferations can aggravate inflammatory manifestations, trigger autoimmune diseases, and lead to severe life-threatening conditions. Current surge in microbiome research is unwinding these complex interplays between disease development and protection against pathobionts. This review summarizes the current knowledge of pathobiont emergence with a focus on Clostridioides difficile and the recent findings on the roles of immune cells such as iTreg cells, Th17 cells, innate lymphoid cells, and cytokines in protection against pathobionts. The review calls for adoption of innovative tools and cutting-edge technologies in clinical diagnostics and therapeutics to provide insights in identification and quantification of pathobionts.

Predictive modelling of Fe(III) precipitation in iron removal process for bioleaching circuits.

In this study, the applicability of three modelling approaches was determined in an effort to describe complex relationships between process parameters and to predict the performance of an integrated process, which consisted of a fluidized bed bioreactor for Fe(3+) regeneration and a gravity settler for precipitative iron removal. Self-organizing maps were used to visually evaluate the associations between variables prior to the comparison of two different modelling methods, the multiple regression modelling and artificial neural network (ANN) modelling, for predicting Fe(III) precipitation. With the ANN model, an excellent match between the predicted and measured data was obtained (R (2) = 0.97). The best-fitting regression model also gave a good fit (R (2) = 0.87). This study demonstrates that ANNs and regression models are robust tools for predicting iron precipitation in the integrated process and can thus be used in the management of such systems.

Extracellular enzyme activities and nutrient availability during artificial groundwater recharge.

Natural organic matter (NOM) removal is the main objective of artificial groundwater recharge (AGR) for drinking water production and biodegradation plays a substantial role in this process. This study focused on the biodegradation of NOM and nutrient availability for microorganisms in AGR by the determination of extracellular enzyme activities (EEAs) and nutrient concentrations along a flow path in an AGR aquifer (Tuusula Water Works, Finland). Natural groundwater in the same area but outside the influence of recharge was used as a reference. Determination of the specific alpha-d-glucosidase (alpha-Glu), beta-d-glucosidase (beta-Glu), phosphomonoesterase (PME), leucine aminopeptidase (LAP) and acetate esterase (AEST) activities by fluorogenic model substrates revealed major increases in the enzymatic hydrolysis rates in the aquifer within a 10m distance from the basin. The changes in the EEAs along the flow path occurred simultaneously with decreases in nutrient concentrations. The results support the assumption that the synthesis of extracellular enzymes in aquatic environments is up and down regulated by nutrient availability. The EEAs in the basin sediment and pore water samples (down to 10cm) were in the same order of magnitude as in the basin water, suggesting similar nutritional conditions. Phosphorus was likely to be the limiting nutrient at this particular AGR site. Furthermore, the extracellular enzymes functioned in a synergistic and cooperative way.

Can Sulfate Be the First Dominant Aqueous Sulfur Species Formed in the Oxidation of Pyrite by Acidithiobacillus ferrooxidans?

According to the literature, pyrite (FeS2) oxidation has been previously determined to involve thiosulfate as the first aqueous intermediate sulfur product, which is further oxidized to sulfate. In the present study, pyrite oxidation by Acidithiobacillus ferrooxidans was studied using electrochemical and metabolic approaches in an effort to extend existing knowledge on the oxidation mechanism. Due to the small surface area, the reaction rate of a compact pyrite electrode in the form of polycrystalline pyrite aggregate in A. ferrooxidans suspension was very slow at a spontaneously formed high redox potential. The slow rate made it possible to investigate the oxidation process in detail over a term of 100 days. Using electrochemical parameters from polarization curves and levels of released iron, the number of exchanged electrons per pyrite molecule was estimated. The values close to 14 and 2 electrons were determined for the oxidation with and without bacteria, respectively. These results indicated that sulfate was the dominant first aqueous sulfur species formed in the presence of bacteria and elemental sulfur was predominantly formed without bacteria. The stoichiometric calculations are consistent with high iron-oxidizing activities of bacteria that continually keep the released iron in the ferric form, resulting in a high redox potential. The sulfur entity of pyrite was oxidized to sulfate by Fe3+ without intermediate thiosulfate under these conditions. Cell attachment on the corroded pyrite electrode surface was documented although pyrite surface corrosion by Fe3+ was evident without bacterial participation. Attached cells may be important in initiating the oxidation of the pyrite surface to release iron from the mineral. During the active phase of oxidation of a pyrite concentrate sample, the ATP levels in attached and planktonic bacteria were consistent with previously established ATP content of iron-oxidizing cells. No significant upregulation of three essential genes involved in energy metabolism of sulfur compounds was observed in the planktonic cells, which represented the dominant biomass in the pyrite culture. The study demonstrated the formation of sulfate as the first dissolved sulfur species with iron-oxidizing bacteria under high redox potential conditions. Minor aqueous sulfur intermediates may be formed but as a result of side reactions.

Decolorization of Reactive Black 5 and Reactive Blue 4 Dyes in Microbial Fuel Cells.

Microbial fuel cells (MFCs) have potential to treat industrial wastewater containing organic compounds and simultaneously generate power. Organic compounds include textile dyes with various chromophore groups, which can be decolorized reductively by microorganisms under anaerobic conditions. In the present study, we examined the decolorization of Reactive Black 5 (RB5) azo dye and Reactive Blue 4 (RBL4) anthraquinone dye under open circuit potential in MFCs with graphite plate and graphite felt electrodes and a microbial consortium originally derived from bovine rumen fluid. RB5 dye was more than 90% decolorized in 120, 165, and 225 min at 50, 100, and 200 mg L-1 concentrations, respectively. RBL4 dye at 50 and 100 mg L-1 took 225 and 300 min to decolorize, while 200 mg L-1 RBL4 dye was not decolorized at all. Under closed circuit conditions, decolorization increased with decrease in external load, whereas current generation increased with external resistance. The results demonstrate that the reductive cleavage of the chromophore was more rapid with RB5 than with RBL4.