Assessment of Full-Scale N2O Emission Characteristics and Testing of Control Concepts in an Activated Sludge Wastewater Treatment Plant with Alternating Aerobic and Anoxic Phases.

This work aims to obtain full-scale N2O emission characteristics translatable into viable N2O control strategies and conduct full-scale testing of the proposed N2O control concepts. Data of a long-term monitoring campaign was first used to quantify full-scale N2O emission and probe into the seasonal pattern. Then trends between N2O production/emission and process variables/conditions during typical operating cycles were revealed to explore the dynamic N2O emission behavior. A multivariate statistical analysis was performed to find the dependency of N2O emission on relevant process variables. The results show for the first time that relatively low/high N2O emission took place in seasons with a decreasing/increasing trend of water temperature, respectively. Aerobic phase contributed to N2O production/emission probably mainly through the hydroxylamine pathway. Comparatively, heterotrophic bacteria had a dual role in the anoxic phase and could be responsible for both net N2O production and consumption. Incomplete denitrification might contribute mainly to the N2O production/emission in the anoxic phase and the accumulation of N2O to be significantly emitted in the following cycle due to the competition between different denitrification steps for electron donors. Therefore, properly extending the length of anoxic phase could serve as a potential control means to regulate N2O accumulation in the anoxic phase. The full-scale testing not only verified the efficacy of reduced dissolved oxygen set-point in reducing N2O emission by 60%, but also confirmed the proposed concepts of control over the aerobic and anoxic phases collectively.

Anaerobic Methanotrophic Archaea of the ANME-2d Cluster Are Active in a Low-sulfate, Iron-rich Freshwater Sediment.

ANaerobic MEthanotrophic (ANME) archaea remove the greenhouse gas methane from anoxic environments and diminish its flux to the atmosphere. High methane removal efficiencies are well documented in marine environments, whereas anaerobic oxidation of methane (AOM) was only recently indicated as an important methane sink in freshwater systems. Freshwater AOM-mediating microorganisms lack taxonomic identification and only little is known about metabolic adaptions to prevailing biogeochemical conditions. One of the first study sites providing information about AOM activity in freshwater sediment is Lake Ørn, a low-sulfate, iron-rich Danish lake. With the aim to identify freshwater AOM-mediating archaea, we incubated AOM-active anoxic, nitrate-free freshwater sediment from Lake Ørn with 13C-labeled methane (13CCH4) and 13C-labeled bicarbonate (13CDIC) and followed the assimilation of 13C into RNA by stable isotope probing. While AOM was active, 13CCH4 and probably also 13CDIC were incorporated into uncultured archaea of the Methanosarcinales-related cluster ANME-2d, whereas other known ANME lineages were not detected. This finding strongly suggests that ANME-2d archaea perform AOM coupled to sulfate and/or iron reduction and may have the capability of mixed assimilation of CH4 and DIC. ANME-2d archaea may thus play an important role in controlling methane emissions from nitrate-depleted and low-sulfate freshwater systems.

Quantitative proteomics of Chlorobaculum tepidum: insights into the sulfur metabolism of a phototrophic green sulfur bacterium.

Chlorobaculum (Cba.) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. To gain insight into the sulfur metabolism, the proteome of Cba. tepidum cells sampled under different growth conditions has been quantified using a rapid gel-free, filter-aided sample preparation (FASP) protocol with an in-solution isotopic labeling strategy. Among the 2245 proteins predicted from the Cba. tepidum genome, approximately 970 proteins were detected in unlabeled samples, whereas approximately 630-640 proteins were detected in labeled samples comparing two different growth conditions. Wild-type cells growing on thiosulfate had an increased abundance of periplasmic cytochrome c-555 and proteins of the periplasmic thiosulfate-oxidizing SOX enzyme system when compared with cells growing on sulfide. A dsrM mutant of Cba. tepidum, which lacks the dissimilatory sulfite reductase DsrM protein and therefore is unable to oxidize sulfur globules to sulfite, was also investigated. When compared with wild type, the dsrM cells exhibited an increased abundance of DSR enzymes involved in the initial steps of sulfur globule oxidation (DsrABCL) and a decreased abundance of enzymes putatively involved in sulfite oxidation (Sat-AprAB-QmoABC). The results show that Cba. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism and other electron-transferring processes in response to the availability of reduced sulfur compounds.

Comparative proteomics and activity of a green sulfur bacterium through the water column of Lake Cadagno, Switzerland.

Primary production in the meromictic Lake Cadagno, Switzerland, is dominated by anoxygenic photosynthesis. The green sulfur bacterium Chlorobium clathratiforme is the dominant phototrophic organism in the lake, comprising more than half of the bacterial population, and its biomass increases 3.8-fold over the summer. Cells from four positions in the water column were used for comparative analysis of the Chl. clathratiforme proteome in order to investigate changes in protein composition in response to the chemical and physical gradient in their environment, with special focus on how the bacteria survive in the dark. Although metagenomic data are not available for Lake Cadagno, proteome analysis was possible based on the completely sequenced genome of an isolated strain of Chl. clathratiforme. Using LC-MS/MS we identified 1321 Chl. clathratiforme proteins in Lake Cadagno and quantitatively compared 621 of these in the four samples. Our results showed that compared with cells obtained from the photic zone, cells collected from the dark part of the water column had the same expression level of key enzymes involved in carbon metabolism and photosynthetic light harvesting. However, most proteins participating in nitrogen and sulfur metabolism were twofold less abundant in the dark. From the proteome analysis we were able to show that Chl. clathratiforme in the photic zone contains enzymes for fixation of N(2) and the complete oxidation of sulfide to sulfate while these processes are probably not active in the dark. Instead we propose that Chl. clathratiforme cells in the dark part of the water column obtain energy for maintenance from the fermentation of polyglucose. Based on the observed protein compositions we have constructed possible pathways for C, N and S metabolism in Chl. clathratiforme.

Dominance of a clonal green sulfur bacterial population in a stratified lake.

For many years, the chemocline of the meromictic Lake Cadagno, Switzerland, was dominated by purple sulfur bacteria. However, following a major community shift in recent years, green sulfur bacteria (GSB) have come to dominate. We investigated this community by performing microbial diversity surveys using FISH cell counting and population multilocus sequence typing [clone library sequence analysis of the small subunit (SSU) rRNA locus and two loci involved in photosynthesis in GSB: fmoA and csmCA]. All bacterial populations clearly stratified according to water column chemistry. The GSB population peaked in the chemocline (c. 8 x 10(6) GSB cells mL(-1)) and constituted about 50% of all cells in the anoxic zones of the water column. At least 99.5% of these GSB cells had SSU rRNA, fmoA, and csmCA sequences essentially identical to that of the previously isolated and genome-sequenced GSB Chlorobium clathratiforme strain BU-1 (DSM 5477). This ribotype was not detected in Lake Cadagno before the bloom of GSB. These observations suggest that the C. clathratiforme population that has stabilized in Lake Cadagno is clonal. We speculate that such a clonal bloom could be caused by environmental disturbance, mutational adaptation, or invasion.

Temperature effect on the sulfur isotope fractionation during sulfate reduction by two strains of the hyperthermophilic Archaeoglobus fulgidus.

Sulfur isotope fractionation during dissimilatory sulfate reduction by two strains of the thermophilic archaeon Archaeoglobus fulgidus (strains VC-16 and Z) was explored over the entire temperature range of growth. The optimal cell-specific sulfate reduction rate (14 fmol cell(-1) h(-1)) was found at 82-84 degrees C but growth was measured as low as 54 degrees C. The fractionation ranged between 0.52 per thousand and 27 per thousand, with largest fractionations were found at intermediate temperatures and the smallest fractionations at the lowest and highest temperatures. There was an inverse relationship between the cell-specific sulfate reduction rate and fractionation, and the cell-specific rate was a good indicator of the expected fractionations regardless of whether temperature or substrate concentrations controlled the rate. Comparison of the fractionation trend found in this study with similar measurements for seven other sulfate-reducers showed that sulfate-reducing organisms respond to temperature in three different ways and this correlated with their maximum fractionation value, but not with the cell-specific sulfate reduction rate. A sulfur isotope model was used to reproduce the observed variation of fractionation with temperature. This approach predicted the rate of internal sulfur transformations as having the major influence on the observed fractionations in the intermediate temperature range, whereas the exchange of sulfate across the cell membrane controls fractionation at low and high temperatures.

High rates of sulfate reduction in a low-sulfate hot spring microbial mat are driven by a low level of diversity of sulfate-respiring microorganisms.

The importance of sulfate respiration in the microbial mat found in the low-sulfate thermal outflow of Mushroom Spring in Yellowstone National Park was evaluated using a combination of molecular, microelectrode, and radiotracer studies. Despite very low sulfate concentrations, this mat community was shown to sustain a highly active sulfur cycle. The highest rates of sulfate respiration were measured close to the surface of the mat late in the day when photosynthetic oxygen production ceased and were associated with a Thermodesulfovibrio-like population. Reduced activity at greater depths was correlated with novel populations of sulfate-reducing microorganisms, unrelated to characterized species, and most likely due to both sulfate and carbon limitation.

Effect of low sulfate concentrations on lactate oxidation and isotope fractionation during sulfate reduction by Archaeoglobus fulgidus strain Z.

The effect of low substrate concentrations on the metabolic pathway and sulfur isotope fractionation during sulfate reduction was investigated for Archaeoglobus fulgidus strain Z. This archaeon was grown in a chemostat with sulfate concentrations between 0.3 mM and 14 mM at 80 degrees C and with lactate as the limiting substrate. During sulfate reduction, lactate was oxidized to acetate, formate, and CO2. This is the first time that the production of formate has been reported for A. fulgidus. The stoichiometry of the catabolic reaction was strongly dependent on the sulfate concentration. At concentrations of more than 300 microM, 1 mol of sulfate was reduced during the consumption of 1 mol of lactate, whereas only 0.6 mol of sulfate was consumed per mol of lactate oxidized at a sulfate concentration of 300 microM. Furthermore, at low sulfate concentrations acetate was the main carbon product, in contrast to the CO2 produced at high concentrations. We suggest different pathways for lactate oxidation by A. fulgidus at high and low sulfate concentrations. At about 300 microM sulfate both the growth yield and the isotope fractionation were limited by sulfate, whereas the sulfate reduction rate was not limited by sulfate. We suggest that the cell channels more energy for sulfate uptake at sulfate concentrations below 300 to 400 microM than it does at higher concentrations. This could explain the shift in the metabolic pathway and the reduced growth yield and isotope fractionation at low sulfate levels.

Calibration of sulfate levels in the archean ocean.

The size of the marine sulfate reservoir has grown through Earth's history, reflecting the accumulation of oxygen into the atmosphere. Sulfur isotope fractionation experiments on marine and freshwater sulfate reducers, together with the isotope record, imply that oceanic Archean sulfate concentrations were <200 microM, which is less than one-hundredth of present marine sulfate levels and one-fifth of what was previously thought. Such low sulfate concentrations were maintained by volcanic outgassing of SO2 gas, and severely suppressed sulfate reduction rates allowed for a carbon cycle dominated by methanogenesis.