Microbial Community Dynamics and Stability during an Ammonia-Induced Shift to Syntrophic Acetate Oxidation

Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, we experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidation. In comparison with control digesters, we observed shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the course of the digesters'' 3-year operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When we perturbed the digesters by increasing the ammonia concentration or temperature, the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidation had allowed the experimental bioreactors to regain stable operation. The emergence of syntrophic acetate oxidation coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. Our 16S rRNA gene analysis also revealed that acetate-fed enrichment experiments resulted in communities that did not represent the bioreactor community. Analysis of shotgun sequencing of community DNA suggests that syntrophic acetate oxidation was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.     

Microbial Community Responses to Atmospheric Carbon Dioxide Enrichment in a Warm-Temperate Forest

Forest productivity depends on nutrient supply, and sustained increases in forest productivity under elevated carbon dioxide (CO₂) may ultimately depend on the response of microbial communities to changes in the quantity and chemistry of plant-derived substrates, We investigated microbial responses to elevated CO₂ in a warm-temperate forest under free-air CO₂ enrichment for 5 years (1997–2001). The experiment was conducted on three 30 m diameter plots under ambient CO₂ and three plots under elevated CO₂ (200 ppm above ambient). To understand how microbial processes changed under elevated CO₂, we assayed the activity of nine extracellular enzymes responsible for the decomposition of labile and recalcitrant carbon (C) substrates and the release of nitrogen (N) and phosphorus (P) from soil organic matter. Enzyme activities were measured three times per year in a surface organic horizon and in the top 15 cm of mineral soil. Initially, we found significant increases in the decomposition of labile C substrates in the mineral soil horizon under elevated CO₂; this overall pattern was present but much weaker in the O horizon. Beginning in the 4th year of this study, enzyme activities in the O horizon declined under elevated CO₂, whereas they continued to be stimulated in the mineral soil horizon. By year 5, the degradation of recalcitrant C substrates in mineral soils was significantly higher under elevated CO₂. Although there was little direct effect of elevated CO₂ on the activity of N- and P-releasing enzymes, the activity of nutrient-releasing enzymes relative to those responsible for C metabolism suggest that nutrient limitation is increasingly regulating microbial activity in the O horizon. Our results show that the metabolism of microbial communities is significantly altered by the response of primary producers to elevated CO₂. We hypothesize that ecosystem responses to elevated CO₂ are shifting from primary production to decomposition as a result of increasing nutrient limitation.     

Soil Microbial Community Response to Land Use Change in an Agricultural Landscape of Western Kenya

Tropical agroecosystems are subject to degradation processes such as losses in soil carbon, nutrient depletion, and reduced water holding capacity that occur rapidly resulting in a reduction in soil fertility that can be difficult to reverse. In this research, a polyphasic methodology has been used to investigate changes in microbial community structure and function in a series of tropical soils in western Kenya. These soils have different land usage with both wooded and agricultural soils at Kakamega and Ochinga, whereas at Ochinga, Leuro, Teso, and Ugunja a replicated field experiment compared traditional continuous maize cropping against an improved N-fixing fallow system. For all sites, principal component analysis of 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) profiles revealed that soil type was the key determinant of total bacterial community structure, with secondary variation found between wooded and agricultural soils. Similarly, phospholipid fatty acid (PLFA) analysis also separated wooded from agricultural soils, primarily on the basis of higher abundance of monounsaturated fatty acids, anteiso- and iso-branched fatty acids, and methyl-branched fatty acids in the wooded soils. At Kakamega and Ochinga wooded soils had between five 5 and 10-fold higher levels of soil carbon and microbial biomass carbon than agricultural soils from the same location, whereas total enzyme activities were also lower in the agricultural sites. Soils with woody vegetation had a lower percentage of phosphatase activity and higher cellulase and chitinase activities than the agricultural soils. BIOLOG analysis showed woodland soils to have the greatest substrate diversity. Throughout the study the two functional indicators (enzyme activity and BIOLOG), however, showed lower specificity with respect to soil type and land usage than did the compositional indicators (DGGE and PLFA). In the field experiment comparing two types of maize cropping, both the maize yields and total microbial biomass were found to increase with the fallow system. Moreover, 16S rRNA gene and PLFA analyses revealed shifts in the total microbial community in response to the different management regimes, indicating that deliberate management of soils can have considerable impact on microbial community structure and function in tropical soils.     

Anaerobic Oxidation of n-Dodecane by an Addition Reaction in a Sulfate-Reducing Bacterial Enrichment Culture

We identified trace metabolites produced during the anaerobic biodegradation of H₂₆- and D₂₆-n-dodecane by an enrichment culture that mineralizes these compounds in a sulfate-dependent fashion. The metabolites are dodecylsuccinic acids that, in the case of the perdeuterated substrate, retain all of the deuterium atoms. The deuterium retention and the gas chromatography-mass spectrometry fragmentation patterns of the derivatized metabolites suggest that they are formed by C—H or C—D addition across the double bond of fumarate. As trimethylsilyl esters, two nearly coeluting metabolites of equal abundance with nearly identical mass spectra were detected from each of H₂₆- and D₂₆-dodecane, but as methyl esters, only a single metabolite peak was detected for each parent substrate. An authentic standard of protonated n-dodecylsuccinic acid that was synthesized and derivatized by the two methods had the same fragmentation patterns as the metabolites of H₂₆-dodecane. However, the standard gave only a single peak for each ester type and gas chromatographic retention times different from those of the derivatized metabolites. This suggests that the succinyl moiety in the dodecylsuccinic acid metabolites is attached not at the terminal methyl group of the alkane but at a subterminal position. The detection of two equally abundant trimethylsilyl-esterified metabolites in culture extracts suggests that the analysis is resolving diastereomers which have the succinyl moiety located at the same subterminal carbon in two different absolute configurations. Alternatively, there may be more than one methylene group in the alkane that undergoes the proposed fumarate addition reaction, giving at least two structural isomers in equal amounts.     

Microbial Community Response to Seawater Amendment in Low-Salinity Tidal Sediments

Rising sea levels and excessive water withdrawals upstream are making previously freshwater coastal ecosystems saline. Plant and animal responses to variation in the freshwater–saline interface have been well studied in the coastal zone; however, microbial community structure and functional response to seawater intrusion remains relatively unexplored. Here, we used molecular approaches to evaluate the response of the prokaryotic community to controlled changes in porewater salinity levels in freshwater sediments from the Altamaha River, Georgia, USA. This work is a companion to a previously published study describing results from an experiment using laboratory flow-through sediment core bioreactors to document biogeochemical changes as porewater salinity was increased from 0 to 10 over 35 days. As reported in Weston et al. (Biogeochemistry, 77:375–408, 62), porewater chemistry was monitored, and cores were sacrificed at 0, 9, 15, and 35 days, at which time we completed terminal restriction fragment length polymorphism and 16S rRNA clone library analyses of sediment microbial communities. The biogeochemical study documented changes in mineralization pathways in response to artificial seawater additions, with a decline in methanogenesis, a transient increase in iron reduction, and finally a dominance of sulfate reduction. Here, we report that, despite these dramatic and significant changes in microbial activity at the biogeochemical level, no significant differences were found between microbial community composition of control vs. seawater-amended treatments for either Bacterial or Archaeal members. Further, taxa in the seawater-amended treatment community did not become more “marine-like” through time. Our experiment suggests that, as seawater intrudes into freshwater sediments, observed changes in metabolic activity and carbon mineralization on the time scale of weeks are driven more by shifts in gene expression and regulation than by changes in the composition of the microbial community.     

Response of Microbial Community Composition and Function to Soil Climate Change

Soil microbial communities mediate critical ecosystem carbon and nutrient cycles. How microbial communities will respond to changes in vegetation and climate, however, are not well understood. We reciprocally transplanted soil cores from under oak canopies and adjacent open grasslands in a California oak–grassland ecosystem to determine how microbial communities respond to changes in the soil environment and the potential consequences for the cycling of carbon. Every 3 months for up to 2 years, we monitored microbial community composition using phospholipid fatty acid analysis (PLFA), microbial biomass, respiration rates, microbial enzyme activities, and the activity of microbial groups by quantifying ¹³C uptake from a universal substrate (pyruvate) into PLFA biomarkers. Soil in the open grassland experienced higher maximum temperatures and lower soil water content than soil under the oak canopies. Soil microbial communities in soil under oak canopies were more sensitive to environmental change than those in adjacent soil from the open grassland. Oak canopy soil communities changed rapidly when cores were transplanted into the open grassland soil environment, but grassland soil communities did not change when transplanted into the oak canopy environment. Similarly, microbial biomass, enzyme activities, and microbial respiration decreased when microbial communities were transplanted from the oak canopy soils to the grassland environment, but not when the grassland communities were transplanted to the oak canopy environment. These data support the hypothesis that microbial community composition and function is altered when microbes are exposed to new extremes in environmental conditions; that is, environmental conditions outside of their “life history” envelopes.     

Soil Microbial Community Response to Nitrobenzene Exposure for a Spartina Wetland

To explore the response of the soil microbial community to nitrobenzene (NB) exposure in a Spartina marsh, a short-term (45 d) mesocosm study was conducted at three NB concentrations of (10, 50, and 100) mg kg^?1. Dry soil, sterile and unsterile controls were also compared. The ability of the microbes to biodegrade NB was studied in an effort to predict the outcome of NB in the mesocosm. The results indicated that a microbial community is capable of doing so. Microbial enumeration and enzyme assays showed that the fluctuations in microbial communities and polyphenol oxidase activities are related to the initial NB concentration. Moreover, cluster analyses through denaturing gradient gel electrophoresis (DGGE) revealed very similar patterns (95.5%) throughout the 45 d term, indicating that the microbial community regenerates when NB is exhausted. Although volatilization and photolysis were the major processes responsible for the reduction in NB in contaminated mesocosms and the microbial community regenerated at the end of incubation, the data indicate potential ecological risks in outfall areas even if the discharged wastewater complies with the national wastewater discharge standards.     

Enrichment of an Endosulfan-Degrading Mixed Bacterial Culture

An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored the alpha isomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.     

Microbial Response to Organic Matter Enrichment in the Oligotrophic Levantine Basin (Eastern Mediterranean)

The response of a deep-water benthic microbial community to organic matter (OM) enrichment was studied in the unexplored region of the SW Cretan margin (E. Mediterranean). A food pulse of 0.5 g C m^?2 was simulated by adding ¹³C-labelled diatoms to sediment cores retrieved from 1079 m depth. The diatom addition resulted in a significant increase in the sediment community oxygen consumption (SCOC). After 6 days, ～50 mg C m^?2 of the added material was processed by the microbial community. The major carbon sink was respiration, which accounted for ～96% of the total processed material. The carbon uptake rate (12 mg C m^?2 d^?1) was considerably lower than previously published values in the E. Mediterranean at similar depths. The microbial community in our study site is distinct, as revealed by the unusually high presence of branched phospholipid fatty acids (PLFA). Previous studies have revealed that the slope under investigation may act as a conduit of OM from the shallow shelf to the deep basins, resulting in the prevalence of relatively refractory OM at mid-slope depths. We postulate that sedimentary processes affect the amount of bioavailable sedimentary OM and consequently the structure and physiological state of bacterial community in our study site. The distinct microbial community composition at our station compared to more stable adjacent slopes could explain the limited response of the microbial community to the addition of labile OM. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental file.     

Resource Availability and Spatial Heterogeneity Control Bacterial Community Response to Nutrient Enrichment in Lakes

The diversity and composition of ecological communities often co-vary with ecosystem productivity. However, the relative importance of productivity, or resource abundance, versus the spatial distribution of resources in shaping those ecological patterns is not well understood, particularly for the bacterial communities that underlie most important ecosystem functions. Increasing ecosystem productivity in lakes has been shown to influence the composition and ecology of bacterial communities, but existing work has only evaluated the effect of increasing resource supply and not heterogeneity in how those resources are distributed. We quantified how bacterial communities varied with the trophic status of lakes and whether community responses differed in surface and deep habitats in response to heterogeneity in nutrient resources. Using ARISA fingerprinting, we found that bacterial communities were more abundant, richer, and more distinct among habitats as lake trophic state and vertical heterogeneity in nutrients increased, and that spatial resource variation produced habitat specific responses of bacteria in response to increased productivity. Furthermore, changes in communities in high nutrient lakes were not produced by turnover in community composition but from additional taxa augmenting core bacterial communities found in lower productivity lakes. These data suggests that bacterial community responses to nutrient enrichment in lakes vary spatially and are likely influenced disproportionately by rare taxa.     

Anaerobic Sulfide Oxidation with Nitrate by a Freshwater Beggiatoa Enrichment Culture

A lithotrophic freshwater Beggiatoa strain was enriched in O₂-H₂S gradient tubes to investigate its ability to oxidize sulfide with NO₃⁻ as an alternative electron acceptor. The gradient tubes contained different NO₃⁻ concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O₂, H₂S, pH, and NO₃⁻ were determined with microsensors. The more NO₃⁻ that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO₃⁻ to oxidize sulfide at depths below the depth that O₂ penetrated. In the presence of NO₃⁻ Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO₃⁻ was added. These thick mats spatially separated O₂ and sulfide but not NO₃⁻ and sulfide, and therefore NO₃⁻ must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O₂ flux and a twofold-higher sulfide flux into the NO₃⁻-exposed mats compared to the fluxes for controls without NO₃⁻. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S⁰ with NO₃⁻ as the electron acceptor.     

Microbial Community Response during the Iron Fertilization Experiment LOHAFEX

Iron fertilization experiments in high-nutrient, low-chlorophyll areas are known to induce phytoplankton blooms. However, little is known about the response of the microbial community upon iron fertilization. As part of the LOHAFEX experiment in the southern Atlantic Ocean, Bacteria and Archaea were monitored within and outside an induced bloom, dominated by Phaeocystis-like nanoplankton, during the 38 days of the experiment. The microbial production increased 1.6-fold (thymidine uptake) and 2.1-fold (leucine uptake), while total cell numbers increased only slightly over the course of the experiment. 454 tag pyrosequencing of partial 16S rRNA genes and catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) showed that the composition and abundance of the bacterial and archaeal community in the iron-fertilized water body were remarkably constant without development of typical bloom-related succession patterns. Members of groups usually found in phytoplankton blooms, such as Roseobacter and Gammaproteobacteria, showed no response or only a minor response to the bloom. However, sequence numbers and total cell numbers of the SAR11 and SAR86 clades increased slightly but significantly toward the end of the experiment. It seems that although microbial productivity was enhanced within the fertilized area, a succession-like response of the microbial community upon the algal bloom was averted by highly effective grazing. Only small-celled members like the SAR11 and SAR86 clades could possibly escape the grazing pressure, explaining a net increase of those clades in numbers.     

Microbial Oxidation of sec-Alcohols to Ketones

The major anthocyanin compound in buckwheat sprouts was determined to be cyanidin 3-O-rutinoside (C3R), based on HPLC data and MS/MS spectra. Investigation of the content of phenolic compounds in commercial buckwheat sprouts indicated that hypocotyls are abundant in C3R and rutin, whereas all of the detected flavonoids are abundant in cotyledons. The superoxide anion radical-scavenging activities (SOD-like activities) of phenolic compounds in buckwheat sprouts and their contents indicated that rutin, isoorientin, and orientin contributed mainly to the SOD-like activity of the extract from buckwheat sprouts. In contrast, the contribution of C3R was substantially lower than that of flavonoids.     

Soil Microbial Community Response to Drought and Precipitation Variability in the Chihuahuan Desert

Increases in the magnitude and variability of precipitation events have been predicted for the Chihuahuan Desert region of West Texas. As patterns of moisture inputs and amounts change, soil microbial communities will respond to these alterations in soil moisture windows. In this study, we examined the soil microbial community structure within three vegetation zones along the Pine Canyon Watershed, an elevation and vegetation gradient in Big Bend National Park, Chihuahuan Desert. Soil samples at each site were obtained in mid-winter (January) and in mid-summer (August) for 2 years to capture a component of the variability in soil temperature and moisture that can occur seasonally and between years along this watershed. Precipitation patterns and amounts differed substantially between years with a drought characterizing most of the second year. Soils were collected during the drought period and following a large rainfall event and compared to soil samples collected during a relatively average season. Structural changes within microbial community in response to site, season, and precipitation patterns were evaluated using fatty acid methyl ester (FAME) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses. Fungal FAME amounts differed significantly across seasons and sites and greatly outweighed the quantity of bacterial and actinomycete FAME levels for all sites and seasons. The highest fungal FAME levels were obtained in the low desert scrub site and not from the high elevation oak–pine forests. Total bacterial and actinomycete FAME levels did not differ significantly across season and year within any of the three locations along the watershed. Total bacterial and actinomycete FAME levels in the low elevation desert-shrub and grassland sites were slightly higher in the winter than in the summer. Microbial community structure at the high elevation oak–pine forest site was strongly correlated with levels of NH₄ ⁺–N, % soil moisture, and amounts of soil organic matter irrespective of season. Microbial community structure at the low elevation desert scrub and sotol grasslands sites was most strongly related to soil pH with bacterial and actinobacterial FAME levels accounting for site differences along the gradient. DGGE band counts of amplified soil bacterial DNA were found to differ significantly across sites and season with the highest band counts found in the mid-elevation grassland site. The least number of bands was observed in the high elevation oak–pine forest following the large summer-rain event that occurred after a prolonged drought. Microbial responses to changes in precipitation frequency and amount due to climate change will differ among vegetation zones along this Chihuahuan Desert watershed gradient. Soil bacterial communities at the mid-elevation grasslands site are the most vulnerable to changes in precipitation frequency and timing, while fungal community structure is most vulnerable in the low desert scrub site. The differential susceptibility of the microbial communities to changes in precipitation amounts along the elevation gradient reflects the interactive effects of the soil moisture window duration following a precipitation event and differences in soil heat loads. Amounts and types of carbon inputs may not be as important in regulating microbial structure among vegetation zones within in an arid environment as is the seasonal pattern of soil moisture and the soil heat load profile that characterizes the location.     

响应面法优化Fe~(2+)的微生物氧化条件

氧化亚铁钩端螺旋菌(Leptospirillum ferrooxidans,L.f)是一种极端嗜酸,专性自养氧化铁的细菌,能够耐受较低pH和较高的温度,被广泛应用于生物浸矿和环境治理。氧化亚铁钩端螺旋体菌的生物浸矿效率与其对Fe~(2+)氧化速率相关,因此,本文采用响应面法,通过建立二次多项式回归方程考察pH、温度、Fe~(2+)浓度及转速四个培养因素对Fe~(2+)氧化速率的影响。结果显示在pH为2.25、温度为32℃、初始Fe~(2+)浓度为175.36 mmol/L、转数为165 r/min时,Fe~(2+)最高氧化速率为0.2911 g/Lh。     

Response of the Soil Microbial Community to Changes in Precipitation in a Semiarid Ecosystem

Microbial communities regulate many belowground carbon cycling processes; thus, the impact of climate change on the structure and function of soil microbial communities could, in turn, impact the release or storage of carbon in soils. Here we used a large-scale precipitation manipulation (+18%, −50%, or ambient) in a piñon-juniper woodland (Pinus edulis-Juniperus monosperma) to investigate how changes in precipitation amounts altered soil microbial communities as well as what role seasonal variation in rainfall and plant composition played in the microbial community response. Seasonal variability in precipitation had a larger role in determining the composition of soil microbial communities in 2008 than the direct effect of the experimental precipitation treatments. Bacterial and fungal communities in the dry, relatively moisture-limited premonsoon season were compositionally distinct from communities in the monsoon season, when soil moisture levels and periodicity varied more widely across treatments. Fungal abundance in the drought plots during the dry premonsoon season was particularly low and was 4.7 times greater upon soil wet-up in the monsoon season, suggesting that soil fungi were water limited in the driest plots, which may result in a decrease in fungal degradation of carbon substrates. Additionally, we found that both bacterial and fungal communities beneath piñon pine and juniper were distinct, suggesting that microbial functions beneath these trees are different. We conclude that predicting the response of microbial communities to climate change is highly dependent on seasonal dynamics, background climatic variability, and the composition of the associated aboveground community.     

Continuous Enrichment Culture and Molecular Monitoring to Investigate the Microbial Diversity of Thermophiles Inhabiting Deep-Sea Hydrothermal Ecosystems

The microflora developing during a continuous enrichment culture from a hydrothermal chimney sample was investigated by molecular methods. The culture was performed in a gas-lift bioreactor under anaerobic conditions, at 90°C and pH 6.5, on a complex medium containing sulfur as the terminal electron acceptor. Archaeal and bacterial diversity was studied. Microorganisms affiliated with the genera Pyrococcus, Marinitoga, and Bacillus were detected through DGGE analysis of 16S rDNA. Additional sequences phylogenetically related to Thermococcus and -Proteobacteria were detected by cloning and sequencing of 16S rDNA from two samples of the enrichment culture. In comparison, the sequences retrieved from cloning analysis from an enrichment culture performed in a flask (batch condition) using the same culture medium showed that only members of the genus Thermococcus were cultivated. Therefore, continuous enrichment culture using the gas-lift bioreactor can be considered as an efficient and improved method for investigating microbial communities originating from deep-sea hydrothermal vents.     

Sulfide Oxidation Coupled to Arsenate Reduction by a Diverse Microbial Community in a Soda Lake

We characterized the arsenate-reducing, sulfide-oxidizing population of Mono Lake, California, by analyzing the distribution and diversity of rrnA, cbbL, and dissimilatory arsenate reductase (arrA) genes in environmental DNA, arsenate-plus sulfide-amended lake water, mixed cultures, and isolates. The arsenate-reducing community was diverse. An organism represented by an rrnA sequence previously retrieved from Mono Lake and affiliated with the Desulfobulbaceae (Deltaproteobacteria) appears to be an important member of the arsenate-reducing, sulfide-oxidizing community. Sulfide oxidation coupled with arsenate reduction appears to proceed via a two-electron transfer, resulting in the production of arsenite and an intermediate S compound that is subsequently disproportionated. A realgar-like As/S mineral was formed in some experiments.