Carbohydrate-dependent sulfur respiration in halo(alkali)philic archaea

Archaea are environmentally ubiquitous on Earth, and their extremophilic and metabolically versatile phenotypes make them useful as model systems for astrobiology. Here, we reveal a new functional group of halo(natrono)archaea able to utilize alpha-d -glucans (amylopectin, amylose and glycogen), sugars, and glycerol as electron donors and carbon sources for sulfur respiration. They are facultative anaerobes enriched from hypersaline sediments with either amylopectin, glucose or glycerol as electron/carbon sources and elemental sulfur as the terminal electron acceptor. They include 10 strains of neutrophilic haloarchaea from circum pH-neutral lakes and one natronoarchaeon from soda-lake sediments. The neutrophilic isolates can grow by fermentation, although addition of S⁰ or dimethyl sulfoxide increased growth rate and biomass yield (with a concomitant decrease in H₂). Natronoarchaeal isolate AArc-S grew only by respiration, either anaerobically with S⁰ or thiosulfate as the terminal electron acceptor, or aerobically. Through genome analysis of five representative strains, we detected the full set of enzymes required for the observed catabolic and respiratory phenotypes. These findings provide evidence that sulfur-respiring haloarchaea partake in biogeochemical sulfur cycling, linked to terminal anaerobic carbon mineralization in hypersaline anoxic habitats. We discuss the implications for life detection in analogue environments such as the polar subglacial brine-lakes of Mars.     

Metabolism of hyperthermophiles

Hyperthermophiles are characterized by a temperature optimum for growth between 80 and 110°C. They are considered to represent the most ancient phenotype of living organisms and thus their metabolic design might reflect the situation at an early stage of evolution. Their modes of metabolism are diverse and include chemolithoautotrophic and chemoorganoheterotrophic. No extant phototrophic hyperthermophiles are known. Lithotrophic energy metabolism is mostly anaerobic or microaerophilic and based on the oxidation of H₂ or S coupled to the reduction of S, SO _inf4 ^sup2- , CO₂ and NO _inf3 ^sup- but rarely to O₂. the substrates are derived from volcanic activities in hyperthermophilic habitats. The lithotrophic energy metabolism of hyperthermophiles appears to be similar to that of mesophiles. Autotrophic CO₂ fixation proceeds via the reductive citric acid cycle, considered to be one of the first metabolic cycles, and via the reductive acetyl-CoA/carbon monoxide dehydrogenase pathway. The Calvin cycle has not been found in hyperthermophiles (or any Archaea). Organotrophic metabolism mainly involves peptides and sugars as substrates, which are either oxidized to CO₂ by external electron acceptors or fermented to acetate and other products. Sugar catabolism in hyperthermophiles involves non-phosphorylated versions of the Entner-Doudoroff pathway and modified versions of the Embden-Meyerhof pathway. The classical Embden-Meyerhof pathway is present in hyperthermophilic Bacteria (Thermotoga) but not in Archaea. All hyperthermophiles (and Archaea) tested so far utilize pyruvate:ferredoxin oxidoreductase for acetyl-CoA formation from pyruvate. Acetyl-CoA oxidation in anaerobic sulphur-reducing and aerobic hyperthermophiles proceeds via the citric acid cycle; in the hyperthermophilic sulphate-reducer Archaeoglobus an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway is operative. Acetate formation from acetyl-CoA in Archaea, including hyperthermophiles, is catalysed by acetyl-CoA synthetase (ADP-forming), a novel prokarvotic enzyme involved in energy conservation. In Bacteria, including the hyperthermophile Thermotoga, acetyl-CoA conversion to acetate involves two enzymes, phosphate acetyltransferase and acetate kinase.The authors are with the Institut für Pflanzenphysiologie und Mikrobiologie. Fachbereich Biologie, Freie Universität Berlin, Königin-Luise-Strasse 12–16 a, D-14195 Berlin, Germany     

The bioenergetic basis for the decrease in metabolic diversity at increasing salt concentrations: implications for the functioning of salt lake ecosystems

Examination of the microbial diversity in hypersaline lakes of increasing salt concentrations shows that certain types of dissimilatory metabolism do not occur at the highest salinities. Examples are methanogenesis from hydrogen and carbon dioxide or from acetate, dissimilatory sulfate reduction with oxidation of acetate, and autotrophic nitrification. The observations can be explained on the basis of the energetic cost of haloadaptation used by the different metabolic groups and the free-energy change associated with the dissimilatory reactions. All halophilic microorganisms spend large amounts of energy to maintain steep gradients of Na⁺ and K⁺concentrations across their cytoplasmic membrane. Most Bacteria and also the methanogenic Archaea produce high intracellular concentrations of organic osmotic solutes at a high energetic cost. The halophilic aerobic Archaea (order Halobacteriales) and the halophilic fermentative Bacteria (order Halanaerobiales) use KCl as the main intracellular solute. This strategy, while requiring far-reaching adaptations of the intracellular machinery, is energetically more favorable than production of organic compatible solutes. By combining information on the amount of energy available to each physiological group and the strategy used to cope with salt stress, a coherent model emerges that provides explanations for the upper salinity limit at which the different microbial conversions occur in hypersaline lakes.     

Anammox bacterial populations in deep marine hypersaline gradient systems

To extend the knowledge of anaerobic ammonium oxidation (anammox) habitats, bacterial communities were examined in two hypersaline sulphidic basins in Eastern Mediterranean Sea. The 2 m thick seawater–brine haloclines of the deep anoxic hypersaline basins Bannock and L’Atalante were sampled in intervals of 10 cm with increasing salinity. ¹⁵N isotope pairing incubation experiments showed the production of ²⁹N₂ and ³⁰N₂ gases in the chemoclines, ranging from 6.0 to 9.2 % salinity of the L’Atalante basin. Potential anammox rates ranged from 2.52 to 49.65 nmol N₂ L^?1 day^?1 while denitrification was a major N₂ production pathway, accounting for more than 85.5 % of total N₂ production. Anammox-related 16S rRNA genes were detected along the L’Atalante and Bannock haloclines up to 24 % salinity, and the amplification of the hydrazine synthase genes (hzsA) further confirmed the presence of anammox bacteria in Bannock. Fluorescence in situ hybridisation and sequence analysis of 16S rRNA genes identified representatives of the marine anammox genus ‘Candidatus Scalindua’ and putatively new operational taxonomic units closely affiliated to sequences retrieved in marine environments that have documented anammox activity. ‘Scalindua brodae’ like sequences constituted up to 84.4 % of the sequences retrieved from Bannock. The anammox community in L’Atalante was different than in Bannock and was stratified according to salinity increase. This study putatively extends anammox bacterial habitats to extremely saline sulphidic ecosystems.     

Labelling halophilic Archaea using 13C and 15N stable isotopes: a potential tool to investigate haloarchaea consumption by metazoans

The use of stable isotope (SI) labelling and tracing of live diets is currently considered one of the most comprehensive tools to detect their uptake and assimilation by aquatic organisms. These techniques are indeed widely used in nutritional studies to follow the fate of specific microbial dietary components, unraveling trophic interactions. Nevertheless, to the current date our understanding of aquatic trophic relationships has yet to include a whole domain of life, the Archaea. The aim of the present research was, therefore, to describe a halophilic Archaea (haloarchaea) labelling procedure, using the SI ¹³C and ¹⁵N, to enable the application of SI tracing in future studies of haloarchaea consumption by aquatic metazoans. To this end, three ¹³C enriched carbon sources and two ¹⁵N enriched nitrogen sources were tested as potential labels to enrich cells of three haloarchaea strains when supplemented to the culture medium. Our overall results indicate ¹³C-glycerol as the most effective carbon source to achieve an efficient ¹³C enrichment in haloarchaea cells, with Δδ¹³C values above 5000‰ in all tested haloarchaea strains. As for ¹⁵N enriched nitrogen sources, both (¹⁵NH₄)₂SO₄ and ¹⁵NH₄Cl seem to be readily assimilated, also resulting in efficient ¹⁵N enrichment in haloarchaea cells, with Δδ¹⁵N values higher than 20,000‰. We believe that the proposed methodology will allow for the use of SI labelled haloarchaea biomass in feeding tests, potentially providing unambiguous confirmation of the assimilation of haloarchaea biomass by aquatic metazoans.

     

Carbon assimilation pathways in sulfate reducing bacteria. Formate,carbon dioxide,carbon monoxide,and acetate assimilation by Desulfovibrio baarsii

Desulfovibrio baarsii is a sulfate reducing bacterium, which can grown on formate plus sulfate as sole energy source and formate and CO₂ as sole carbon sources. It is shown by ¹⁴C labelling studies that more than 60% of the cell carbon is derived from CO₂ and the rest from formate. The cells thus grow autotrophically. Labelling studies with [¹⁴C]acetate, ¹⁴CO and [¹⁴C]formate indicate that CO₂ fixation does not proceed via the Calvin cycle. The labelling patterns of alanine, aspartate, glutamate, and glucosamine indicate that acetate (or activated acetic acid) is an early intermediate in formate and CO₂ assimilation; the methyl group of acetate is derived from formate, and the carboxyl group from CO₂ via CO; pyruvate is formed from acetyl-CoA by reductive carboxylation. The capacity to synthesize an acetate unit from two C₁-compounds obviously distinguishes D. baarsii from those Desulfovibrio species, which require acetate as a carbon source in addition to CO₂.     

Acetate and Bicarbonate Assimilation and Metabolite Formation in Chlamydomonas reinhardtii: A 13C-NMR Study

Cellular metabolite analyses by ¹³C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO₂ ^aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly ¹³C-labelled acetate (¹³CH₃-COOH or CH₃-¹³COOH) supported that both the ¹³C nuclei give rise to bicarbonate and CO₂ ^aq. The observed metabolite(s) upon further incubation led to the production of starch and triacylglycerol (TAG) in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO₂ ^aq in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO₂ ^aq, which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO₂ ^aq pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.     

New insights into the energy metabolism and taxonomy of Deferribacteres revealed by the characterization of a new isolate from a hypersaline microbial mat

Strain L21-Ace-BES^T, isolated from a lithifying cyanobacterial mat, could be assigned to a novel species and genus within the class Deferribacteres. It is an important model organism for the study of anaerobic acetate degradation under hypersaline conditions. The metabolism of strain L21-Ace-BES^T was characterized by biochemical studies, comparative genome analyses, and the evaluation of gene expression patterns. The central metabolic pathway is the citric acid cycle, which is mainly controlled by the enzyme succinyl-CoA:acetate-CoA transferase. The potential use of a reversed oxidative citric acid cycle to fix CO₂ has been revealed through genome analysis. However, no autotrophic growth was detected in this strain, whereas sulfide and H₂ can be used mixotrophically. Preferred electron acceptors for the anaerobic oxidation of acetate are nitrate, fumarate and dimethyl sulfoxide, while oxygen can be utilized only under microoxic conditions. Aerotolerant growth by fermentation was observed at higher oxygen concentrations. The redox cycling of sulfur/sulfide enables the generation of reducing power for the assimilation of acetate during growth and could prevent the over-reduction of cells in stationary phase. Extracellular electron transfer appears to be an essential component of the respiratory metabolism in this clade of Deferribacteres and may be involved in the reduction of nitrite to ammonium.     

Halorhabdus tiamatea: proteogenomics and glycosidase activity measurements identify the first cultivated euryarchaeon from a deep‐sea anoxic brine lake as potential polysaccharide degrader

Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensis AX‐2^T from the shallow Great Salt Lake of Utah, and Halorhabdus tiamatea SARL4B^T from the Shaban deep‐sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H. tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H. tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant‐derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low‐oxygen conditions. Likewise, proteomics indicated quinone‐mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light‐dependent enzymes (e.g. bacteriorhodopsin), suggesting that H. tiamatea evolution was mostly not governed by a cold, dark, anoxic deep‐sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase‐rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments.     

A Study of the Mechanism of Acetate Assimilation in Purple Nonsulfur Bacteria Lacking the Glyoxylate Shunt: Enzymes of the Citramalate Cycle in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The mechanism of acetate assimilation by the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shunt, has been studied. In a previous work, proceeding from data on acetate assimilation by Rba. sphaeroides cell suspensions, a suggestion was made regarding the operation, in this bacterium, of the citramalate cycle. This cycle was earlier found in Rhodospirillum rubrum in the form of an anaplerotic reaction sequence that operates during growth on acetate instead of the glyoxylate shunt, which is not present in the latter bacterium. The present work considers the enzymes responsible for acetate assimilation in Rba. sphaeroides. It is shown that this bacterium possesses the key enzymes of the citramalate cycle: citramalate synthase, which catalyzes condensation of acetyl-CoA and pyruvate and, as a result, forms citramalate, and 3-methylmalyl-CoA lyase, which catalyzes the cleavage of 3-methylmalyl-CoA to glyoxylate and propionyl-CoA. The regeneration of pyruvate, which is the acetyl-CoA acceptor in the citramalate cycle, involves propionyl- CoA and occurs via the following reaction sequence: propionyl-CoA (+CO₂) å methylmalonyl-CoA å succinyl-CoA å succinate å fumarate malate å oxaloacetate (−CO₂) å phosphoenolpyruvate å pyruvate. The independence of the cell growth and the acetate assimilation of CO₂ is due to the accumulation of CO₂/HCO ₃ ⁻ (released during acetate assimilation) in cells to a level sufficient for the effective operation of propionyl-CoA carboxylase.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 319–328.Original Russian Text Copyright © 2005 by Filatova, Berg, Krasil’nikova, Ivanovsky.     

Enhanced growth of Acidovorax sp. strain 2AN during nitrate-dependent Fe(II) oxidation in batch and continuous-flow systems

Microbial nitrate-dependent, Fe(II) oxidation (NDFO) is a ubiquitous biogeochemical process in anoxic sediments. Since most microorganisms that can oxidize Fe(II) with nitrate require an additional organic substrate for growth or sustained Fe(II) oxidation, the energetic benefits of NDFO are unclear. The process may also be self-limiting in batch cultures due to formation of Fe-oxide cell encrustations. We hypothesized that NDFO provides energetic benefits via a mixotrophic physiology in environments where cells encounter very low substrate concentrations, thereby minimizing cell encrustations. Acidovorax sp. strain 2AN was incubated in anoxic batch reactors in a defined medium containing 5 to 6 mM NO₃⁻, 8 to 9 mM Fe²⁺, and 1.5 mM acetate. Almost 90% of the Fe(II) was oxidized within 7 days with concomitant reduction of nitrate and complete consumption of acetate. Batch-grown cells became heavily encrusted with Fe(III) oxyhydroxides, lost motility, and formed aggregates. Encrusted cells could neither oxidize more Fe(II) nor utilize further acetate additions. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced, and further additions of acetate and Fe(II)-EDTA could be oxidized. Experiments using a novel, continuous-flow culture system with low concentrations of substrate, e.g., 100 μM NO₃⁻, 20 μM acetate, and 50 to 250 μM Fe²⁺, showed that the growth yield of Acidovorax sp. strain 2AN was always greater in the presence of Fe(II) than in its absence, and electron microscopy showed that encrustation was minimized. Our results provide evidence that, under environmentally relevant concentrations of substrates, NDFO can enhance growth without the formation of growth-limiting cell encrustations.     

Iron cycling in the anoxic cryo-ecosystem of Antarctic Lake Vida

Iron redox cycling in metal-rich, hypersaline, anoxic brines plays a central role in the biogeochemical evolution of life on Earth, and similar brines with the potential to harbor life are thought to exist elsewhere in the solar system. To investigate iron biogeochemical cycling in a terrestrial analog we determined the iron redox chemistry and isotopic signatures in the cryoencapsulated liquid brines found in frozen Lake Vida, East Antarctica. We used both in situ voltammetry and the spectrophotometric ferrozine method to determine iron speciation in Lake Vida brine (LVBr). Our results show that iron speciation in the anoxic LVBr was, unexpectedly, not free Fe(II). Iron isotope analysis revealed highly depleted values of ?2.5‰ for the ferric iron of LVBr that are similar to iron isotopic signatures of Fe(II) produced by dissimilatory iron reduction. The presence of Fe(III) in LVBr therefore indicates dynamic iron redox cycling beyond iron reduction. Furthermore, extremely low δ¹⁸O–SO₄ ^2? values (?9.7‰) support microbial iron-sulfur cycling reactions. In combination with evidence for chemodenitrification resulting in iron oxidation, we conclude that coupled abiotic and biotic redox reactions are driving the iron cycle in Lake Vida brine. Our findings challenge the current state of knowledge of anoxic brine chemistry and may serve as an analogue for icy brines found in the outer reaches of the solar system.     

Neutrophilic, nitrate-dependent, Fe(II) oxidation by a Dechloromonas species

A species of Dechloromonas, strain UWNR4, was isolated from a nitrate-reducing, enrichment culture obtained from Wisconsin River (USA) sediments. This strain was characterized for anaerobic oxidation of both aqueous and chelated Fe(II) coupled to nitrate reduction at circumneutral pH. Dechloromonas sp. UWNR4 was incubated in anoxic batch reactors in a defined medium containing 4.5–5 mM NO₃ ^?, 6 mM Fe²⁺ and 1–1.8 mM acetate. Strain UWNR4 efficiently oxidized Fe²⁺ with 90 % oxidation of Fe²⁺ after 3 days of incubation. However, oxidation of Fe²⁺ resulted in Fe(III)-hydroxide-encrusted cells and loss of metabolic activity, suggested by inability of the cells to utilize further additions of acetate. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced and further additions of acetate and Fe(II)-EDTA could be oxidized. Although members of the genus Dechloromonas are primarily known as perchlorate and nitrate reducers, our findings suggest that some species could be members of microbial communities influencing iron redox cycling in anoxic, freshwater sediments. Our work using Fe(II)-EDTA also demonstrates that Fe(II) oxidation was microbially catalyzed rather than a result of abiotic oxidation by biogenic NO₂ ^?.     

Kohlenstoffassimilation von Chlamydobotrys (Volvocales)

Zusammenfassung Mit Hilfe C¹⁴-markierten Acetats und Bicarbonats wurde die Kohlenstoffassimilation aus Acetat und Kohlendioxyd von Chlamydobotrys unter verschiedenen Bedingungen untersucht. Der überwiegende Teil des aus Acetat in zelleigenes Material eingebauten Kohlenstoffs stammt aus der Methylgruppe, während entwickeltes Kohlendioxyd vorwiegend aus der Carboxylgruppe freigesetzt wird. Verglichen mit der Kohlenstofffixierung unter Anaerobiose steigern aerobe Bedingungen die Kohlenstoffassimilation aus Acetat im Licht um rund 25%. Im Dunkeln wird nur 10% des Kohlenstoffs aus Acetat in zelleigene Substanz eingebaut, das sind nur 3% des unter gleichen Bedingungen im Licht assimilierten.Zur Zeit optimaler Acetatassimilation findet nur eine sehr schwache Kohlendioxydfixierung statt, diese steigt mit dem Alter der Kultur. Die CO₂-Assimilation nimmt bei Acetatmangel zu; der aus CO₂ unter optimalen Bedingungen fixierte Kohlenstoff beträgt nur 5% des unter optimalen Verhältnissen aus Acetat assimilierten.

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Summary The carbon assimilation from radioactive labeled acetate and from NaH¹⁴CO₃ by Chlamydobotrys was studied under different conditions. Most of the carbon assimilated from acetate in the light comes from the methyl group, while the CO₂ produced was derived mainly from the carboxyl carbon. In light under aerobic conditions the carbon assimilation from acetate is higher by 25% than under anaerobic conditions. In the dark only 10% of the total acetate-carbon utilized are incorporated into cell material; that is only 3% of the total acetate carbon incorporated in light. During the period of high acetate assimilation the photosynthetic fixation of CO₂ is extremely weak. At acetate deficiency CO₂-fixation rises, but reaches only 5% of the carbon assimilated from acetate under optimum conditions.

     

Identification of Methanoculleus spp. as Active Methanogens during Anoxic Incubations of Swine Manure Storage Tank Samples

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-¹³C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of ¹³C into DNA was detectable at in situ acetate concentrations (∼7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the ¹³C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.     

Suppression of methanogenesis by dissimilatory Fe(III)-reducing bacteria in tropical rain forest soils: implications for ecosystem methane flux

Tropical forests are an important source of atmospheric methane (CH₄), and recent work suggests that CH₄ fluxes from humid tropical environments are driven by variations in CH₄ production, rather than by bacterial CH₄ oxidation. Competition for acetate between methanogenic archaea and Fe(III)‐reducing bacteria is one of the principal controls on CH₄ flux in many Fe‐rich anoxic environments. Upland humid tropical forests are also abundant in Fe and are characterized by high organic matter inputs, steep soil oxygen (O₂) gradients, and fluctuating redox conditions, yielding concomitant methanogenesis and bacterial Fe(III) reduction. However, whether Fe(III)‐reducing bacteria coexist with methanogens or competitively suppress methanogenic acetate use in wet tropical soils is uncertain. To address this question, we conducted a process‐based laboratory experiment to determine if competition for acetate between methanogens and Fe(III)‐reducing bacteria influenced CH₄ production and C isotope composition in humid tropical forest soils. We collected soils from a poor to moderately drained upland rain forest and incubated them with combinations of ¹³C‐bicarbonate, ¹³C‐methyl labeled acetate (¹³CH₃COO^?), poorly crystalline Fe(III), or fluoroacetate. CH₄ production showed a greater proportional increase than Fe²⁺ production after competition for acetate was alleviated, suggesting that Fe(III)‐reducing bacteria were suppressing methanogenesis. Methanogenesis increased by approximately 67 times while Fe²⁺ production only doubled after the addition of ¹³CH₃COO^?. Large increases in both CH₄ and Fe²⁺ production also indicate that the two process were acetate limited, suggesting that acetate may be a key substrate for anoxic carbon (C) metabolism in humid tropical forest soils. C isotope analysis suggests that competition for acetate was not the only factor driving CH₄ production, as ¹³C partitioning did not vary significantly between ¹³CH₃COO^? and ¹³CH₃COO^?+Fe(III) treatments. This suggests that dissimilatory Fe(III)‐reduction suppressed both hydrogenotrophic and aceticlastic methanogenesis. These findings have implications for understanding the CH₄ biogeochemistry of highly weathered wet tropical soils, where CH₄ efflux is driven largely by CH₄ production.     

Both Forward and Reverse TCA Cycles Operate in Green Sulfur Bacteria

The anoxygenic green sulfur bacteria (GSBs) assimilate CO₂ autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO₂ or HCO₃⁻. It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO₂ is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO₂ is essential for assimilating acetate and pyruvate through the CO₂-anaplerotic pathway and pyruvate synthesis from acetyl-CoA.     

Prokaryotic diversity and community composition in the Salar de Uyuni,a large scale,chaotropic salt flat

Salar de Uyuni (SdU), with a geological history that reflects 50 000 years of climate change, is the largest hypersaline salt flat on Earth and is estimated to be the biggest lithium reservoir in the world. Its salinity reaches saturation levels for NaCl, a kosmotropic salt, and high concentrations of MgCL₂ and LiCl, both salts considered important chaotrophic stressors. In addition, extreme temperatures, anoxic conditions, high UV irradiance, high albedo and extremely low concentrations of phosphorous, make SdU a unique natural extreme environment in which to contrast hypotheses about limiting factors of life diversification. Geophysical studies of brines from different sampling stations show that water activity is rather constant along SdU. Geochemical measurements show significant differences in magnesium concentration, ranging from 0.2 to 2M. This work analyses the prokaryotic diversity and community structure at four SdU sampling stations, selected according to their location and ionic composition. Prokaryotic communities were composed of both Archaea (with members of the classes Halobacteria, Thermoplasmata and Nanohaloarchaea, from the Euryarchaeota and Nanohaloarcheota phyla respectively) and Bacteria (mainly belonging to Bacteroidetes and Proteobacteria phyla). The important differences in composition of microbial communities inversely correlate with Mg²⁺ concentration, suggesting that prokaryotic diversity at SdU is chaotropic dependent.