Studies on the substrate range of Clostridium kluyveri; the use of propanol and succinate

The metabolism of Clostridium kluyveri has been extensively studied, but the range of substrates C. kluyveri can use for growth has not been fully explored. The use of propanol and succinate as growth substrates were established. C. kluyveri grows on acetate with propanol replacing ethanol. The principle carbon containing products were propionate, valerate, butyrate and hexanoate with traces of heptanoate. When grown with ethanol and succinate the principle carbon-containing products were acetate, butyrate and hexanoate. Hexanol was found as a product when incubated with ethanol and succinate 4-hydroxybutyrate or 3-butenoate. 5-Hexenoate was also a product of 3-butenoate and ethanol metabolism. The splitting of succinate into 2 acetates was indicated with ethanol providing the necessary reducing equivalents. Hydrogen was also found as a source of reducing equivalents but could not replace ethanol. A mechanism of succinate metabolism to acetate was proposed which accounts for growth yield, energetics considerations, carbon balances, production of side products and intermediates.Contribution No. 3619     

Chlorophenol dechlorination and subsequent degradation in denitrifying microcosms fed low concentrations of nitrate

The potential of using nitrate as a terminal electron acceptor to stimulate anaerobic degradation of mixtures of monochlorophenols (MCPs) or dichlorophenols (DCPs) was evaluated. Contaminated and non-contaminated soils were added to water saturated anaerobic microcosms supplemented with 1 mM or 5 mM nitrate. Denitrification and dechlorination activity were present in three diverse soil types and were maintained upon refeeding both nitrate and the appropriate chlorophenol. However, dechlorination activity could only be serially transferred in enrichments with an added electron donor such as acetate. Dehalogenation activity in enrichments from four of the primary microcosms showed at least five different dechlorination reactions, each mediated by different microbial communities. Three of these are distinct ortho-dechlorinating paths; two are meta-dechlorinating and one is the para-dechlorination of 3,4-DCP. Simultaneous dechlorination and denitrification was observed and both activities could be maintained in microcosms but only in the presence of low nitrate concentrations. Dechlorination and denitrification were mediated by two separate microbial communities; one that dechlorinates without use of nitrate and one that denitrifies while oxidizing the dechlorinated aromatic ring. There was no evidence that dechlorination is mediated by the denitrifying community, however the maintenance of a denitrification potential using low (< 1 mM) nitrate concentrations may be useful for completing the food chain by stimulating the mineralization of phenol and benzoate.     

克氏梭菌硫解酶的鉴定及其功能研究

【目的】硫解酶是梭菌属微生物合成短中链脂肪酸的关键酶。克氏梭菌(Clostridium kluyveri)具有3个高度同源的硫解酶编码基因,对这3个基因的功能鉴定是解析克氏梭菌高己酸合成能力的关键。【方法】通过发酵动力学分析确定克氏梭菌的己酸和丁酸生成动力学特征;转录组测序结合反转录-荧光定量RCR分析克氏梭菌3个硫解酶编码基因的表达水平和时序表达特征;在大肠杆菌中异源表达这3个硫解酶,并对其硫解酶动力学参数进行测定。【结果】克氏梭菌生成丁酸、己酸、辛酸,其中己酸为主要代谢产物;转录组数据显示,在乙酸消耗完全之前,thlA1基因维持恒定表达,thlA2基因表达时序上调,thlA3基因表达时序下调,转录组测序表明3个硫解酶编码基因均具有较高水平的转录活性,thlA2和thlA3的最高表达量分别约为thlA1的29%和43%;硫解酶动力学参数测定结果表明,克氏梭菌3个硫解酶对于四碳底物均显示出相似的底物亲和力(K_m),但ThlA1对四碳底物的催化效率(k_(cat)/K_m)略低于ThlA2和ThlA3。【结论】克氏梭菌的3个硫解酶均具有催化活性,在克氏梭菌体内均呈活跃表达,表明克氏梭菌拥有3个具有催化活性的硫解酶,这为后续深入研究克氏梭菌己酸合成机理奠定了基础。     

Fatty Acid-Oxidizing Consortia along a Nutrient Gradient in the Florida Everglades

The Florida Everglades is one of the largest freshwater marshes in North America and has been subject to eutrophication for decades. A gradient in P concentrations extends for several kilometers into the interior of the northern regions of the marsh, and the structure and function of soil microbial communities vary along the gradient. In this study, stable isotope probing was employed to investigate the fate of carbon from the fermentation products propionate and butyrate in soils from three sites along the nutrient gradient. For propionate microcosms, 16S rRNA gene clone libraries from eutrophic and transition sites were dominated by sequences related to previously described propionate oxidizers, such as Pelotomaculum spp. and Syntrophobacter spp. Significant representation was also observed for sequences related to Smithella propionica, which dismutates propionate to butyrate. Sequences of dominant phylotypes from oligotrophic samples did not cluster with known syntrophs but with sulfate-reducing prokaryotes (SRP) and Pelobacter spp. In butyrate microcosms, sequences clustering with Syntrophospora spp. and Syntrophomonas spp. dominated eutrophic microcosms, and sequences related to Pelospora dominated the transition microcosm. Sequences related to Pelospora spp. and SRP dominated clone libraries from oligotrophic microcosms. Sequences from diverse bacterial phyla and primary fermenters were also present in most libraries. Archaeal sequences from eutrophic microcosms included sequences characteristic of Methanomicrobiaceae, Methanospirillaceae, and Methanosaetaceae. Oligotrophic microcosms were dominated by acetotrophs, including sequences related to Methanosarcina, suggesting accumulation of acetate.     

Preparation and physical properties of chitin fatty acids esters

Trifluoroacetic anhydride is an effective promoter for the preparation of chitin single- and mixed-acid esters. Complete dissolution is achieved within 30 min when powdered chitin is heated at 70 °C in a mixed solution of carboxylic acid(s) and trifluoroacetic anhydride. Chitin esters prepared are chitin acetate, chitin butyrate, chitin hexanoate and chitin octanoate, chitin co-acetate/butyrate, chitin co-acetate/hexanoate, chitin co-acetate/octanoate, chitin co-acetate/palmitate, each from a solution of the respective reactants. The products have degrees of O-acyl substitution in a range of DS 1-2 depending on the nature of acyl group, as analyzed by gas-liquid and high-pressure liquid chromatography. Acetic acid as a mutual component for the mixed-acid esters increases the total degree of substitution, and the acetyl substitution is close to the relative distribution in the reaction mixture for chitin co-acetate/butyrate. It is favored over hexanoate, octanoate, and palmitate. The parent molecules, as calculated by the composition of the chitin esters and their molecular weights by light-scattering spectroscopy, are 30 kDa for the smallest and 150-151 kDa for the largest. Films of these chitin derivatives when cast from solution are strong and flexible with limited extensibility. By dynamic mechanical analysis of the ester film, it was found that both the glass transition temperature (T_g) and the tensile modulus (E′ at 25 °C) are highest for chitin acetate (218 °C and 5.8 GPa), and lowest for chitin octanoate (182 °C and 1.5 GPa). For the other esters, these values lie between the above-cited values, where the T_g and the E′ decrease with an increase in the chain length of the acyl constituent.     

Production of caproic acid by cocultures of ruminal cellulolytic bacteria and Clostridium kluyveri grown on cellulose and ethanol

Ruminal cellulolytic bacteria (Fibrobacter succinogenes S85 or Ruminococcus flavefaciens FD-1) were combined with the non-ruminal bacterium Clostridium kluyveri and grown together on cellulose and ethanol. Succinate and acetate produced by the cellulolytic organisms were converted to butyrate and caproate only when the culture medium was supplemented with ethanol. Ethanol (244 mM) and butyrate (30 mM at pH 6.8) did not inhibit cellulose digestion or product formation by S85 or FD-1; however caproate (30 mM at pH 6.8) was moderately inhibitory to FD-1. Succinate consumption and caproate production were sensitive to culture pH, with more caproic acid being produced when the culture was controlled at a pH near neutrality. In a representative experiment under conditions of controlled pH (at 6.8) 6.0 g cellulose 1^–1 and 4.4 g ethanol 1^–1 were converted to 2.6 g butyrate 1^–1 and 4.6 g caproate 1^–1. The results suggest that bacteria that efficiently produce low levels of ethanol and acetate or succinate from cellulose should be useful in cocultures for the production of caproic acid, a potentially useful industrial chemical and bio-fuel precursor.Mention of specific products is intended only to provide information and does not contitute an endorsement by the U.S. Department of Agriculture over other products not mentioned.     

Carbon flow from volcanic CO2 into soil microbial communities of a wetland mofette

Effects of extremely high carbon dioxide (CO₂) concentrations on soil microbial communities and associated processes are largely unknown. We studied a wetland area affected by spots of subcrustal CO₂ degassing (mofettes) with focus on anaerobic autotrophic methanogenesis and acetogenesis because the pore gas phase was largely hypoxic. Compared with a reference soil, the mofette was more acidic (ΔpH ∼0.8), strongly enriched in organic carbon (up to 10 times), and exhibited lower prokaryotic diversity. It was dominated by methanogens and subdivision 1 Acidobacteria, which likely thrived under stable hypoxia and acidic pH. Anoxic incubations revealed enhanced formation of acetate and methane (CH₄) from hydrogen (H₂) and CO₂ consistent with elevated CH₄ and acetate levels in the mofette soil. ¹³CO₂ mofette soil incubations showed high label incorporations with ∼512 ng ¹³C g (dry weight (dw)) soil⁻¹ d⁻¹ into the bulk soil and up to 10.7 ng ¹³C g (dw) soil⁻¹ d⁻¹ into almost all analyzed bacterial lipids. Incorporation of CO₂-derived carbon into archaeal lipids was much lower and restricted to the first 10 cm of the soil. DNA-SIP analysis revealed that acidophilic methanogens affiliated with Methanoregulaceae and hitherto unknown acetogens appeared to be involved in the chemolithoautotrophic utilization of ¹³CO₂. Subdivision 1 Acidobacteriaceae assimilated ¹³CO₂ likely via anaplerotic reactions because Acidobacteriaceae are not known to harbor enzymatic pathways for autotrophic CO₂ assimilation. We conclude that CO₂-induced geochemical changes promoted anaerobic and acidophilic organisms and altered carbon turnover in affected soils.     

Acetogenic Capacities and the Anaerobic Turnover of Carbon in a Kansas Prairie Soil

To assess the anaerobic capacities of a temperate grassland soil, a Kansas prairie soil was incubated anaerobically as either soil-water (1:2) suspensions or as soil microcosms at 78% soil water-holding capacity. Prairie soil formed acetate and CO(inf2) as the two main initial carbonaceous products from the anaerobic turnover of endogenous organic matter. Metabolic capacities of soil suspensions and microcosms were similar. Rates of acetate formation from endogenous organic matter in soil-water suspensions incubated at 40, 30, and 15(deg)C approximated 3.3, 2.4, and 1.1 (mu)g of acetate per g (dry weight) of soil per h, respectively. Supplemental H(inf2) and CO(inf2) were subject to consumption with the apparent concomitant synthesis of acetate in both soil suspensions and soil microcosms. In soil microcosms, rates of H(inf2)-dependent acetogenesis at 30 and 55(deg)C were nearly equivalent. The uptake of supplemental H(inf2) was not coupled to methanogenesis under any condition examined. These anaerobic activities were relatively stable when soils were subjected to either aerobic drying or alternating periods of O(inf2) enrichment. On the basis of the formation of nitrogen (N(inf2)), denitrification was engaged during anaerobic incubation periods; nitrous oxide (N(inf2)O) was also formed under certain conditions. Although extended incubation of soil induced the delayed methanogenic turnover of acetate, acetate was subject to immediate turnover under either O(inf2)- or nitrate-enriched conditions. These studies support the following concepts: (i) obligately anaerobic bacteria such as acetogenic bacteria are stable to periods of aerobiosis and are active in the anaerobic microsites of oxic soils, and (ii) acetate synthesized in anaerobic microsites of oxic terrestrial soils constitutes a trophic link to both aerobic and anaerobic microbial communities.     

Inhibition of adenosine 3':k'-monophosphate accumulation white fat acids, lactate, and beta-hydroxybutyrate.

The large increase in cyclic AMP accumulation by rat white fat cells seen in the presence of lipolytic agents plus methylxanthines and adenosine deaminase was markedly inhibited by lactate. However, lipolysis was unaffected by lactate. Octanoate, hexanoate, heptanoate, and beta-hydroxybutyrate inhibited both cyclic AMP accumulation and lipolysis by rat fat cells. The mechanism by which these acids inhibit lipolysis differs from that for long chain fatty acids such as oleate. Oleate directly inhibited triglyceride lipase activity of homogenized rat adipose tissue. In contrast, octanoate, beta-hydroxybutyrate, and lacatate had no effect on triglyceride lipase activity. Hormone-stimulated adenylate cyclase activity of rat fat cell ghosts was inhibited by oleate and 4mM octanoate but not by 1.6 mM octanoate, heptanoate, hexanoate, beta-hydroxybutyrate or lactate. None of the acids affected the soluble protein kinase activity of rat adipose tissue. There was no stimulation by lactate, butyrate, beta-hydroxybutyrate, or octanoate of the soluble or particulate cyclic AMP antilipolytic action of a short chain acid such as octanoate or hexanoate was not accompanied by any drop in total fat cell ATP. The mechanism by which lactate lowers cyclic AMP but not lipolysis remains to be established.     

Investigation of putative genes for the production of medium-chained acids and alcohols in autotrophic acetogenic bacteria

Gas fermentation is a technology for producing platform chemicals as well as fuels and one of the most promising alternatives to petrochemicals. Medium-chained acids and alcohols such as hexanoate and hexanol are particularly interesting due to their versatile application. This study elucidated the pathway of chain elongation in native C6 compound-producing acetogens. Essential genes of Clostridium carboxidivorans for synthesis of medium-chained acids and alcohols were identified in order to demonstrate their catalytic activity in the acetogenic model organism Acetobacterium woodii. Two such gene clusters were identified, which are responsible for conversion of acetyl-CoA to butyryl-CoA by reverse β-oxidation. Using RT-PCR it could be demonstrated that only genes of cluster 1 are expressed constitutively with simultaneous formation of C6 compounds. Based on genes from C. carboxidivorans, a modular hexanoyl-CoA synthesis (hcs) plasmid system was constructed and transferred into A. woodii. With the recombinant A. woodii strains AWO [pPta_hcs1], AWO [pPta_hcs2], AWO [pTet_hcs1], and AWO [pTet_hcs2] butyrate and hexanoate production under heterotrophic (1.22–4.15 mM hexanoate) and autotrophic conditions (0.48–1.56 mM hexanoate) with both hcs clusters could be detected. hcs Cluster 1 from C. carboxidivorans was transferred into the ABE-fermenting strain Clostridium saccharoperbutylacetonicum as well. For further analysis, genes were also cloned into the hcs plasmid system individually. The resulting recombinant C. saccharoperbutylacetonicum strains with just individual genes neither produced hexanoate nor hexanol, but the strains containing the entire gene cluster were capable of chain elongation. A production of 0.8 mM hexanoate and 5.2 mM hexanol in the fermentation with glucose could be observed.     

Effects of Environmental Parameters on the Formation and Turnover of Acetate by Forest Soils

The capacity to form acetate from endogenous matter was a common property of diverse forest soils when incubated under anaerobic conditions. At 15 to 20(deg)C, acetate synthesis occurred without appreciable delay when forest soils were incubated as buffered suspensions or in microcosms at various percentages of their maximum water holding capacity. Rates for acetate formation with soil suspensions ranged from 35 to 220 (mu)g of acetate per g (dry weight) of soil per 24 h, and maximal acetate concentrations obtained in soil suspensions were two- to threefold greater than those obtained with soil microcosms at the average water holding capacity of the soil. Cellobiose degradation in soil suspensions yielded H(inf2) as a transient product. Under anaerobic conditions, supplemental H(inf2) and CO(inf2) were directed towards the acetogenic synthesis of acetate, and enrichments yielded a syringate-H(inf2)-consuming acetogenic consortium. At in situ temperatures, acetate was a relatively stable anaerobic end product; however, extended incubation periods induced acetoclastic methanogenesis and sulfate reduction. Higher mesophilic and thermophilic temperatures greatly enhanced the capacity of soils to form methane. Although methanogenic and sulfate-reducing activities under in situ-relevant conditions were negligible, these findings nonetheless demonstrated the occurrence of methanogens and sulfate-reducing bacteria in these aerated terrestrial soils. In contrast to the protracted stability of acetate under anaerobic conditions at 15 to 20(deg)C with unsupplemented soils, acetate formed by forest soils was rapidly consumed in the presence of oxygen and nitrate, and substrate-product stoichiometries indicated that acetate turnover was coupled to oxygen-dependent respiration and denitrification. The collective results suggest that acetate formed under anaerobic conditions might constitute a trophic link between anaerobic and aerobic processes in forest soils.     

Peroxisomal and mitochondrial oxidation of fatty acids in the heart, assessed from the 13C labeling of malonyl-CoA and the acetyl moiety of citrate

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13C labeling ratio (malonyl-CoA)/(acetyl moiety of citrate) was >1.0 with 13C-fatty acids, which yields [13C]acetyl-CoA in both mitochondria and peroxisomes and < 1.0 with substrates, which yields [13C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. Hearts were perfused with increasing concentrations of labeled docosanoate, oleate, octanoate, hexanoate, butyrate, acetate, or dodecanedioate. In contrast to the liver, peroxisomal oxidation of 1-13C-fatty acids in heart does not form [1-13C]acetate. With [1-13C]docosanoate and [1,12-13C2]dodecanedioate, malonyl-CoA enrichment plateaued at 11 and 9%, respectively, with no detectable labeling of the acetyl moiety of citrate. Thus, in the intact rat heart, docosanoate and dodecanedioate appear to be oxidized only in peroxisomes. With [1-13C]oleate or [1-13C]octanoate, the labeling ratio >1 indicates the partial peroxisomal oxidation of oleate and octanoate. In contrast, with [3-13C]octanoate, [1-13C]hexanoate, [1-13C]butyrate, or [1,2-13C2]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal beta-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C2-C18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length.     

High throughput,colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z‐factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.     

Metabolic activity of subterranean microbial communities in deep granitic groundwater supplemented with methane and H2

It was previously concluded that opposing gradients of sulphate and methane, observations of 16S ribosomal DNA sequences displaying great similarity to those of anaerobic methane-oxidizing Archaea and a peak in sulphide concentration in groundwater from a depth of 250–350 m in Olkiluoto, Finland, indicated proper conditions for methane oxidation with sulphate. In the present research, pressure-resistant, gas-tight circulating systems were constructed to enable the investigation of attached and unattached anaerobic microbial populations from a depth of 327 m in Olkiluoto under in situ pressure (2.4 MPa), diversity, dissolved gas and chemistry conditions. Three parallel flow cell cabinets were configured to allow observation of the influence on microbial metabolic activity of 11 mℳ methane, 11 mℳ methane plus 10 mℳ H₂ or 2.1 mℳ O₂ plus 7.9 mℳ N₂ (that is, air). The concentrations of these gases and of organic acids and carbon, sulphur chemistry, pH and E_h, ATP, numbers of cultivable micro-organisms, and total numbers of cells and bacteriophages were subsequently recorded under batch conditions for 105 days. The system containing H₂ and methane displayed microbial reduction of 0.7 mℳ sulphate to sulphide, whereas the system containing only methane resulted in 0.2 mℳ reduced sulphate. The system containing added air became inhibited and displayed no signs of microbial activity. Added H₂ and methane induced increasing numbers of lysogenic bacteriophages per cell. It appears likely that a microbial anaerobic methane-oxidizing process coupled to acetate formation and sulphate reduction may be ongoing in aquifers at a depth of 250–350 m in Olkiluoto.     

Kinetics of Butyrate, Acetate, and Hydrogen Metabolism in a Thermophilic, Anaerobic, Butyrate-Degrading Triculture

Kinetics of butyrate, acetate, and hydrogen metabolism were determined with butyrate-limited, chemostat-grown tricultures of a thermophilic butyrate-utilizing bacterium together with Methanobacterium thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic rod. Kinetic parameters were determined from progress curves fitted to the integrated form of the Michaelis-Menten equation. The apparent half-saturation constants, K_m, for butyrate, acetate, and dissolved hydrogen were 76 μM, 0.4 mM, and 8.5 μM, respectively. Butyrate and hydrogen were metabolized to a concentration of less than 1 μM, whereas acetate uptake usually ceased at a concentration of 25 to 75 μM, indicating a threshold level for acetate uptake. No significant differences in K_m values for butyrate degradation were found between chemostat- and batch-grown tricultures, although the maximum growth rate was somewhat higher in the batch cultures in which the medium was supplemented with yeast extract. Acetate utilization was found to be the rate-limiting reaction for complete degradation of butyrate to methane and carbon dioxide in continuous culture. Increasing the dilution rate resulted in a gradual accumulation of acetate. The results explain the low concentrations of butyrate and hydrogen normally found during anaerobic digestion and the observation that acetate is the first volatile fatty acid to accumulate upon a decrease in retention time or increase in organic loading of a digestor.     

Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra

Cryoturbated peat circles (that is, bare surface soil mixed by frost action; pH 3–4) in the Russian discontinuous permafrost tundra are nitrate-rich ‘hotspots'' of nitrous oxide (N₂O) emissions in arctic ecosystems, whereas adjacent unturbated peat areas are not. N₂O was produced and subsequently consumed at pH 4 in unsupplemented anoxic microcosms with cryoturbated but not in those with unturbated peat soil. Nitrate, nitrite and acetylene stimulated net N₂O production of both soils in anoxic microcosms, indicating denitrification as the source of N₂O. Up to 500 and 10 μ nitrate stimulated denitrification in cryoturbated and unturbated peat soils, respectively. Apparent maximal reaction velocities of nitrite-dependent denitrification were 28 and 18 nmol N₂O g_DW⁻¹ h⁻¹, for cryoturbated and unturbated peat soils, respectively. Barcoded amplicon pyrosequencing of narG, nirK/nirS and nosZ (encoding nitrate, nitrite and N₂O reductases, respectively) yielded ≈49 000 quality-filtered sequences with an average sequence length of 444 bp. Up to 19 species-level operational taxonomic units were detected per soil and gene, many of which were distantly related to cultured denitrifiers or environmental sequences. Denitrification-associated gene diversity in cryoturbated and in unturbated peat soils differed. Quantitative PCR (inhibition-corrected per DNA extract) revealed higher copy numbers of narG in cryoturbated than in unturbated peat soil. Copy numbers of nirS were up to 1000 × higher than those of nirK in both soils, and nirS nirK⁻¹ copy number ratios in cryoturbated and unturbated peat soils differed. The collective data indicate that the contrasting N₂O emission patterns of cryoturbated and unturbated peat soils are associated with contrasting denitrifier communities.     

Influence of External pH and Fermentation Products on Clostridium acetobutylicum Intracellular pH and Cellular Distribution of Fermentation Products

Clostridium acetobutylicum ATCC 824 cells harvested from a phosphate-limited chemostat culture maintained at pH 4.5 had intracellular concentrations of acetate, butyrate, and butanol which were 13-, 7-, and 1.3-fold higher, respectively, than the corresponding extracellular concentrations. Cells from a culture grown at pH 6.5 had intracellular concentrations of acetate and butyrate which were only 2.2-fold higher than the respective external concentrations. The highest intracellular concentrations of these acids were attained at ca. pH 5.5. When cells were suspended in anaerobic citrate-phosphate buffer at pH 4.5, exogenous acetate and butyrate caused a concentration-dependent decrease in the intracellular pH, while butanol had relatively little effect until the external concentration reached 150 mM. Acetone had no effect at concentrations up to 200 mM. These data demonstrate that acetate and butyrate are concentrated within the cell under acidic conditions and thus tend to lower the intracellular pH. The high intracellular butyrate concentration presumably leads to induction of solvent production, thereby circumventing a decrease in the intracellular pH great enough to be deleterious to the cell.     

Formation and Fate of Fermentation Products in Hot Spring Cyanobacterial Mats

The fate of representative fermentation products (acetate, propionate, butyrate, lactate, and ethanol) in hot spring cyanobacterial mats was investigated. The major fate during incubations in the light was photoassimilation by filamentous bacteria resembling Chloroflexus aurantiacus. Some metabolism of all compounds occurred under dark aerobic conditions. Under dark anaerobic conditions, only lactate was oxidized extensively to carbon dioxide. Extended preincubation under dark anaerobic conditions did not enhance anaerobic catabolism of acetate, propionate, or ethanol. Acetogenesis of butyrate was suggested by the hydrogen sensitivity of butyrate conversion to acetate and by the enrichment of butyrate-degrading acetogenic bacteria. Accumulation of fermentation products which were not catabolized under dark anaerobic conditions revealed their importance. Acetate and propionate were the major fermentation products which accumulated in samples collected at temperatures ranging from 50 to 70°C. Other organic acids and alcohols accumulated to a much lesser extent. Fermentation occurred mainly in the top 4 mm of the mat. Exposure to light decreased the accumulation of acetate and presumably of other fermentation products. The importance of interspecies hydrogen transfer was investigated by comparing fermentation product accumulation at a 65°C site, with naturally high hydrogen levels, and a 55°C site, where active methanogenesis prevented significant hydrogen accumulation. There was a greater relative accumulation of reduced products, notably ethanol, in the 65°C mat.     

Degradation of cyanobacterial biomass in anoxic tidal-flat sediments: a microcosm study of metabolic processes and community changes

To follow the anaerobic degradation of organic matter in tidal-flat sediments, a stimulation experiment with ¹³C-labeled Spirulina biomass (130 mg per 21 g sediment slurry) was conducted over a period of 24 days. A combination of microcalorimetry to record process kinetics, chemical analyses of fermentation products and RNA-based stable-isotope probing (SIP) to follow community changes was applied. Different degradation phases could be identified by microcalorimetry: Within 2 days, heat output reached its maximum (55 μW), while primary fermentation products were formed (in μmol) as follows: acetate 440, ethanol 195, butyrate 128, propionate 112, H₂ 127 and smaller amounts of valerate, propanol and butanol. Sulfate was depleted within 7 days. Thereafter, methanogenesis was observed and secondary fermentation proceeded. H₂ and alcohols disappeared completely, whereas fatty acids decreased in concentration. Three main degraders were identified by RNA-based SIP and denaturant gradient gel electrophoresis. After 12 h, two phylotypes clearly enriched in ¹³C: (i) Psychrilyobacter atlanticus, a fermenter known to produce hydrogen and acetate and (ii) bacteria distantly related to Propionigenium. A Cytophaga-related bacterium was highly abundant after day 3. Sulfate reduction appeared to be performed by incompletely oxidizing species, as only sulfate-reducing bacteria related to Desulfovibrio were labeled as long as sulfate was available.     

Metal-induced inhibition of anaerobic metabolism of volatile fatty acids and hydrogen

The effects of copper (Cu), chromium (Cr), cadmium (Cd), lead (Pb) and zinc (Zn) on the biotransformation of organic acids (acetate, propionate and butyrate) and H₂ were assessed in serum-bottle microcosms. Experiments were performed over a range of metal concentrations (20–200 mg/1) using biomass from an anaerobic bioreactor fed continuously with ethanol distillery waste as inoculum. In general, the added metals inhibited the biotransformation of organic acids with increasing metal concentration. However, the extent of inhibition varied for the different biotransformations and for the different metals tested. For example, the concentration of CuCl₂ effecting a 50% reduction in the rate constant for biotransformation of acetate, propionate and butyrate was 60, 75 and 30 mg/1, respectively. Cu and Cr (VI) were the most inhibitory metals in organic acid transformation, whereas Pb was the least toxic. The rate of biotransformation of acetate was reduced by half at Cu and Cr concentrations of 60 and 40 gm/1 respectively, whereas Cd, Pb, and Zn concentrations of 160 to 200 mg/l had little effect. The activities of hydrogenotrophic methanogens were much less affected by the same metals and metal concentrations.