The occurrence and ecology of microbial chain elongation of carboxylates in soils

Chain elongation is a growth-dependent anaerobic metabolism that combines acetate and ethanol into butyrate, hexanoate, and octanoate. While the model microorganism for chain elongation, Clostridium kluyveri, was isolated from a saturated soil sample in the 1940s, chain elongation has remained unexplored in soil environments. During soil fermentative events, simple carboxylates and alcohols can transiently accumulate up to low mM concentrations, suggesting in situ possibility of microbial chain elongation. Here, we examined the occurrence and microbial ecology of chain elongation in four soil types in microcosms and enrichments amended with chain elongation substrates. All soils showed evidence of chain elongation activity with several days of incubation at high (100 mM) and environmentally relevant (2.5 mM) concentrations of acetate and ethanol. Three soils showed substantial activity in soil microcosms with high substrate concentrations, converting 58% or more of the added carbon as acetate and ethanol to butyrate, butanol, and hexanoate. Semi-batch enrichment yielded hexanoate and octanoate as the most elongated products and microbial communities predominated by C. kluyveri and other Firmicutes genera not known to undergo chain elongation. Collectively, these results strongly suggest a niche for chain elongation in anaerobic soils that should not be overlooked in soil microbial ecology studies.Subject terms: Soil microbiology, Microbial ecology     

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。     

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.     

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.     

On the specifity and stereospecificity of the conversion of different 2,3-unsaturated acids by Clostridium kluyveri (author's transl)]

Further 2,3-unsaturated acids are revealed which can be reduced by Clostridium kluyveri with crotonate or butyrate as hydrogen donors. Unsaturated and saturated 3-halogenated acids are transformed into the saturated halogen-free acids. The following reaction sequence is proposed: a) hydrogenation, b) elumination of HX and c) again hydrogenation. Tiglinate ((E)-2-methyl-2-butenoate) and (E)-2-methyl-2-pentenoate are stereospecifically reduced to the (S)-2-methyl substituted acids. C. kluyveri contains endogenous material; in the presence of hydrogen acceptors such as 2,3-unsaturated acids this is degraded to acetate, and the reducing equivalents liberated hydrogenate the unsaturated acid. In a transient phase the hydration products of the unsaturated acids are present in the non-activated form in appreciable amounts. Tiglinate as well as crotonate is partially converted to ethyl methyl ketone and aceton and/or propanol, respectively.     

Glycerol fermentation by (open) mixed cultures: a chemostat study

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.     

Isolation,characterization, and quantification of <Emphasis Type="Italic">Clostridium kluyveri</Emphasis> from the bovine rumen

A strain of Clostridium kluyveri was isolated from the bovine rumen in a medium containing ethanol as an electron donor and acetate and succinate (common products of rumen fermentation) as electron acceptors. The isolate displayed a narrow substrate range but wide temperature and pH ranges atypical of ruminal bacteria and a maximum specific growth rate near the typical liquid dilution rate of the rumen. Quantitative real-time PCR revealed that C. kluyveri was widespread among bovine ruminal samples but was present at only very low levels (0.00002% to 0.0002% of bacterial 16S rRNA gene copy number). However, the species was present in much higher levels (0.26% of bacterial 16S rRNA gene copy number) in lucerne silage (but not maize silage) that comprised much of the cows’ diet. While C. kluyveri may account for several observations regarding ethanol utilization and volatile fatty acid production in the rumen, its population size and growth characteristics suggest that it is not a significant contributor to ruminal metabolism in typical dairy cattle, although it may be a significant contributor to silage fermentation. The ability of unadapted cultures to produce substantial levels (12.8 g L⁻¹) of caproic (hexanoic) acid in vitro suggests that this strain may have potential for industrial production of caproic acid.     

The influence of carbon substrate on the activity of the periplasmic nitrate reductase in aerobically grown Thiosphaera pantotropha

The periplasmic nitrate reductase was assayed in intact cells of Thiosphaera pantotropha, after aerobic growth with either malate, succinate, acetate, butyrate or caproate present as sole carbon source. The level of enzyme activity was largely dependent upon carbon source and was lowest on malate and succinate, intermediate on acetate and highest on butyrate and caproate. The presence or absence of nitrate did not effect enzyme activity. The results indicate that, during aerobic growth, activity of the periplasmic nitrate reductase increases with the extent of reduction of the carbon substrate.Abbreviation MV⁺ reduced methylviologen     

Modeling of growth, lactate consumption, and volatile fatty acid production by Megasphaera elsdenii cultivated in minimal and complex media

Use of the Pirt and Luedeking-Piret equations permits the determination of the effect of medium composition on the metabolic patterns of Megasphaera elsdenii grown in minimal and complex media with lactate as the major carbon source. To establish the significance of the parameters involved in the Pirt and Luedeking-Piret equations, a quantitative statistical criterion was proposed. In the complex medium, lactate was completely used for growth and product formation, whereas in the minimal medium a fraction of the energy obtained from lactate was used for maintenance purposes. Modeling of VFA production by the Luedeking-Piret equation showed that, independent of the type of medium, acetate and propionate are growth-associated products, while butyrate and valerate are only partially growth-associated. The growth-associated products are related to energy-yielding metabolism and the non-growth-associated products are related to the consumption of reducing equivalents.     

Adh4, an alcohol dehydrogenase controls alcohol formation within bacterial microcompartments in the acetogenic bacterium Acetobacterium woodii

Acetobacterium woodii utilizes the Wood-Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non-acetogenic growth on 1,2-propanediol (1,2-PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2-PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC-encapsulated propanol-generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH-dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2-PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.     

Butyrate production in the acetogen Eubacterium limosum is dependent on the carbon and energy source

Eubacterium limosum KIST612 is one of the few acetogenic bacteria that has the genes encoding for butyrate synthesis from acetyl-CoA, and indeed, E. limosum KIST612 is known to produce butyrate from CO but not from H₂ + CO₂. Butyrate production from CO was only seen in bioreactors with cell recycling or in batch cultures with addition of acetate. Here, we present detailed study on growth of E. limosum KIST612 on different carbon and energy sources with the goal, to find other substrates that lead to butyrate formation. Batch fermentations in serum bottles revealed that acetate was the major product under all conditions investigated. Butyrate formation from the C1 compounds carbon dioxide and hydrogen, carbon monoxide or formate was not observed. However, growth on glucose led to butyrate formation, but only in the stationary growth phase. A maximum of 4.3 mM butyrate was observed, corresponding to a butyrate:glucose ratio of 0.21:1 and a butyrate:acetate ratio of 0.14:1. Interestingly, growth on the C1 substrate methanol also led to butyrate formation in the stationary growth phase with a butyrate:methanol ratio of 0.17:1 and a butyrate:acetate ratio of 0.33:1. Since methanol can be produced chemically from carbon dioxide, this offers the possibility for a combined chemical-biochemical production of butyrate from H₂ + CO₂ using this acetogenic biocatalyst. With the advent of genetic methods in acetogens, butanol production from methanol maybe possible as well.     

Propanol as an end product of threonine fermentation

Clostridium sp. strain 17cr1 was able to ferment l-threonine to propionate and propanol. Electrons arising in the oxidation of 2-oxobutyrate to propionyl-CoA were apparently used in reductive pathway leading to propanol formation. Part of the propionyl-CoA was used to form propionate in an ATP-forming pathway via a propionate kinase, so that the final ATP yield was 0.5 mol per mol of l-threonine metabolised. Other growth substrates were fermented mainly to acetate and butyrate, and the reductive formation of butyrate, from 2 mol of acetyl-CoA or from crotonate or 3-hydroxybutyrate, was the main route for recycling reduced electron carriers arising during oxidative pathways for most substrates.     

Carbon dioxide fixation and mixotrophic metabolism by strain DCB-1, a dehalogenating anaerobic bacterium

Fixation by strain DCB-1 of CO2 carbon into cell material and organic acids occurred during growth on pyruvate both with and without thiosulfate. By using sodium [14C]bicarbonate and sodium [2-14C]pyruvate, the isotopic composition of products and cells was investigated. Up to 70% of cell carbon was derived from CO2. CO2 carbon was also incorporated into succinate, formate, and acetate. Both carbons of acetate underwent exchange reactions with CO2, although the carboxyl-group exchange was twice as fast. Because strain DCB-1 uses CO2 as its major but not sole carbon source while deriving energy from pyruvate metabolism, we describe its metabolism as mixotrophic. Other mixotrophic conditions also supported growth. Lactate or butyrate, which could not support growth in mineral medium, could replace pyruvate as the oxidizable substrate only when acetate was added to the medium.     

Carbon dioxide fixation and mixotrophic metabolism by strain DCB-1, a dehalogenating anaerobic bacterium.

Fixation by strain DCB-1 of CO2 carbon into cell material and organic acids occurred during growth on pyruvate both with and without thiosulfate. By using sodium [14C]bicarbonate and sodium [2-14C]pyruvate, the isotopic composition of products and cells was investigated. Up to 70% of cell carbon was derived from CO2. CO2 carbon was also incorporated into succinate, formate, and acetate. Both carbons of acetate underwent exchange reactions with CO2, although the carboxyl-group exchange was twice as fast. Because strain DCB-1 uses CO2 as its major but not sole carbon source while deriving energy from pyruvate metabolism, we describe its metabolism as mixotrophic. Other mixotrophic conditions also supported growth. Lactate or butyrate, which could not support growth in mineral medium, could replace pyruvate as the oxidizable substrate only when acetate was added to the medium.     

Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z

Actinobacillus sp. 130Z fermented glucose to the major products succinate, acetate, and formate. Ethanol was formed as a minor fermentation product. Under CO₂-limiting conditions, less succinate and more ethanol were formed. The fermentation product ratio remained constant at pH values from 6.0 to 7.4. More succinate was produced when hydrogen was present in the gas phase. Actinobacillus sp. 130Z grew at the expense of fumarate and l-malate reduction, with hydrogen as an electron donor. Other substrates such as more-reduced carbohydrates (e.g., d-sorbitol) resulted in higher succinate and/or ethanol production. Actinobacillus sp. 130Z contained the key enzymes involved in the Embden-Meyerhof-Parnas and the pentose-phosphate pathways and contained high levels of phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, pyruvate kinase, pyruvate formate-lyase, phosphotransacetylase, acetate kinase, malic enzyme, and oxaloacetate decarboxylase. The levels of PEP carboxykinase, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. 130Z than in Escherichia coli K-12 and accounted for the differences in succinate production. Key enzymes in end product formation in Actinobacillus sp. 130Z were regulated by the energy substrates. Received: 2 September 1996 / Accepted: 10 January 1997     

Parameters Affecting Solvent Production by Clostridium pasteurianum

The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100 mol), lactate (32 mol/100 mol), and butanol (36 mol/100 mol) in addition to the usual products, acetate (38 mol/100 mol) and butyrate (17 mol/100 mol). During glycerol fermentation, a completely different product pattern was found. In continuous culture under phosphate limitation, acetate and butyrate were produced only in trace amounts, whereas ethanol (30 mol/100 mol), butanol (18 mol/100 mol), and 1,3-propanediol (18 mol/100 mol) were the major products. Under iron limitation, the ratio of these products could be changed in favor of 1,3-propanediol (34 mol/100 mol). In addition, lactate was produced in significant amounts (25 mol/100 mol). The tolerance of C. pasteurianum to glycerol was remarkably high; growth was not inhibited by glycerol concentrations up to 17% (wt/vol). Increasing glycerol concentrations favored the production of 1,3-propanediol.     

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.     

Physiology of Geobacter metallireducens under excess and limitation of electron donors. Part I. Batch cultivation with excess of carbon sources

For microorganisms that play an important role in bioremediation, the adaptation to swift changes in the availability of various substrates is a key for survival. The iron-reducing bacterium Geobacter metallireducens was hypothesized to repress utilization of less preferred substrates in the presence of high concentrations of easily degradable compounds. In our experiments, acetate and ethanol were preferred over benzoate, but benzoate was co-consumed with toluene and butyrate. To reveal overall physiological changes caused by different single substrates and a mixture of acetate plus benzoate, a nano-liquid chromatography–tandem mass spectrometry-based proteomic approach (nano-LC–MS/MS) was performed using label-free quantification. Significant differential expression during growth on different substrates was observed for 155 out of 1477 proteins. The benzoyl-CoA pathway was found to be subjected to incomplete repression during exponential growth on acetate in the presence of benzoate and on butyrate as a single substrate. Peripheral pathways of toluene, ethanol, and butyrate degradation were highly expressed only during growth on the corresponding substrates. However, low expression of these pathways was detected in all other tested conditions. Therefore, G. metallireducens seems to lack strong carbon catabolite repression under high substrate concentrations, which might be advantageous for survival in habitats rich in fatty acids and aromatic hydrocarbons.     

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ3-Δ2-isomerase

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg^-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.     

Clostridium grantii sp. nov., a new obligately anaerobic,alginolytic bacterium isolated from mullet gut

A gram-positive, motile, rod-shaped, strictly anaerobic, sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C and pH6.5, and at a salinity of 1–10³. Out of a variety of polysaccharides tested as growth substrates, only alginate supported growth in either semidefined or complex culture medium. The organism also grew on a variety of mono- and disaccharides. Moles product per 100mol of alginate monomer degraded were: acetate, 186; ethanol, 19; formate, 54; and CO₂, 0.19. Moles product per 100mol of hexose in cellobiose or glucose degraded were: acetate, 135; ethanol,61; formate, 63: and CO₂, 61. Hydrogen was not detectable during the incubations (detection limit, <10^-5atm) and propionate, butyrate, lactate, or succinate were not produced as fermentation end products (<2 mol per 100 mol of monomer). The G+C content of DNA from the bacterium was 30.2±0.3 mol%, and the cell walls contained the peptidoglycan component meso-diaminopimelic acid. A phylogenetic analysis of the 16S rDNA sequence indicated that the organism grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. However, it differed from other species of the genus with regard to morphology, growth temperature optimum, substrate range, and fermentation pattern and is therefore designated as a new species of Clostridium; the type strain is A-1 (DSM 8605).