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Climate changes significantly impact greenhouse gas emissions from wetland soil. Specifically, wetland soil may be exposed to oxygen (O 2 ) during droughts, or to sulfate (SO 4 2- ) as a result of sea level rise. How these stressors - separately and together - impact microbial food webs driving carbon cycling in the wetlands is still not understood. To investigate this, we integrated geochemical analysis, proteogenomics, and stoichiometric modeling to characterize the impact of elevated SO 4 2- and O 2 levels on microbial methane (CH 4 ) and carbon dioxide (CO 2 ) emissions. The results uncovered the adaptive responses of this community to changes in SO 4 2- and O 2 availability and identified altered microbial guilds and metabolic processes driving CH 4 and CO 2 emissions. Elevated SO 4 2- reduced CH 4 emissions, with hydrogenotrophic methanogenesis more suppressed than acetoclastic. Elevated O 2 shifted the greenhouse gas emissions from CH 4 to CO 2 . The metabolic effects of combined SO 4 2- and O 2 exposures on CH 4 and CO 2 emissions were similar to those of O 2 exposure alone. The reduction in CH 4 emission by increased SO 4 2- and O 2 was much greater than the concomitant increase in CO 2 emission. Thus, greater SO 4 2- and O 2 exposure in wetlands is expected to reduce the aggregate warming effect of CH 4 and CO 2 . Metaproteomics and stoichiometric modeling revealed a unique subnetwork involving carbon metabolism that converts lactate and SO 4 2- to produce acetate, H 2 S, and CO 2 when SO 4 2- is elevated under oxic conditions. This study provides greater quantitative resolution of key metabolic processes necessary for the prediction of CH 4 and CO 2 emissions from wetlands under future climate scenarios., (© 2024. The Author(s).)

Microbial CO 2 utilization reduces the carbon footprint, providing economic potential. Biochar, rich in minerals and trace metals, can enhance microbial activity. This study investigates poultry litter and switchgrass biochars produced at 350 and 700 °C (PLB350, PLB700, SGB350 and SGB700, respectively) affect CO 2 conversion to C2-C6 alcohols and acids by Clostridium muellerianum P21, C. ragsdalei P11 and C. carboxidivorans P7. Fermentations were in 250-mL bottles containing H 2 :CO 2 :N 2 (60:20:20) shaken at 125 rpm and 37 °C. SGB350 increased alcohol titers by 1.1-2.1 fold, and PLB350 enhanced acid concentrations by 1.2-1.7 fold compared to the control without biochar. About 2.0-3.3 fold more ethanol was formed by strain P11 compared to strains P7 and P21 with SGB350. However, strain P21 produced 2.4-fold more butanol than strain P7 with SGB350, including unique hexanol production. These results highlight the potential of biochar in enhancing C2-C6 alcohol production from CO 2 , thereby boosting process feasibility., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Atiyeh reports financial support, equipment, or supplies, statistical analysis, and travel were provided by U.S. Department of Agriculture, under award number 2019-38502-30120 through South Central Sun Grant Program. Atiyeh has patents # 10,053,711; 10,017,789; 10,640,792; 11,180,779 issued to NA. Tanner have patent # 7,704,723 issued to NA., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Enterolert, a fluorogenic substrate test, is used as a quantitative method for determining freshwater concentrations of Enterococcus for water quality indicators. However, there is some evidence from recent studies suggesting that Enterolert may not suppress false positives due to pollution sources in waterbodies. In this study, we evaluated this method by analyzing field water and sediment samples from four freshwater streams. We also performed a laboratory microcosm study from two of the stream sediments. The Enterolert method was investigated by phenotypic and genomic analyses for accuracy of isolating and quantifying Enterococcus and/or Streptococcus. Additionally, we tested isolates from Enterolert panels for antibiotic resistance. Results from the field and microcosm studies from initial to final time points indicated that false positives were predominantly Paenibacillus spp. and other non-fecal indicator bacteria. Furthermore, the microcosm study indicated shifts from lactic acid to non-lactic acid bacteria between initial to final time points, but Enterococcus concentrations from Enterolert panels remained stable for the duration of the study for both stream sediments. Antibiotic resistance indicated no distinct pattern of resistance or susceptibility to a suite of antibiotics. However, all isolates tested were resistant to bacitracin and nalidixic acid. In conclusion, we found that Enterolert was not exclusively selective for Enterococcus from freshwater environments and that sediment and polluted waterbodies have the potential to skew the presumed concentrations. More research is needed to evaluate the effectiveness and selectivity of the medium used for the fluorogenic substrate test for Enterococcus enumeration., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Complex interactions exist among microorganisms in a community to carry out ecological processes and adapt to changing environments. Here, we constructed a quad-culture consisting of a cellulolytic bacterium (Ruminiclostridium cellulolyticum), a hydrogenotrophic methanogen (Methanospirillum hungatei), an acetoclastic methanogen (Methanosaeta concilii), and a sulfate-reducing bacterium (Desulfovibrio vulgaris). The four microorganisms in the quad-culture cooperated via cross-feeding to produce methane using cellulose as the only carbon source and electron donor. The community metabolism of the quad-culture was compared with those of the R. cellulolyticum-containing tri-cultures, bi-cultures, and mono-culture. Methane production was higher in the quad-culture than the sum of the increases in the tri-cultures, which was attributed to a positive synergy of four species. In contrast, cellulose degradation by the quad-culture was lower than the additive effects of the tri-cultures which represented a negative synergy. The community metabolism of the quad-culture was compared between a control condition and a treatment condition with sulfate addition using metaproteomics and metabolic profiling. Sulfate addition enhanced sulfate reduction and decreased methane and CO 2 productions. The cross-feeding fluxes in the quad-culture in the two conditions were modeled using a community stoichiometric model. Sulfate addition strengthened metabolic handoffs from R. cellulolyticum to M. concilii and D. vulgaris and intensified substrate competition between M. hungatei and D. vulgaris. Overall, this study uncovered emergent properties of higher-order microbial interactions using a four-species synthetic community. IMPORTANCE A synthetic community was designed using four microbial species that together performed distinct key metabolic processes in the anaerobic degradation of cellulose to methane and CO 2 . The microorganisms exhibited expected interactions, such as cross-feeding of acetate from a cellulolytic bacterium to an acetoclastic methanogen and competition of H 2 between a sulfate reducing bacterium and a hydrogenotrophic methanogen. This validated our rational design of the interactions between microorganisms based on their metabolic roles. More interestingly, we also found positive and negative synergies as emergent properties of high-order microbial interactions among three or more microorganisms in cocultures. These microbial interactions can be quantitatively measured by adding and removing specific members. A community stoichiometric model was constructed to represent the fluxes in the community metabolic network. This study paved the way toward a more predictive understanding of the impact of environmental perturbations on microbial interactions sustaining geochemically significant processes in natural systems.

An acid/alcohol-producing, Gram-stain-positive, obligately anaerobic, rod-shaped, non-motile, non-spore forming acetogen, designated as strain P21 T , was isolated from old hay after enrichment with CO as the substrate. Spores not observed even after prolonged incubation (30 days). Phylogenetic analysis of the 16S rRNA gene sequence of strain P21 T showed it was closely related to Clostridium carboxidivorans DSM 15243 T (97.9%), Clostridium scatologenes DSM 757 T (97.7 %) and Clostridium drakei DSM 12750 T (97.7 %). The genome is 5.6 Mb and the G+C content is 29.4 mol%. Average nucleotide identity between strain P21 T , C. carboxidivorans , C. scatologenes and C. drakei was 87.1, 86.4, 86.4 %, respectively. Strain P21 T grew on CO:CO 2 , H 2 :CO 2 , l-arabinose, ribose, xylose, fructose, galactose, glucose, lactose, mannose, cellobiose, sucrose, cellulose, starch, pyruvate, choline, glutamate, histidine, serine, threonine and casamino acids. End products of metabolism were acetate, butyrate, caproate, ethanol and hexanol. Dominant cellular fatty acids (>10 %) were C 16 : 0 (41.5 %), C 16 : 1  ω7 c /C 16 : 1  ω6 c (10.0 %), and a summed feature containing cyclo C 17 : 1 /C 18 : 0 (17.3 %). Based on the phenotypic, chemotaxonomic, phylogenetic and phylogenomic analyses, strain P21 T represents a new species in the genus Clostridium , for which the name Clostridium muellerianum sp. nov. is proposed. The type strain is P21 T (=DSM 111390 T =NCIMB 15261 T ).

Here, we report the genome sequence of Clostridium sp. strain P21, isolated from old hay from Stillwater, Oklahoma. This announcement describes the generation and annotation of the 5.6-Mb genomic sequence of strain P21, which will aid in studies targeting genes involved in the enhancement of acid-alcohol production., (Copyright © 2021 Doyle et al.)

Biochar has functional groups, pH buffering capacity and cation exchange capacity (CEC) that can be beneficial in syngas fermentation. This study examined the properties of biochar made from switchgrass (SGBC), forage sorghum (FSBC), red cedar (RCBC) and poultry litter (PLBC), and their effects on ethanol and butanol production from syngas using Clostridium carboxidivorans. Experiments were performed in 250 mL bottle reactors with a 50 mL working volume at 37 °C fed syngas containing CO:H 2 :CO 2 (40:30:30 by volume). Results showed that PLBC and SGBC enhanced ethanol production by 90% and 73%, respectively, and butanol production by fourfold compared to standard yeast extract medium without biochar (control). CO and H 2 utilization in PLBC and SGBC media increased compared to control. PLBC had the highest pH buffering capacity, CEC and total amount of cations compared with SGBC, FSBC and RCBC, which could have contributed to its highest enhancement of ethanol and butanol production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Microbially influenced corrosion (MIC), also known as biocorrosion, has significant impacts on the environment and economy. Typical systems to study biocorrosion are either dynamic (once-through flow) or static (serum bottle incubations). Dynamic systems can be materials and personnel intensive, while static systems quickly become nutrient limiting and exhibit long incubations. A semi-continuous biocorrosion cell was developed to address these issues. Low carbon shim steel was used as a test surface. Initial results revealed that 50 ppm glutaraldehyde (GLT), a common oil field biocide, in an abiotic cell was 3.6 times more corrosive (24.5 × 10 -3  mm/y) than a biocorrosion cell inoculated with a sulfate-reducing bacteria (SRB) enrichment (6.73 × 10 -3  mm/y). The SRB inoculated cell treated with GLT (50 ppm) reduced the corrosion rate from 6.73 × 10 -3  mm/y to 3.68 × 10 -3  mm/y. It was hypothesized that a biocide-surfactant combination would enhance biocide activity, thereby lowering corrosion in a semi-continuous biocorrosion cell. The biocide and surfactant were GLT (30 ppm) and Tween 80 (TW80; 100 ppm). MIC of SRB increased in the presence of a non-inhibitory concentration of GLT (23.4 × 10 -3  mm/y), compared to the untreated +SRB condition (8.29 × 10 -3  mm/y). The non-ionic surfactant alone reduced MIC (4.57 × 10 -3  mm/y) and even more so in combination with GLT (3.69 × 10 -3  mm/y). Over 50% of 16S rDNA sequences in the biofilm on the test surface were identified as belonging to the genera Desulfovibrio and Desulfomicrobium. The utility of a semi-continuous system for MIC studies and biocide testing was demonstrated. The concept of regular partial medium replacement is applicable to different corrosion cell and corrosion coupon geometries. Biocide-surfactant combinations may have the potential to reduce the concentration of biocides used in the field. In addition, a semi-defined medium for enumerating Acid-Producing Bacteria (APB) was developed, resulting in higher recoveries compared to a standard phenol red medium (e.g., 1.1 × 10 4 APB/cm 2 vs < 4 × 10 -1 APB/cm 2 )., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Acetomicrobium hydrogeniformans , an obligate anaerobe of the phylum Synergistetes , was isolated from oil production water. It has the unusual ability to produce almost 4 molecules H 2 /molecule glucose. The draft genome of A. hydrogeniformans OS1 (DSM 22491 T ) is 2,123,925 bp, with 2,068 coding sequences and 60 RNA genes., (Copyright © 2018 Cook et al.)

The present study evaluated butanol production from switchgrass based on hydrothermolysis pretreatment. The inhibitors present in the hydrolyzates were measured. Results showed poor butanol production (1g/L) with non-detoxified hydrolyzate. However, adjusting the pH of the non-detoxified hydrolyzate to 6 and adding 4 g/L CaCO3 increased butanol formation to about 6g/L. There was about 1g/L soluble lignin content (SLC), and various levels of furanic and phenolic compounds found in the non-detoxified hydrolyzate. Detoxification of hydrolyzates with activated carbon increased the butanol titer to 11 g/L with a total acetone, butanol and ethanol (ABE) concentration of 17 g/L. These results show the potential of butanol production from hydrothermolysis pretreated switchgrass., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Clostridium carboxidivorans was grown on model syngas (CO:H2:CO2 [70:20:10]) in a defined nutrient medium with concentrations of nitrogen, phosphate and trace metals formulated to enhance production of higher alcohols. C. carboxidivorans was successfully grown in a limited defined medium (no yeast extract, no MES buffer and minimal complex chemical inputs) using an improved fermentation protocol. Low partial pressure of CO in the headspace, coupled with restricted mass transfer for CO and H2, was required for successful fermentation. In the absence of substrate inhibition (particularly from CO), growth limitation increased production of alcohols, especially butanol and hexanol. Concentrations of butanol (over 1.0g/L), hexanol (up to 1.0g/L) and ethanol (over 3.0g/L) were achieved in bottle fermentations. Minimal medium and controlled supply of CO and H2 should be used in characterizing candidate butanol and hexanol producing strains to select for commercial potential., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Eastern redcedar is an invasive softwood species in Oklahoma and across grasslands in the Central Plains of the United States and potential feedstock for butanol production. Butanol has higher energy content than ethanol and can be upgraded to jet and diesel fuels. The objective of this study was to develop a process for production of butanol from redcedar. Results showed that Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 did not grow in fermentation medium with citrate buffer. However, both strains grew in the medium with acetate buffer, resulting in 3-4g/L greater butanol than without acetate. Detoxification of redcedar hydrolyzate was required to increase butanol concentration from 1 to 13g/L. Hydrolyzate was detoxified by activated carbon to remove inhibitors. Fermentations in detoxified redcedar hydrolyzate reached 13g/L butanol and 19g/L total ABE, comparable to glucose control. This shows the potential for redcedar use in butanol production., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

"Clostridium ragsdalei" is an acetogen that ferments synthesis gas (syngas, predominantly H2:CO2:CO) to ethanol, acetate, and cell mass. Previous research showed that C. ragsdalei could also convert propionic acid to 1-propanol and butyric acid to 1-butanol at conversion efficiencies of 72.3 and 21.0 percent, respectively. Our research showed that C. ragsdalei can also reduce pentanoic and hexanoic acid to the corresponding primary alcohols. This reduction occurred independently of growth in an optimized medium with headspace gas exchange (vented and gassed with CO) every 48 h. Under these conditions, conversion efficiencies increased to 97 and 100 % for propionic and butyric acid, respectively. The conversion efficiencies for pentanoic and hexanoic acid to 1-pentanol and 1-hexanol, respectively, were 82 and 62 %. C. ragsdalei also reduced acetone to 2-propanol at a conversion efficiency of 100 %. Further, we showed that C. ragsdalei uses an aldehyde oxidoreductase-like enzyme to reduce n-fatty acids to the aldehyde intermediates in a reaction that requires ferredoxin and exogenous CO.

Syngas fermentation to fuels is a technology on the verge of commercialization. Low cost of fermentation medium is important for process feasibility. The use of corn steep liquor (CSL) instead of yeast extract (YE) in Alkalibaculum bacchi strain CP15 bottle fermentations reduced the medium cost by 27% and produced 78% more ethanol. When continuous fermentation was performed in a 7-L fermentor, 6g/L ethanol was obtained in the YE and YE-free media. When CSL medium was used in continuous fermentation, the maximum produced concentrations of ethanol, n-propanol and n-butanol were 8 g/L, 6 g/L and 1 g/L, respectively. n-Propanol and n-butanol were not typical products of strain CP15. A 16S rRNA gene-based survey revealed a mixed culture in the fermentor dominated by A. bacchi strain CP15 (56%) and Clostridium propionicum (34%). The mixed culture presents an opportunity for higher alcohols production from syngas., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Higher alcohols such as n-butanol and n-hexanol have higher energy density than ethanol, are more compatible with current fuel infrastructure, and can be upgraded to jet and diesel fuels. Several organisms are known to convert syngas to ethanol, but very few can produce higher alcohols alone. As a potential solution, mixed culture fermentation between the syngas fermenting Alkalibaculum bacchi strain CP15 and propionic acid producer Clostridium propionicum was studied. The monoculture of CP15 produced only ethanol from syngas without initial addition of organic acids to the fermentation medium. However, the mixed culture produced ethanol, n-propanol and n-butanol from syngas. The addition of propionic acid, butyric acid and hexanoic acid to the mixed culture resulted in a 50% higher conversion efficiency of these acids to their respective alcohols compared to CP15 monoculture. These findings illustrate the great potential of mixed culture syngas fermentation in production of higher alcohols., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A taxonomic study employing a polyphasic approach was performed on a novel anaerobic bacterium isolated from natural gas production-water. The bacterium stained Gram-negative and consisted of non-motile, non-spore-forming, rod-shaped cells. Products of glucose or starch fermentation were ethanol, CO2, formate, acetate and H2. The predominant fatty acids were C16 : 0 ALDE and summed feature 3 comprising C16 : 1ω7c and/or C16 : 1ω6c. The DNA G+C content was 45.5 mol%. 16S rRNA gene sequence analysis demonstrated that the nearest phylogenetic neighbours of the novel strain were Acetivibrio multivorans DSM 6139(T) (98.5 %) and Proteiniclasticum ruminis JCM 14817(T) (95.4 %). The DNA-DNA hybridization value between the novel organism and Acetivibrio multivorans PeC1 DSM 6139(T) was determined to be only 30.2 %, demonstrating the separateness of the two species. Based on phylogenetic, phenotypic and chemotaxonomic evidence that clearly distinguished strain 232.1(T) from Proteiniclasticum ruminis and other close relatives, it is proposed that the novel isolate be classified as representing a novel species of a new genus within the family Clostridiaceae, Youngiibacter fragilis gen. nov., sp. nov. The type strain of the type species is 232.1(T) ( = ATCC BAA-2257(T) = DSM 24749(T)). In addition, Acetivibrio multivorans is proposed to be reclassified as Youngiibacter multivorans comb. nov.

Clostridium carboxidivorans P7 is one of three microbial catalysts capable of fermenting synthesis gas (mainly CO, CO(2) , and H(2) ) to produce the liquid biofuels ethanol and butanol. Gasification of feedstocks to produce synthesis gas (syngas), followed by microbial conversion to solvents, greatly expands the diversity of suitable feedstocks that can be used for biofuel production beyond commonly used food and energy crops to include agricultural, industrial, and municipal waste streams. C. carboxidivorans P7 uses a variation of the classic Wood-Ljungdahl pathway, identified through genome sequence-enabled approaches but only limited direct metabolic analyses. As a result, little is known about gene expression and enzyme activities during solvent production. In this study, we measured cell growth, gene expression, enzyme activity, and product formation in autotrophic batch cultures continuously fed a synthetic syngas mixture. These cultures exhibited an initial phase of growth, followed by acidogenesis that resulted in a reduction in pH. After cessation of growth, solventogenesis occurred, pH increased and maximum concentrations of acetate (41 mM), butyrate (1.4 mM), ethanol (61 mM), and butanol (7.1 mM) were achieved. Enzyme activities were highest during the growth phase, but expression of carbon monoxide dehydrogenase (CODH), Fe-only hydrogenases and two tandem bi-functional acetaldehyde/alcohol dehydrogenases were highest during specific stages of solventogenesis. Several amino acid substitutions between the tandem acetaldehyde/alcohol dehydrogenases and the differential expression of their genes suggest that they may have different roles during solvent formation. The data presented here provide a link between the expression of key enzymes, their measured activities and solvent production by C. carboxidivorans P7. This research also identifies potential targets for metabolic engineering efforts designed to produce higher amounts of ethanol or butanol from syngas. ., (Copyright © 2012 Wiley Periodicals, Inc.)

Fermentation of biomass derived synthesis gas to ethanol is a sustainable approach that can provide more usable energy and environmental benefits than food-based biofuels. The effects of various medium components on ethanol production by Clostridium ragsdalei utilizing syngas components (CO:CO(2)) were investigated, and corn steep liquor (CSL) was used as an inexpensive nutrient source for ethanol production by C. ragsdalei. Elimination of Mg(2+), NH(4) (+) and PO(4) (3-) decreased ethanol production from 38 to 3.7, 23 and 5.93 mM, respectively. Eliminating Na(+), Ca(2+), and K(+) or increasing Ca(2+), Mg(2+), K(+), NH(4) (+) and PO(4) (3-) concentrations had no effect on ethanol production. However, increased Na(+) concentration (171 mM) inhibited growth and ethanol production. Yeast extract (0.5 g l(-1)) and trace metals were necessary for growth of C. ragsdalei. CSL alone did not support growth and ethanol production. Nutrients limiting in CSL were trace metals, NH(4) (+) and reducing agent (Cys: cysteine sulfide). Supplementation of trace metals, NH(4) (+) and CyS to CSL (20 g l(-1), wet weight basis) yielded better growth and similar ethanol production as compared to control medium. Using 10 g l(-1), the nutritional limitation led to reduced ethanol production. Higher concentrations of CSL (50 and 100 g l(-1)) were inhibitory for cell growth and ethanol production. The CSL could replace yeast extract, vitamins and minerals (excluding NH(4) (+)). The optimized CSL medium produced 120 and 50 mM of ethanol and acetate, respectively. The CSL could provide as an inexpensive source of most of the nutrients required for the syngas fermentation, and thus could improve the economics of ethanol production from biomass derived synthesis gas by C. ragsdalei.

A novel anaerobic, moderately thermophilic, NaCl-requiring fermentative bacterium, strain OS1T, was isolated from oil production water collected from Alaska, USA. Cells were Gram-negative, non-motile, non-spore-forming rods (1.7-2.7×0.4-0.5 µm). The G+C content of the genomic DNA of strain OS1T was 46.6 mol%. The optimum temperature, pH and NaCl concentration for growth of strain OS1T were 55 °C, pH 7 and 10 g l(-1), respectively. The bacterium fermented D-fructose, D-glucose, maltose, D-mannose, α-ketoglutarate, L-glutamate, malonate, pyruvate, L-tartrate, L-asparagine, Casamino acids, L-cysteine, L-histidine, L-leucine, L-phenylalanine, L-serine, L-threonine, L-valine, inositol, inulin, tryptone and yeast extract. When grown on D-glucose, 3.86 mol hydrogen and 1.4 mol acetate were produced per mol substrate. Thiosulfate, sulfur and L-cystine were reduced to sulfide, and crotonate was reduced to butyrate with glucose as the electron donor. 16S rRNA gene sequence analysis indicated that strain OS1T was related to Anaerobaculum thermoterrenum (99.7 % similarity to the type strain), a member of the phylum Synergistetes. DNA-DNA hybridization between strain OS1T and A. thermoterrenum DSM 13490T yielded 68 % relatedness. Unlike A. thermoterrenum, strain OS1T fermented malonate, maltose, tryptone, L-leucine and L-phenylalanine, but not citrate, fumarate, lactate, L-malate, glycerol, pectin or starch. The major cellular fatty acid of strain OS1T was iso-C15:0 (91 % of the total). Strain OS1T also contained iso-C13:0 3-OH (3 %), which was absent from A. thermoterrenum, and iso-C13:0 (2 %), which was absent from Anaerobaculum mobile. On the basis of these results, strain OS1T represents a novel species of the genus Anaerobaculum, for which the name Anaerobaculum hydrogeniformans sp. nov. is proposed. The type strain is OS1T (=DSM 22491T=ATCC BAA-1850T). An emended description of the genus Anaerobaculum is also given.

Ethanol production from syngas using three moderately alkaliphilic strains of a novel genus and species Alkalibaculum bacchi CP11(T), CP13 and CP15 was investigated in 250 ml bottle fermentations containing 100ml of yeast extract medium at 37 °C and pH 8.0. Two commercial syngas mixtures (Syngas I: 20% CO, 15% CO(2), 5% H(2), 60% N(2)) and (Syngas II: 40% CO, 30% CO(2), 30% H(2)) were used. Syngas I and Syngas II represent gasified biomass and coal, respectively. The maximum ethanol concentration (1.7 g l(-1)) and yield from CO (76%) were obtained with strain CP15 and Syngas II after 360 h. CP15 produced over twofold more ethanol with Syngas I compared to strains CP11(T) and CP13. In addition, CP15 produced 18% and 71% more ethanol using Syngas II compared to strains CP11(T) and CP13, respectively. These results show that CP15 is the most promising for ethanol production because of its higher growth and ethanol production rates and yield compared to CP11(T) and CP13., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A polyphasic taxonomic study was performed on a strain of an unknown Gram-negative, non-motile, saccharolytic, facultatively anaerobic bacterium, strain OCF 7(T), isolated from anoxic freshwater sediment. The strain grew optimally at 22 °C and pH 7.5, and was able to grow under strictly anaerobic conditions. Major fermentation products from glucose metabolism were formate, acetate, ethanol and lactate. Comparative 16S rRNA gene sequence analysis indicated that strain OCF 7(T) was phylogenetically related to the type strain of Tolumonas auensis (97.2 % similarity) within the family Aeromonadaceae of the Gammaproteobacteria. However, OCF 7(T) did not produce toluene from phenylacetate, phenylalanine, phenoxyacetate, phenylsuccinate or phenylbutyrate in the presence of glucose. Phenol was not produced from tyrosine or phenoxyacetate in the presence of glucose. Dominant fatty acids of this micro-organism included C(16 : 0), C(18 : 1)ω7c and C(16 : 1)ω7c (and/or iso-C(15 : 0) 2-OH). Major polar lipids were phosphatidylglycerol and phosphatidylethanolamine, and the respiratory quinone was menaquinone MK-8. The genomic DNA G+C content of strain OCF 7(T) was 52.1 mol%. Based on phylogenetic and phenotypic evidence, strain OCF 7(T) should be classified as a representative of a novel species of Tolumonas, for which the name Tolumonas osonensis sp. nov. is proposed; the type strain is OCF 7(T) ( = DSM 22975(T) = ATCC BAA-1908(T)). An emended description of the genus Tolumonas is also given.

Although much newsprint is devoted to the subject of reducing the United States and other major developed countries dependence on their respective foreign energy sources; the most challenging issues for society is to provide long-term, sustainable energy sources to accommodate the global population as a whole. The projected population of planet Earth for the year 2050 is estimated to be in excess of 9 billion. With hydrocarbon-based energy becoming limiting it is unlikely that one type of energy will alone replace our dependence on this source. So-called "green" technologies that include solar, wind and wave powers are now being explored to reduce on traditional hydrocarbon-based fuel sources. The diverse and functional properties of microbes, and in particular anaerobes, are now being utilized in the production of biofuels and may provide one piece of the jigsaw for future energy requirements. Here we present some results of a screening program to identify and characterize a number of carbon monoxide oxidizing, ethanol-producing acetogenic anaerobes phylogenetically located within the Clostridiales., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effect of trace metal ions (Co²+, Cu²+, Fe²+, Mn²+, Mo⁶+, Ni²+, Zn²+, SeO₄⁻ and WO₄⁻) on growth and ethanol production by an ethanologenic acetogen, Clostridium ragsdalei was investigated in CO:CO₂-grown cells. A standard acetogen medium (ATCC medium no. 1754) was manipulated by varying the concentrations of trace metals in the media. Increasing the individual concentrations of Ni²+, Zn²+, SeO₄⁻ and WO₄⁻ from 0.84, 6.96, 1.06, and 0.68 μM in the standard trace metals solution to 8.4, 34.8, 5.3, and 6.8 μM, respectively, increased ethanol production from 35.73 mM under standard metals concentration to 176.5, 187.8, 54.4, and 72.3 mM, respectively. Nickel was necessary for growth of C. ragsdalei. Growth rate (μ) of C. ragsdalei improved from 0.34 to 0.49 (day⁻¹), and carbon monoxide dehydrogenase (CODH) and hydrogenase (H₂ase)-specific activities improved from 38.45 and 0.35 to 48.5 and 1.66 U/mg protein, respectively, at optimum concentration of Ni²+. At optimum concentrations of WO₄⁻ and SeO₄⁻, formate dehydrogenase (FDH) activity improved from 32.3 to 42.6 and 45.4 U/mg protein, respectively. Ethanol production and the activity of FDH reduced from 35 mM and 32.3 U/mg protein to 1.14 mM and 8.79 U/mg protein, respectively, upon elimination of WO₄⁻ from the medium. Although increased concentration of Zn²+ enhanced growth and ethanol production, the activities of CODH, FDH, H₂ase and alcohol dehydrogenase (ADH) were not affected by varying the Zn²+ concentration. Omitting Fe²+ from the medium decreased ethanol production from 35.7 to 6.30 mM and decreased activities of CODH, FDH, H₂ase and ADH from 38.5, 32.3, 0.35, and 0.68 U/mg protein to 9.07, 7.01, 0.10, and 0.24 U/mg protein, respectively. Ethanol production improved from 35 to 54 mM when Cu²+ was removed from the medium. The optimization of trace metals concentration in the fermentation medium improved enzyme activities (CODH, FDH, and H₂ase), growth and ethanol production by C. ragsdalei.

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.

Phenotypic and phylogenetic studies were performed on three strains of an acetogenic bacterium isolated from livestock-impacted soil. The bacterium stained Gram-negative and was a non-spore-forming rod that was motile by peritrichous flagella. The novel strains had an optimum pH for growth of 8.0-8.5 and utilized H₂ : CO₂, CO : CO₂, glucose, fructose, mannose, turanose, ribose, trimethylamine, pyruvate, methanol, ethanol, n-propanol and n-butanol as growth substrates. Acetate was produced from glucose. Acetate, CO₂ and ethanol were produced from CO : CO₂. 16S rRNA gene sequence analysis indicated that the novel strains formed a new subline in the family Eubacteriaceae (rRNA cluster XV) of the low G+C-containing Gram-positive bacteria of the class Clostridia. The DNA G+C base composition was 34 mol%. Cell wall analysis revealed the existence of a novel B-type peptidoglycan similar to the B2α-type (B4) configuration with a variation containing aspartic acid. Based on phylogenetic and phenotypic evidence, it is proposed that the new isolates represent a novel genus and species, for which the name Alkalibaculum bacchi gen. nov., sp. nov. is proposed. The type strain of the type species is CP11(T) (=ATCC BAA-1772(T)=DSM 22112(T)).

Corrosion of metallic oilfield pipelines by microorganisms is a costly but poorly understood phenomenon, with standard treatment methods targeting mesophilic sulfate-reducing bacteria. In assessing biocorrosion potential at an Alaskan North Slope oil field, we identified thermophilic hydrogen-using methanogens, syntrophic bacteria, peptide- and amino acid-fermenting bacteria, iron reducers, sulfur/thiosulfate-reducing bacteria, and sulfate-reducing archaea. These microbes can stimulate metal corrosion through production of organic acids, CO2, sulfur species, and via hydrogen oxidation and iron reduction, implicating many more types of organisms than are currently targeted. Micromolar quantities of putative anaerobic metabolites of C1-C4 n-alkanes in pipeline fluids were detected, implying that these low molecular weight hydrocarbons, routinely reinjected into reservoirs for oil recovery purposes, are biodegraded and can provide biocorrosive microbial communities with an important source of nutrients.

Ninety-six used personal care and topical OTC drug items collected from consumers in the USA were examined for the presence of microbial contaminants. Of the eye and face product type containing global preservative chemistries (i.e., acceptable for use in Japan without major restrictions), 55% yielded numbers of microorganisms in excess of 500 CFU/g (P < 0.1814). For the mascara products with global preservative chemistries, 79% yielded numbers of microorganisms in excess of 500 CFU/g (P < 0.024). Products containing global preservative chemistries accounted for 88% (n = 14) of the products that had microbial contents above 10(4) CFU/g (P < 0.001). Prominent contaminants were species of Staphylococcus, Pseudomonas, Klebsiella, Streptococcus, Lactobacillus, Bacillus, Corynebacterium, and yeast. In general, under the stress of consumer use, products preserved with global preservative chemistries did not maintain as adequate preservation as products with non-global preservatives.

A moderately acid-tolerant, malodorous bacterium, strain FP, was isolated from peat that had a pore water pH of c. 4.2. The 16S rRNA gene sequence of FP was closely related to that of acetogens Clostridium drakei, Clostridium scatologenes, and Clostridium carboxidivorans. The DNA-DNA reassociation values obtained with DNA from FP and that of these three acetogens approximated 80%, 64%, and 59%, respectively, indicating that FP was a new strain of C. drakei. FP had broad pH and temperature ranges (3.6-7.4 and 5-40 degrees C, respectively), and metabolized a wide range of substrates, including cellobiose, glucose, xylose, vanillate, ferulate, lactate, propanol, formate, H(2)-CO(2), and CO-CO(2). Acetate was the primary reduced end product, and substrate/product stoichiometries were indicative of acetogenesis at circumneutral pH. Butyrate and H(2) became significant products from glucose at low pH. FP tolerated and could consume moderate amounts of O(2). These results (1) demonstrate that peat can harbor acetogens with a broad substrate range and tolerance to transient exposure to O(2), and (2) confirm that C. drakei, the type strain of which was originally isolated from an acidic coal mine pond, occurs in moderately acidic habitats.

Phenotypic and phylogenetic studies were performed on a novel sulfate-reducing bacterium, strain D41(T), isolated as part of a methanogenic syntrophic culture from a gas condensate-contaminated aquifer undergoing intrinsic bioremediation. The bacterium was a Gram-negative, non-spore-forming, curved rod, motile by a single polar flagellum, which oxidized several alcohols incompletely, including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 3-methyl-1-butanol (isoamyl alcohol), ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, phenylethanol and benzyl alcohol. Additionally, the strain oxidized H(2)/CO(2), formate, lactate, pyruvate, maleate, malate and fumarate. Sulfate, thiosulfate and sulfite were used as electron acceptors. The DNA G+C content was 63 mol%. Based on phylogenetic and phenotypic evidence, the novel species Desulfovibrio carbinoliphilus sp. nov. is proposed. The type strain is D41(T) (=ATCC BAA-1241(T) =DSM 17524(T)).

Phenotypic and phylogenetic studies were performed on three isolates of an unknown Gram-negative, facultatively anaerobic, non-motile, yellow-pigmented, rod-shaped organism isolated from raw sewage. 16S rRNA gene sequence analysis indicated that these strains were members of the Bergeyella-Chryseobacterium-Riemerella branch of the family Flavobacteriaceae. The unknown bacterium was readily distinguished from reference strains by 16S rRNA gene sequencing and biochemical tests. The organism contained menaquinone MK-6 as the predominant respiratory quinone and had a DNA G+C content of 31 mol%. A most probable number-PCR approach was developed to detect, and estimate the numbers of, this organism. Untreated wastewater from one plant yielded an estimated count of 1.4 x 10(5) cells ml(-1), and untreated wastewater from a second plant yielded an estimated count of 1.4 x 10(4) cells ml(-1). Signal was not detected from treated effluent or from human stool specimens. On the basis of the results of the study presented, it is proposed that the unknown bacterium be classified in a novel genus Cloacibacterium, as Cloacibacterium normanense gen. nov., sp. nov., which is also the type species. The type strain of Cloacibacterium normanense is strain NRS1(T) (=CCUG 46293(T) = CIP 108613(T) = ATCC BAA-825(T) = DSM 15886(T)).

A novel solvent-producing, anaerobic clostridium, strain P7(T), was isolated from sediment from an agricultural settling lagoon after enrichment with CO as the substrate. The metabolism of this Gram-positive, motile, spore-forming rod was primarily acetogenic. Acetate, ethanol, butyrate and butanol were the end-products of metabolism. Strain P7(T) grew on CO, H(2)/CO(2), glucose, galactose, fructose, xylose, mannose, cellobiose, trehalose, cellulose, starch, pectin, citrate, glycerol, ethanol, propanol, 2-propanol, butanol, glutamate, aspartate, alanine, histidine, asparagine, serine, betaine, choline and syringate as sole substrates. Growth was not supported by methanol, formate, D-arabinose, fucose, lactose, melibiose, amygdalin, gluconate, lactate, malate, arginine, glutamine or vanillate. Nitrate reduction, production of indole, gelatin hydrolysis and aesculin hydrolysis were not observed. Analysis of the 16S rRNA gene sequence of the isolate showed that it was closely related to Clostridium scatologenes ATCC 25775(T) (99.7% sequence similarity) and clostridial strain SL1(T) (99.8% sequence similarity). Strain SL1 had been classified as a strain of C. scatologenes. However, DNA-DNA reassociation analysis showed that both strain P7(T) and strain SL1 represented novel clostridial species. It is proposed that strain P7(T) (=ATCC BAA-624(T)=DSM 15243(T)) be classified as the type strain of Clostridium carboxidivorans sp. nov. and that strain SL1(T) (=ATCC BAA-623(T)=DSM 12750(T)) be reclassified as the type strain of Clostridium drakei sp. nov.

Analyses of 165 rRNA gene sequences, restriction endonuclease digestion fingerprints of 16S-23S intergenic regions, DNA base compositions, fatty-acid profiles, cell-wall chemistry, cell physiology and fermentation end-product composition, along with other biochemical and phenotypic properties, supported the view that Trichococcus flocculiformis EchtT (DSM 2094T), Lactosphaera pasteurii KoTa2T (DSM 2381T), Ruminococcus palustris Z-7189T (DSM 9172T) and an isolate named 'Carnococcus allantoicus' NDP were all very similar and should be merged into a single genus. Detailed characterization of strains Ben 77, Ben 200 and Ben 201 described previously as 'Nostocoida limicola' I, a filamentous bacterium which causes bulking in activated sludge systems, revealed that these strains also belonged to the same genus as T. flocculiformis EchtT, L. pasteurii KoTa2T, R. palustris Z-7189T and 'C allantoicus' NDP. In fact, their shared properties suggested that these strains all belonged to a single species. However, DNA-DNA hybridization data indicated that T. flocculiformis EchtT, all of the 'N. limicola' I isolates and 'C allantoicus' NDP belonged to the same species, whereas L. pasteurii KoTa2T, R. palustris Z-7189T and two new isolates, 37AN3*T and 45AN2, represented three distinct species within the same genus. The priority of the genus name Trichococcus is established and since its validation predates the description of the genus Lactosphaera this name should take precedence. Under certain culture conditions, all of the strains mentioned above could produce chains of cocci. Furthermore, the morphology of T. flocculiformis EchtT could change to a non-filamentous form on certain media. This study proposes that the above strains be reclassified as members of the genus Trichococcus as four species, namely Trichococcus flocculiformis emend. (type strain EchtT = DSM 2094T), Trichococcus pasteurii comb. nov. (type strain KoTa2T = DSM 2381T = ATCC 35945T), Trichococcus collinsii sp. nov. (type strain 37AN3*T = DSM 14526T = ATCC BAA-296T, and Trichococcus palustris comb. nov. (type strain Z-7189T = DSM 9172T).

Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in pore water (A. Vairavamurthy and M. Mopper, Anal. Chim. Acta 78:363-370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples. The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample. CoM was not adsorbed to sediments. The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 +/- 0.07 fmol of CoM/cell. Environmental samples contained an average of 0.41 +/- 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-(N)-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass.

Three strains, designated VS-751T, VS-511 and VS-732, of a strictly anaerobic, moderately halophilic, Gram-negative, rod-shaped bacterium were isolated from a highly saline (15-20%) brine from an oil reservoir in central Oklahoma, USA. The optimal concentration of NaCl for growth of these three strains was 2 M (12%), and the strains also grew in the presence of an additional 1 M MgCl2. The strains were mesophilic and grew at a pH range of 6-8. Carbohydrates used by all three strains included glucose, fructose, arabinose, galactose, maltose, mannose, cellobiose, sucrose and inulin. Glucose fermentation products included ethanol, acetate, H2 and CO2, with formate produced by two of the three strains. Differences were noted among strains in the optimal temperature and pH for growth, the maximum and minimum NaCl concentration that supported growth, substrate utilization and cellular fatty acid composition. Despite the phenotypic differences among the three strains, analysis of the 16S rRNA gene sequences and DNA-DNA hybridizations showed that these three strains were members of the same genospecies which belonged to the genus Haloanaerobium. The phenotypic and genotypic characteristics of strains VS-751T, VS-511 and VS-732 are different from those of previously described species of Haloanaerobium. It is proposed that strain VS-751T (ATCC 700103T) be established as the type strain of a new species, Haloanaerobium kushneri.

The metabolism of cresols under sulfate-reducing conditions was investigated in Desulfotomaculum sp. strain Groll. This strain grows on a variety of aromatic compounds, including para- and meta- but not ortho-cresol. Degradation of p-cresol proceeded by oxidation reactions of the methyl group to yield p-hydroxybenzoate, which was then dehydroxylated to benzoate. The aromatic intermediates expected for this pathway, p-hydroxybenzyl alcohol, p-hydroxybenzaldehyde, p-hydroxybenzoate, and benzoate, were readily metabolized by strain Groll. Utilization of these intermediates generally preceded and inhibited the degradation of p-cresol. p-Hydroxybenzoate and benzoate were detected in culture fluid as metabolites of p-cresol. p-Hydroxybenzaldehyde and p-hydroxybenzoate were detected in cultures degrading p-hydroxybenzyl alcohol. Enzyme activities responsible for utilization of p- and m-cresol, induced by growth on the respective cresol, were detected in cell-free extracts of strain Groll. The compounds detected in culture fluids and the enzyme activities detected in cell-free extracts indicate that the pathways for the degradation of p- and m-cresol converge on benzoate, followed by metabolism to benzoyl-coenzyme A (CoA). Strain Groll can utilize both cresol isomers under sulfate-reducing conditions by similar reactions, but the enzyme activities catalyzing these transformations of the two isomers appear distinct.

Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 +/- 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the delta-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

Two new diterpenes possessing a 9-methyladeninium substituent, agelasines H (5) and I (6), along with five related known ones, 1-4 and 7, were isolated from a sponge, Agelas sp. The structures of the new compounds were determined from spectral data. Compounds 2-4, 6, and 7 exhibited varying degrees of antimicrobial activity.

Four new imidazole alkaloids, 2-5, along with the known isonaamidine B (1) were isolated from a Pacific sponge, Leucetta cf. chagosensis, collected from Yap, Federated States of Micronesia. Among these, 4 and 5 are zinc complexes derived from isonaamidine B and isonaamidine D. The structures of the new compounds were elucidated from spectral data. Isonaamidine D (3) showed weakly antifungal activity against Aspergillus niger with MIC = 100 micrograms/mL.

Two new nitrotyramine derivatives, 1 and 2, along with five known aromatic compounds, were isolated from the culture broth of a facultatively anaerobic, halophilic bacterium isolated from a sediment from the Great Salt Plains, Alfalfa County, Oklahoma. The structures of the new compounds were determined from spectral data and were confirmed by synthesis from tyramine hydrochloride. Compound 1 showed cytotoxicity against the murine leukemia P-388 cell line (IC50 3 micrograms/ml).

A new cellulolytic anaerobic clostridium was isolated from the intestinal tract of pigs. The single isolate was a gram-positive, motile rod, formed terminal to subterminal swollen sporangia, and required a fermentable carbohydrate for growth. Cellulose, cellobiose, maltose, starch, and glycogen supported growth, but glucose and fructose did not. The major end products from the fermentation of cellobiose were butyrate and formate; minor amounts of hydrogen and ethanol were also formed. Ruminal fluid (15%) or yeast extract (1%) was required for good growth. The optimum temperature for growth was 39 to 42 degrees C, and the optimum pH was 6.8 to 7.2. Cell lysis occurred rapidly once stationary growth was reached. A 16S rRNA sequence analysis showed that the strain was related to a group of gram-positive anaerobes that includes Clostridium oroticum and the cellulolytic species Clostridium polysaccharolyticum and Clostridium populeti. The DNA base composition of the isolate is 38 mol% G + C. We propose the name Clostridium herbivorans for this organism; strain 54408 (= ATCC 49925) is the type strain.

A strictly anaerobic, moderately halophilic, gram-negative bacterium was isolated from a highly saline oil field brine. The bacterium was a non-spore-forming, nonmotile rod, appearing singly, in pairs, or occasionally as long chains, and measured 0.3 to 0.4 by 2.6 to 4 microns. The bacterium had a specific requirement for NaCl and grew at NaCl concentrations of between 6 and 24%, with optimal growth at 9% NaCl. The isolate grew at temperatures of between 22 and 51 degrees C and pH values of between 5.6 and 8.0. The doubling time in a complex medium containing 10% NaCl was 9 h. Growth was inhibited by chloramphenicol, tetracycline, and penicillin but not by cycloheximide or azide. Fermentable substrates were predominantly carbohydrates. The end products of glucose fermentation were acetate, ethanol, CO2, and H2. The major components of the cellular fatty acids were C14:0, C16:0, C16:1, and C17:0 cyc acids. The DNA base composition of the isolate was 34 mol% G+C. Oligonucleotide catalog and sequence analyses of the 16S rRNA showed that strain VS-752T was most closely related to Haloanaerobium praevalens GSLT (ATCC 33744), the sole member of the genus Haloanaerobium. We propose that strain VS-752 (ATCC 51327) be established as the type strain of a new species, Haloanaerobium salsugo, in the genus Haloanaerobium.

Arhodomonas aquaeolei gen. nov., sp. nov., isolated from a petroleum reservoir production fluid, is described. The single isolate was an obligately halophilic, aerobic, gram-negative, oval rod-shaped bacterium that was actively motile by means of a single polar flagellum. It was catalase and oxidase positive. The isolate had a specific requirement for NaCl; growth occurred at NaCl concentrations between 6 and 20%, and optimal growth occurred in the presence of 15% NaCl. This species metabolized primarily organic acids and required biotin for growth. The name Arhodomonas is proposed for the new genus, which was placed in the gamma subclass of the Proteobacteria on the basis of the results of a 16S rRNA sequence analysis. Although A. aquaeolei is most closely related to purple sulfur bacteria (the genera Ectothiorhodospira and Chromatium), it is not a phototrophic microorganism, which is consistent with its isolation from a subterranean environment. The major components of its cellular fatty acids were C16:0, C18:1, C19:0, C16:1, and C18:0 acids. The DNA base composition of the type strain is 67 mol% G+C. The type and only strain is strain HA-1 (= ATCC 49307).

Clostridium ljungdahlii sp. nov. strain ATCC 49587T (T = type strain) was isolated from chicken yard waste for its ability to produce ethanol from synthesis gas. This gram-positive, motile, sporeforming rod's metabolism was primarily acetogenic. C. ljungdahlii grew with carbon monoxide, hydrogen and carbon dioxide, ethanol, pyruvate, arabinose, xylose, fructose, or glucose. Methanol, ferulic acid, lactate, galactose, and mannose did not support growth. The G+C content was 22 to 23 mol%. C. ljungdahlii is the first acetogen in clostridial 23S rRNA homology group I.

Methanomicrobium mobile requires a heat-stable factor present in ruminal fluid and in boiled cell extract from Methanobacterium thermoautotrophicum for growth. By comparing the growth of M. mobile with boiled cell extract with that observed with various methanogenic cofactors, we found that 7-mercaptoheptanoylthreonine phosphate (HS-HTP) supported sustained growth of M. mobile, at an optimal concentration of 100 microM. No derivatives or possible biosynthetic precursors of HS-HTP could replace HS-HTP as the sole source of growth factor. Results suggest that the growth requirement might be satisfied by 7-mercaptoheptanoic acid plus a second, unidentified heat-stable factor.

The 5S rRNA sequences of eubacteria and mycoplasmas have been analyzed and a phylogenetic tree constructed. We determined the sequences of 5S rRNA from Clostridium innocuum, Acholeplasma laidlawii, Acholeplasma modicum, Anaeroplasma bactoclasticum, Anaeroplasma abactoclasticum, Ureaplasma urealyticum, Mycoplasma mycoides mycoides, Mycoplasma pneumoniae, and Mycoplasma gallisepticum. Analysis of these and published sequences shows that mycoplasmas form a coherent phylogenetic group that, with C. innocuum, arose as a branch of the low G+C Gram-positive tree, near the lactobacilli and streptococci. The initial event in mycoplasma phylogeny was formation of the Acholeplasma branch; hence, loss of cell wall probably occurred at the time of genome reduction to approximately to 1000 MDa. A subsequent branch produced the Spiroplasma. This branch appears to have been the origin of sterol-requiring mycoplasmas. During development of the Spiroplasma branch there were several independent genome reductions, each to approximately 500 MDa, resulting in Mycoplasma and Ureaplasma species. Mycoplasmas, particularly species with the smallest genomes, have high mutation rates, suggesting that they are in a state of rapid evolution.

Acetate synthesis from CO2 by Acetobacterium woodii may occur as in homoacetate-fermenting clostridia, as indicated by high levels of enzymes of the tetrahydrofolate pathway and by pyruvate-dependent formation of acetate from methyl-B12 and methyltetrahydrofolate.

A formate-requiring auxotroph of Methanobacterium thermoautotrophicum Marburg was isolated after hydroxylamine mutagenesis and bacitracin selection. The requirement for formate is unique and specific; combined pools of other volatile fatty acids, amino acids, vitamins, and nitrogen bases did not substitute for formate. Compared with those of the wild type, cell extracts of the formate auxotroph were deficient in formate dehydrogenase activity, but cells of all of the strains examined catalyzed a formate-carbon dioxide exchange activity. All of the strains examined took up a small amount (200 to 260 mumol/liter) of formate (3 mM) added to medium. The results of the study of this novel auxotroph indicate a role for formate in biosynthetic reactions in this methanogen. Moreover, because methanogenesis from H2-CO2 is not impaired in the mutant, free formate is not an intermediate in the reduction of CO2 to CH4.

A defined medium was developed for Methanomicrobium mobile BP. M. mobile required acetate for growth; the optimal concentration was 30 mM. Other requirements and their optimal concentrations included isobutyrate (0.65 mM), isovalerate (0.73 mM), and 2-methylbutyrate (1.5 mM). The appropriate branched-chain amino acids did not substitute for these branched-chain fatty acids. M. mobile required tryptophan at an optimal concentration of 24 microM. Indole substituted for tryptophan, but the possible precursor compounds shikimic acid and anthranilic acid and the degradation compound skatole did not. Vitamin requirements and their optimal concentrations included pyridoxine (0.49 microM), thiamine (0.15 microM), biotin (0.04 microM), and vitamin B12 (0.04 microM); p-aminobenzoic acid (0.18 microM) was required for optimal growth, but folic acid did not replace p-aminobenzoic acid. M. mobile required an unidentified growth factor found in ruminal fluid or extracts of Methanobacterium thermoautotrophicum for growth. M. mobile has a complex nutrition compared with that of other methanogens, but not an unusual nutrition in the context of organisms from the ruminal ecosystem.