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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.)

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.)

"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.

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.

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.

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.

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.

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 °C, respectively), and metabolized a wide range of substrates, including cellobiose, glucose, xylose, vanillate, ferulate, lactate, propanol, formate, H2–CO2, and CO–CO2. Acetate was the primary reduced end product, and substrate/product stoichiometries were indicative of acetogenesis at circumneutral pH. Butyrate and H2 became significant products from glucose at low pH. FP tolerated and could consume moderate amounts of O2. These results (1) demonstrate that peat can harbor acetogens with a broad substrate range and tolerance to transient exposure to O2, 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. [ABSTRACT FROM AUTHOR]