Clostridium Ragsdalei Research Articles

(S)-2-Hydroxyisovalerate Production from d-Xylose with CO-Converting Clostridium ragsdalei

Clostridium ragsdalei was found to produce (S)-2-hydroxyisovalerate (2-HIV) as a novel product in addition to acetate, ethanol, and d-2,3-butanediol in heterotrophic (d-xylose), autotrophic (CO), and mixotrophic (d-xylose + CO) conditions. Mixotrophic batch processes in stirred-tank bioreactors with continuous gassing resulted in improved production of this alpha-hydroxy acid compared to batch processes solely with either d-xylose or CO. The maximal CO uptake rate was considerably reduced in mixotrophic compared to autotrophic processes, resulting in a concomitant decreased total CO2 production. Simultaneous conversion of 9.5 g L−1 d-xylose and 320 mmol CO enabled the production of 1.8 g L−1 2-HIV in addition to 1.1 g L−1 d-2,3-butanediol, 2.0 g L−1 ethanol, and 1.8 g L−1 acetate. With reduced initial d-xylose (3.1 g L−1), l-valine production started when d-xylose was depleted, reaching a maximum of 0.4 g L−1 l-valine. Using l-arabinose or d-glucose instead of d-xylose in mixotrophic batch processes reduced the 2-HIV production by C. ragsdalei. Considerable amounts of meso-2,3-butanediol (0.9–1.3 g L−1) were produced instead, which was not observed with d-xylose. The monomer 2-HIV can form polyesters that make the molecule attractive for application as bioplastic (polyhydroxyalkanoates) or new composite material.

Simultaneous Formate and Syngas Conversion Boosts Growth and Product Formation by Clostridium ragsdalei.

Electrocatalytic CO2 reduction to CO and formate can be coupled to gas fermentation with anaerobic microorganisms. In combination with a competing hydrogen evolution reaction in the cathode in aqueous medium, the in situ, electrocatalytic produced syngas components can be converted by an acetogenic bacterium, such as Clostridium ragsdalei, into acetate, ethanol, and 2,3-butanediol. In order to study the simultaneous conversion of CO, CO2, and formate together with H2 with C. ragsdalei, fed-batch processes were conducted with continuous gassing using a fully controlled stirred tank bioreactor. Formate was added continuously, and various initial CO partial pressures (pCO0) were applied. C. ragsdalei utilized CO as the favored substrate for growth and product formation, but below a partial pressure of 30 mbar CO in the bioreactor, a simultaneous CO2/H2 conversion was observed. Formate supplementation enabled 20-50% higher growth rates independent of the partial pressure of CO and improved the acetate and 2,3-butanediol production. Finally, the reaction conditions were identified, allowing the parallel CO, CO2, formate, and H2 consumption with C. ragsdalei at a limiting CO partial pressure below 30 mbar, pH 5.5, n = 1200 min-1, and T = 32 °C. Thus, improved carbon and electron conversion is possible to establish efficient and sustainable processes with acetogenic bacteria, as shown in the example of C. ragsdalei.

Continuous sulfide supply enhanced autotrophic production of alcohols with Clostridium ragsdalei

Autotrophic syngas fermentation with clostridia enables the conversion of CO, CO2, and H2 into organic acids and alcohols. The batch process performance of Clostridium ragsdalei was studied in fully controlled and continuously gassed (600 mbar CO, 200 mbar H2, 200 mbar CO2) stirred-tank bioreactors. The final ethanol concentration varied as function of the reaction conditions. Decreasing the pH from pH 6.0–5.5 at a temperature of 37 °C increased the ethanol concentration from 2.33 g L−1 to 3.95 g L−1, whereas lowering the temperature from 37 to 32 °C at constant pH 6.0 resulted in a final ethanol concentration of 5.34 g L−1 after 5 days of batch operation. The sulphur availability was monitored by measuring the cysteine concentration in the medium and the H2S fraction in the exhaust gas. It was found that most of the initially added sulphur was stripped out within the first day of the batch process (first half of the exponential growth phase). A continuous sodium sulfide feed allowed ethanol concentrations to increase more than threefold to 7.67 g L−1 and the alcohol-to-acetate ratio to increase 43-fold to 17.71 g g−1.Graphical

Bioethanol production from C1 gases using alternative media by syngas fermentation

The main aim of the present study was to evaluate the effects of the presence or absence of trace elements and yeast extract on growth and ethanol production by Clostridium ragsdalei grown on C1 gas dominated syngas (60% CO, 10% CO2, 10% CH4, 10% H2, 10% N2). Comparison of whey powder and glucose syrup as low-cost substitutes helped develop a more cost-effective medium formulation to produce ethanol from syngas with significantly higher yields, achieving up to a 3.8-fold improvement with co-utilisation of syngas and glucose syrup. In addition, the results provided further insights into medium manipulation and possible modifications to decrease total costs for increased ethanol production by solventogenic acetogens via syngas fermentation by achieving a 15.6% reduction in the cost of fermentation medium.

Selective butanol production from carbon monoxide by an enriched anaerobic culture

An anaerobic mixed culture able to grow on pure carbon monoxide (CO) as well as syngas (CO, CO2 and H2), that produced unusual high concentrations of butanol, was enriched in a bioreactor with intermittent CO gas feeding. At pH 6.2, it mainly produced acids, generally acetic and butyric acid. After adaptation, under stress conditions of CO exposure at a partial pressure of 1.8 bar and low pH (e.g., 5.7), the enrichment accumulated ethanol, but also high amounts of butanol, up to 6.8 g/L, never reported before, with a high butanol/butyric acid molar ratio of 12.6, highlighting the high level of acid to alcohol conversion. At the end of the assay, both the acetic acid and ethanol concentrations decreased, with concomitant butyric acid production, suggesting C2 to C4 acid bioconversion, though this was not a dominant bioconversion process. The reverse reaction of ethanol oxidation to acetic acid was observed in the presence of CO2 produced during CO fermentation. Interestingly, butanol oxidation with simultaneous butyric acid production occurred upon production of CO2 from CO, which has to the best of our knowledge never been reported. Although the sludge inoculum contained a few known solventogenic Clostridia, the relative taxonomic abundance of the enriched sludge was diverse in Clostridia and Bacilli classes, containing known solventogens, e.g., Clostridium ljungdhalii, Clostridium ragsdalei and Clostridium coskatii, confirming their efficient enrichment. The relative abundance of unassigned Clostridium species amounted to 27% with presumably novel ethanol/butanol producers.

Bioethanol Production Through Syngas Fermentation by a Novel Immobilized Bioreactor Using Clostridium Ragsdalei

Global energy demand has been escalating creating ever increasing pressure on climate crisis caused by fossil-based fuels. Humankind is now desperately in need of alternative and sustainable energy sources. Therefore, biofuels provide promising solution.
 Amongst the various biofuels, bioethanol from syngas, which is a mixture of, mostly, CO, CO2, N2, H2, and CH4 gases has been drawing increasing attention recently. Regarding this, the conversion of syngas to bioethanol, an alternative biofuel to fossil fuels, is considered a promising approach to reduce the negative effects of global warming by reducing greenhouse gas emissions.
 In this study, a novel immobilized cell bioreactor, where Clostridium ragsdalei was grown, was designed and used to achieve an efficient production of ethanol regarding volumetric production. For this purpose, a 300 mL immobilized reactor filled with ceramic balls as immobilization material was set and operated at 30oC throughout the study where CO gas as the main substrate was fed at rate of 6 ml/min continuously. Results showed ethanol and acetic acid concentrations reaching up to 1.4 g/L and 0.2 g/L, respectively, after 600h with a volumetric production rate of 0,0023g ethanol/L/h. We believe, the ceramic ball was used for bioethanol production for syngas for the first time.

Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum

Clostridium autoethanogenum and Clostridium ljungdahlii are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide as sole carbon source. While exact nutritional requirements have not been reported, we observed that for growth, the addition of vitamins to media already containing yeast extract was required, an indication that these are fastidious microorganisms. Elimination of complex components and individual vitamins from the medium revealed that the only organic compounds required for growth were pantothenate, biotin and thiamine. Analysis of the genome sequences revealed that three genes were missing from pantothenate and thiamine biosynthetic pathways, and five genes were absent from the pathway for biotin biosynthesis. Prototrophy in C. autoethanogenum and C. ljungdahlii for pantothenate was obtained by the introduction of plasmids carrying the heterologous gene clusters panBCD from Clostridium acetobutylicum, and for thiamine by the introduction of the thiC-purF operon from Clostridium ragsdalei. Integration of panBCD into the chromosome through allele-coupled exchange also conveyed prototrophy. C. autoethanogenum was converted to biotin prototrophy with gene sets bioBDF and bioHCA from Desulfotomaculum nigrificans strain CO-1-SRB, on plasmid and integrated in the chromosome. The genes could be used as auxotrophic selection markers in recombinant DNA technology. Additionally, transformation with a subset of the genes for pantothenate biosynthesis extended selection options with the pantothenate precursors pantolactone and/or beta-alanine. Similarly, growth was obtained with the biotin precursor pimelate combined with genes bioYDA from C. acetobutylicum. The work raises questions whether alternative steps exist in biotin and thiamine biosynthesis pathways in these acetogens.

Enhanced ethanol production from syngas by Clostridium ragsdalei in continuous stirred tank reactor using medium with poultry litter biochar

Microorganisms used in syngas fermentation require nutrients to grow and convert syngas (CO, H2 and CO2) into various products. Many of the essential nutrients can be provided by biochar. Poultry litter biochar (PLBC) contains minerals and trace metals and has a high pH buffering capacity, making it suitable as a nutrient supplement. The effects of PLBC loadings from 1 to 20 g L−1 on syngas fermentation were determined in 250 ml bottle assays. Results showed that 10 and 20 g L−1 PLBC significantly increased ethanol production compared to standard yeast extract (YE) medium. Fermentations in a 3L continuous stirred tank reactor (CSTR) with 10 g L−1 PLBC with and without 4-morpholineethanesulfonic acid (MES) showed 64% and 36% more ethanol production, respectively, than standard medium. The acetic acid accumulated at the beginning of fermentation was completely converted to ethanol in all media tested in the CSTR. These results demonstrate the feasibility of using PLBC medium without costly MES in the CSTR to enhance ethanol production from syngas for potential use at commercial scale.

Enhanced ethanol production by Clostridium ragsdalei from syngas by incorporating biochar in the fermentation medium

Biochar contains minerals and metals that can serve as nutrients for acetogens to produce ethanol via syngas fermentation. In this study, four fermentation media containing biochar from switchgrass (SGBC), forage sorghum (FSBC), red cedar (RCBC) and poultry litter (PLBC) were compared with standard yeast extract (YE) medium for syngas fermentation using Clostridium ragsdalei. Fermentations were performed in 250mL bottle reactors at 150rpm and 37°C with syngas containing CO:H2:CO2 (40:30:30) by volume. Results showed that media containing RCBC and PLBC improved ethanol production by 16.3% and 58.9%, respectively, compared to YE medium. C. ragsdalei consumed 69% more H2 and 40% more CO in PLBC medium compared to YE medium. However, no enhancement of ethanol production was observed in SGBC and FSBC media. The highest release of Na, K, Ca, Mg, S and P was from PLBC, which was considered to contribute in enhancement of ethanol production.

High efficient ethanol and VFA production from gas fermentation: Effect of acetate, gas and inoculum microbial composition

In bioindustry, syngas fermentation is a promising technology for biofuel production without the use of plant biomass as sugar-based feedstock. The aim of this study was to identify optimal conditions for high efficient ethanol and volatile fatty acids (VFA) production from synthetic gas fermentation. Therefore, the effect of different gases (pure CO, H2, and a synthetic syngas mixture), media (acetate medium and acetate-free medium), and biocatalyst (pure and mixed culture) were studied. Acetate was the most dominant product independent on inoculum type. The maximum concentration of volatile fatty acids and ethanol was achieved by the pure culture (Clostridium ragsdalei). Depending on the headspace gas composition, VFA concentrations were up to 300% higher after fermentation with Clostridium ragsdalei compared to fermentation with mixed culture. The preferred gas composition with respect to highest VFA concentration was pure CO (100%) regardless of microbial composition of the inoculum and media composition. The addition of acetate had a negative impact on the VFA formation which was depending on the initial gas composition in head space.

Continuous Ethanol Production from Synthesis Gas by Clostridium ragsdalei in a Trickle-Bed Reactor

A trickle-bed reactor (TBR) when operated in a trickle flow regime reduces liquid resistance to mass transfer because a very thin liquid film is in contact with the gas phase and results in improved gas–liquid mass transfer compared to continuous stirred tank reactors (CSTRs). In the present study, continuous syngas fermentation was performed in a 1-L TBR for ethanol production by Clostridium ragsdalei. The effects of dilution and gas flow rates on product formation, productivity, gas uptakes and conversion efficiencies were examined. Results showed that CO and H2 conversion efficiencies reached over 90% when the gas flow rate was maintained between 1.5 and 2.8 standard cubic centimeters per minute (sccm) at a dilution rate of 0.009 h−1. A 4:1 molar ratio of ethanol to acetic acid was achieved in co-current continuous mode with both gas and liquid entered the TBR at the top and exited from the bottom at dilution rates of 0.009 and 0.012 h−1, and gas flow rates from 10.1 to 12.2 sccm and 15.9 to 18.9 sccm, respectively.

Ethanol production during semi-continuous syngas fermentation in a trickle bed reactor using Clostridium ragsdalei

An efficient syngas fermentation bioreactor provides a mass transfer capability that matches the intrinsic kinetics of the microorganism to obtain high gas conversion efficiency and productivity. In this study, mass transfer and gas utilization efficiencies of a trickle bed reactor during syngas fermentation by Clostridium ragsdalei were evaluated at various gas and liquid flow rates. Fermentations were performed using a syngas mixture of 38% CO, 28.5% CO2, 28.5% H2 and 5% N2, by volume. Results showed that increasing the gas flow rate from 2.3 to 4.6sccm increased the CO uptake rate by 76% and decreased the H2 uptake rate by 51% up to Run R6. Biofilm formation after R6 increased cells activity with over threefold increase in H2 uptake rate. At 1662h, the final ethanol and acetic acid concentrations were 5.7 and 12.3g/L, respectively, at 200ml/min of liquid flow rate and 4.6sccm gas flow rate.

Method for Design of Production Medium for Fermentation of Synthesis Gas to Ethanol by Acetogenic Bacteria

Abstract. Biomass and waste materials are converted to ethanol by gasification followed by fermentation of the synthesis gas using acetogenic bacteria such as Clostridium ragsdalei. Fuel ethanol is a high-volume, low-cost commodity, so capital and operating expenses must be very low to achieve profitable operation. The fermentation medium supplies minerals, metals, and organic co-factors needed to build cell biomass and the functional enzyme platform that conducts the conversion reactions. Research directed toward commercial deployment of synthesis gas (syngas) fermentation processes must consider cost and availability of medium components, and use economically viable medium. A method based on elemental accounting of cell composition was developed to assess the cost and function of fermentation media and their potential for commercial use for syngas fermentation to produce ethanol using C. ragsdalei. Cost of medium was reduced from $9.87 L -1 to $0.26 L -1 . Additional cost reduction can be achieved by eliminating components that are not required, using commercial components purchased at bulk commodity prices, using H 2 S and NH 3 from the syngas production in lieu of purchased sodium sulfide and ammonium chloride, and retaining the acetate buffer from ethanol distillation. These savings can reduce the medium cost below $0.01 L -1 .

Improved conversion efficiencies for n-fatty acid reduction to primary alcohols by the solventogenic acetogen "Clostridium ragsdalei".

"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.

Influence of switchgrass generated producer gas pre-adaptation on growth and product distribution of Clostridium ragsdalei

Gasification-fermentation is a thermo chemical-biological process for the production of fuels and chemicals. Producer gas cleanup is a major issue that must be addressed for integration of these platforms. Pre-adaptation of producer gas fermenting microbes to gas impurities has improved tolerances to impurities and production of alcohols in certain bacteria. In this research, the effect of switchgrass generated producer gas was studied with adapted and unadapted cultures of C. ragsdalei and compared to fermentations with a control of clean custom producer gas. Results indicated no inhibition to microbial growth with unadapted cells and final cell mass concentrations were 22% higher when cells were exposed to switchgrass-based producer gas compared to control. The ethanol productivity with adapted cells was 1.9 and 2.8 times higher than unadapted and control treatments, respectively. Similarly, the ethanol yield (YETOH/X) of C. ragsdalei adapted to producer gas was 119% more than the control and 35% greater than the unadapted cells used in this study. The presence of switchgrass-based producer gas also induced metabolic shifts resulting in reduction of acetic acid to ethanol that increased ethanol to acetate ratios from 0.7 g/g in control to 4.9 g/g with unadapted cells and 13.7 g/g with adapted cells. Isopropanol was also observed as a product when switchgrass generated producer gas was used. We conclude that cultural adaptation of C. ragsdalei to biomass generated producer gas during preculture stages could be used as an important strategy to enhance ethanol yields for integrating gasification and fermentation platforms using C. ragsdalei.

Development of low cost medium for ethanol production from syngas by Clostridium ragsdalei

The development of a low cost medium for ethanol production is critical for process feasibility. Ten media were formulated for Clostridium ragsdalei by reduction, elimination and replacement of expensive nutrients. Cost analysis and effects of medium components on growth and product formation were investigated. Fermentations were performed in 250 mL bottles using syngas (20% CO, 15% CO2, 5% H2 and 60% N2). The standard medium M1 cost is $9.83/L, of which 93% is attributed to morpholinoethane sulfonic acid (MES) buffer. Statistical analysis of the results showed that MES removal did not affect cell growth and ethanol production (P>0.05). Based on cells' elemental composition, a minimal mineral concentration medium M7 was formulated, which provided 29% higher ethanol yield from CO at 3% of the cost compared to medium M1. Ethanol yield from CO in the completely defined medium M9 was 36% higher than while at 5% the cost of medium M1.

Carbon dioxide conversion to fuels and chemicals using a hybrid green process

A unique hybrid technology that uses renewable hydrogen (H2) and carbon dioxide (CO2) sequestered from large point sources, to produce fuels and chemicals has been proposed and tested. The primary objective of this research was to determine the feasibility of using two acetogenic bacteria to metabolize H2 and CO2 for the production of ethanol. Three experiments were conducted in small scale reactors to select a bacterium, feed gas composition and nutrient medium source to produce ethanol. The results indicated that Clostridium carboxidivorans produced 33% more ethanol and 66% less acetic acid compared to Clostridium ragsdalei, making C. carboxidivorans the better candidate for ethanol production. Furthermore, the removal of morpholinoethanesulfonic acid (MES) buffer from cotton seed extract (CSE) medium offered a low-cost medium for fermentations. Additionally, we observed that corn steep liquor (CSL) in the medium diversified the product range with both bacteria. Maximum concentrations of ethanol, n-butanol, n-hexanol, acetic acid, butyric acid, and hexanoic acid from different fermentation treatments were 2.78gL−1, 0.70gL−1, 0.52gL−1, 4.06gL−1, 0.13gL−1 and 0.42gL−1, respectively. This study highlights the important role that acetogenic microbes can offer for CO2 conversion into valuable fuels and chemicals.

Biocatalytic reduction of short‐chain carboxylic acids into their corresponding alcohols with syngas fermentation

Short-chain carboxylic acids generated by various mixed- or pure-culture fermentation processes have been considered valuable precursors for production of bioalcohols. While conversion of carboxylic acids into alcohols is routinely performed with catalytic hydrogenation or with strong chemical reducing agents, here, a biological conversion route was explored. The potential of carboxydotrophic bacteria, such as Clostridium ljungdahlii and Clostridium ragsdalei, as biocatalysts for conversion of short-chain carboxylic acids into alcohols, using syngas as a source of electrons and energy is demonstrated. Acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, and n-caproic acid were converted into their corresponding alcohols. Furthermore, biomass yields and fermentation stoichiometry from the experimental data were modeled to determine how much metabolic energy C. ljungdahlii generated during syngas fermentation. An ATP yield of 0.4-0.5 mol of ATP per mol CO consumed was calculated in the presence of hydrogen. The ratio of protons pumped across the cell membrane versus electrons transferred from ferredoxin to NAD(+) via the Rnf complex is suggested to be 1.0. Based on these results, we provide suggestions how n-butyric acid to n-butanol conversion via syngas fermentation can be further improved.

Effect of Energetic Gas Composition on Hydrogenase Activity and Ethanol Production in Syngas Fermentation by Clostridium ragsdalei

With the advancement of post-gasification processing, evaluating the effect of gas composition on ethanol production becomes important, as the composition could be shifted prior to fermentation to optimize ethanol yield. Increasing the overall efficiency of syngas conversion to bio-ethanol involves optimizing the use of the hydrogen (H2) component of syngas. This study focuses on the effect of different CO and H2 concentrations in the gaseous substrate on ethanol production and hydrogenase activity using Clostridium ragsdalei. Hydrogenase activity was observed to be the least inhibited by the lowest concentrations of H2 and CO. The maximum hydrogenase activity appeared early in the growth phase. At the lowest concentrations of CO and H2, hydrogenase appeared to recover activity slightly, and coincided with a depletion of acetic acid and an increase in ethanol concentrations. This phenomenon resulted in the highest concentration of ethanol observed (1.4 g L-1) at 10 days.

Optimization of a corn steep medium for production of ethanol from synthesis gas fermentation by Clostridium ragsdalei

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.