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In situ anaerobic groundwater bioremediation of trichloroethene (TCE) to nontoxic ethene is contingent on organohalide-respiring Dehalococcoidia, the most common strictly hydrogenotrophic Dehalococcoides mccartyi (D. mccartyi). The H2 requirement for D. mccartyi is fulfilled by adding various organic substrates (e.g., lactate, emulsified vegetable oil, and glucose/molasses), which require fermenting microorganisms to convert them to H2. The net flux of H2 is a crucial controlling parameter in the efficacy of bioremediation. H2 consumption by competing microorganisms (e.g., methanogens and homoacetogens) can diminish the rates of reductive dechlorination or stall the process altogether. Furthermore, some fermentation pathways do not produce H2 or having H2 as a product is not always thermodynamically favorable under environmental conditions. Here, we report on a novel application of microbial chain elongation as a H2-producing process for reductive dechlorination. In soil microcosms bioaugmented with dechlorinating and chain-elongating enrichment cultures, near stoichiometric conversion of TCE (0.07 ± 0.01, 0.60 ± 0.03, and 1.50 ± 0.20 mmol L-1 added sequentially) to ethene was achieved when initially stimulated by chain elongation of acetate and ethanol. Chain elongation initiated reductive dechlorination by liberating H2 in the conversion of acetate and ethanol to butyrate and caproate. Syntrophic fermentation of butyrate, a chain-elongation product, to H2 and acetate further sustained the reductive dechlorination activity. Methanogenesis was limited during TCE dechlorination in soil microcosms and absent in transfer cultures fed with chain-elongation substrates. This study provides critical fundamental knowledge toward the feasibility of chlorinated solvent bioremediation based on microbial chain elongation.

Medium-chain fatty acids (MCFA) such as caproic, heptanoic, and caprylic acids are monocarboxylic acids, which can be used as precursor molecules to synthesize biodiesel, bioplastics, antimicrobials, and corrosion inhibitors. Anaerobic bacteria transform ethanol and short-chain fatty acids (SCFA) such as acetate, butyrate, and valerate into MCFA through chain elongation. SCFA and MCFA production were evaluated in this study using two types of waste: apple waste (AW) and pumpkin waste (PW). Two kinds of PW, pumpkin pulp waste (PPW) and pumpkin skin waste (PSW), were used. AW and PW were first converted into a paste using a blender, and then into a leachate (liquid extract obtained from the paste) by adding an anaerobic mixed bacterial culture to the paste in a separate anaerobic chamber. The leachate containing SCFA was further transformed to MCFA using a Clostridium kluyveri augmented, especially enriched anaerobic mixed bacterial culture. The production of SCFA, MCFA and biogas was established for 75 days and analyzed throughout the experiment using high-pressure liquid chromatography (HPLC) and gas chromatography (GC), respectively. Bacteria incubated with AW produced significant amounts of SCFA (7.2 g l−1 of butyric acid) and MCFA (1.2 g l−1 of caproic acid). Bacteria incubated with PPW produced 4.4 g l−1 of butyric acid and 6.1 g l−1 of caproic acid, whereas, bacteria incubated with PSW showed lower amounts of SCFA (1.7 g l−1 of butyric acid) and MCFA (2.5 g l−1 of caproic acid). Clean energy in the form of biohydrogen (H2) constituted the highest proportion (75%) of the biogas composition.

Chain elongation is a process that produces medium chain fatty acids such as caproic acid, which is one of the promising products of the carboxylate platform. This study analyzed the impact of bioaugmentation of heat-treated anaerobic digester sludge with Clostridium kluyveri (AS + Ck) on caproic acid production from a mixed substrate (lactose, lactate, acetate, and ethanol). It was compared with processes initiated with non-augmented heat-treated anaerobic digester sludge (AS) and mono-culture of C. kluyveri (Ck). Moreover, stability of the chain elongation process was evaluated by performing repeated batch experiments. All bacterial cultures demonstrated efficient caproate production in the first batch cycle. After 18 days, caproate concentration reached 9.06 ± 0.43, 7.86 ± 0.38, and 7.67 ± 0.37 g/L for AS, Ck, and AS + Ck cultures, respectively. In the second cycle, AS microbiome was enriched toward caproate production and showed the highest caproate concentration of 11.44 ± 0.47 g/L. On the other hand, bioaugmented culture showed the lowest caproate production in the second cycle (4.10 ± 0.30 g/L). Microbiome analysis in both AS and AS + Ck culture samples indicated strong enrichment toward the anaerobic order of Clostridia. Strains belonging to genera Sporanaerobacter, Paraclostridium, Haloimpatiens, Clostridium, and Bacillus were dominating in the bioreactors.

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] In recent years, the possibility of merging technologies for waste recovery such as those based on syngas fermentation and chain elongation has been studied for the production of medium chain fatty acids (MCFAs) and bioalcohols, in an attempt to integrate the concept of circular economy in the industry. Nevertheless, one of the main issues of this approach is the pH mismatch between acetogens and chain elongating microorganisms. This work reports, for the first time, the suitability of a co-culture of C. aceticum and C. kluyveri metabolizing syngas at near neutral pH in stirred tank bioreactors. For this purpose, bioreactor studies were carried out with continuous syngas supply. In the first experiment, maximum concentrations of 𝑛-butyrate and 𝑛-caproate of 7.0 and 8.2 g/L, respectively, were obtained. In the second experiment, considerable amounts of 𝑛-butanol were produced as a result of the reduction, by C. aceticum, of the carboxylates already formed in the broth. In both experiments, ethanol was used as an exogenous electron agent at some point. Finally, batch bottle assays were performed with a pure culture of C. aceticum grown on CO in presence of n-butyrate to assess and confirm its ability to produce 𝑛-butanol, reaching concentrations up to 951 mg/L, with a 𝑛-butyrate conversion efficiency of 96%, which had never been reported before in this species. Therefore, this work contributes to the state of the art, presenting a novel system for the bioproduction of MCFAs by combining syngas fermentation and chain elongation at near neutral pH, as opposed to the acidic pH range used in all previously reported literature. This research was funded through the Spanish Ministry of Science and Innovation and European FEDER funds (PID2020-117805RB-I00). CFB thanks Xunta de Galicia for her doctoral contract (ED481A-2020/028). The authors, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2021/55). Funding for open access charge provided by Universidade da Coruña/CISUG Xunta de Galicia; ED481A-2020/028 Xunta de Galicia; ED431C 2021/55

The regulation of two-stage caproate fermentation from fruit and vegetable waste (FVW) via anaerobic microbial consortia was investigated in this study. The results showed the highest caproate production achieved 14.9 g/L at the optimal inoculum to substrate ratio (ISR) of 2:1, ethanol to acid ratio (E/A) of 4:1, and pH of 7.5. The caproate yield and selectivity respectively reached 0.62 g/g and 80.8% (as COD). In acidification stage, an appropriate ISR provided a high conversion efficiency and more acetate formation, which was beneficial to caproate biosynthesis. In caproate production stage, chain elongation performance was sensitive to E/A and pH condition. Butyrate became the main by-product at low E/A or acidic conditions, while excessive ethanol or alkaline condition seriously suppressed substrate conversion. The caproate fermentation was dominated by Clostridium kluyveri. Furthermore, caproate formation was uncoupled with Clostridium kluyveri proliferation, which was mainly generated during the middle and late stages of growth.

An efficient multienzyme system for the preparative synthesis of d-xylonate, a chemical with versatile industrial applications, is described. The multienzyme system is based on d-xylose oxidation catalyzed by the xylose dehydrogenase from Calulobacter crescentus and the use of catalytic amounts of NAD. The cofactor is regenerated in situ by coupling the reduction of acetaldehyde into ethanol catalyzed by alcohol dehydrogenase from Clostridium kluyveri. Excellent conversions (>95%) were obtained in a process that allows easy product isolation by simple evaporation of the volatile buffer and byproducts., This work was partially supported by the grant MAT2015- 65184-C2-2-R (MINECO/FEDER). We thank the referees for their helpful comments

Biosynthesis of n-caproate from waste streams rich in acetate and ethanol through chain elongation has offered a potentially sustainable way for future production of liquid biofuels. However, most of the waste streams that fit with the purpose (e.g., digestate) are also rich in ammonium which at high concentration may cause toxic effects on the bioconversion process. This study aims to develop a robust, efficient, and cost-effective chain elongation process with high caproate productivity and tolerance to high ammonia concentration, through immobilization of Clostridium kluyveri on biomass particles as immobilization material. The threshold ammonia concentration for suspended cells cultivation was 2.1 g/L, while it was higher than 5.0 g/L for the wheat straw immobilized system. The caproate production process was dependent on the selected carriers and was performing in the order of: wheat straw > grass straw > saw dust. The biofilm immobilized on the wheat straw showed good reuse capability for caproate production under high ammonia concentration. Moreover, the lag phase for caproate production was shortened from 72 to 30 h after 8 times reuse. These results proved that caproate production and tolerance of chain elongation to ammonia toxicity could be enhanced via cell immobilization. This study offers insight into future development of efficient and cost-effective chain elongation system for production of caproate and other value-added products. Keywords: Caproate, Chain elongation, Biofilm, Ammonia, Clostridium kluyveri, Wheat straw

Glycerol is presently being generated in surplus with the rapid growth of the biodiesel industry and seeks ways to be upcycled, rather than to be treated with costs. Glycerol for the co-production of 1,3-propanediol (1,3-PDO) and caproate has a great prospect. Yet, its technical difficulty lies in the enhancement of caproate productivity, which requires the presence of ethanol as a co-substrate and necessitates the co-existence of functional microbes for glycerol fermentation and chain elongation. This study successfully achieved 6.38 mM C 1,3-PDO d−1 and 2.95 mM C caproate d−1 in a 2-L mixed-cultured semi-continuous fermenter with a glycerol-ethanol-acetate stoichiometric ratio of 4:3:1. Such conversions were mainly facilitated by a microbial community of Eubacterium limosum, Clostridium kluyveri and Massilibacterium senegalense. With such a synergistic microbiome, the co-production of 1,3-PDO and caproate was achieved from glycerol without ethanol addition. Based on metagenomics, E. limosum is capable of converting glycerol to 1,3-PDO, ethanol and H2, and also redirecting the electron potential of H2 into acetate via the Wood–Ljungdahl pathway, which is then used for chain elongation. C. kluyveri worked synergistically with E. limosum by consuming ethanol and acetate for caproate production. M. senegalense encodes for ethanol oxidation to acetate and butyrate, facilitating the generation of these intermediates for C. kluyveri elongation to caproate. During the transition between fermentation and elongation, an unexpected observation of poly-β-hydroxybutyrate (PHB) formation and reutilization by M. senegalense may be associated with butyrate formation for further caproate generation. The knowledge gleaned from the substrate constitute, microbial consortium and their synergetic metabolism demonstrates a resource upgrade potential for crude glycerol or glycerol-containing wastewater generated from the biodiesel industry.

Anaerobic microbial communities can produce carboxylic acids of medium chain length (e.g., caproate, caprylate) by elongating short chain fatty acids through reversed β-oxidation. Ethanol is a common electron donor for this process. The influence of environmental conditions on the stoichiometry and kinetics of ethanol-based chain elongation remains elusive. Here, a sequencing batch bioreactor setup with high-resolution off-gas measurements was used to identify the physiological characteristics of chain elongating microbial communities enriched on acetate and ethanol at pH 7.0 ± 0.2 and 5.5 ± 0.2. Operation at both pH-values led to the development of communities that were highly enriched (>50%, based on 16S rRNA gene amplicon sequencing) in Clostridium kluyveri related species. At both pH-values, stably performing cultures were characterized by incomplete substrate conversion and decreasing biomass-specific hydrogen production rates during an operational cycle. The process stoichiometries obtained at both pH-values were different: at pH 7.0, 71 ± 6% of the consumed electrons were converted to caproate, compared to only 30 ± 5% at pH 5.5. Operating at pH 5.5 led to a decrease in the biomass yield, but a significant increase in the biomass-specific substrate uptake rate, suggesting that the organisms employ catabolic overcapacity to deal with energy losses associated to product inhibition. These results highlight that chain elongating conversions rely on a delicate balance between substrate uptake- and product inhibition kinetics.

Poly(d-lactate-co-glycolate-co-4-hydroxybutyrate) [poly(d-LA-co-GA-co-4HB)] and poly(d-lactate-co-glycolate-co-4-hydroxybutyrate-co-d-2-hydroxybutyrate) [poly(d-LA-co-GA-co-4HB-co-d-2HB)] are of interest for their potential applications as new biomedical polymers. Here we report their enhanced production by metabolically engineered Escherichia coli. To examine the polymer properties, poly(d-LA-co-GA-co-4HB) polymers having various monomer compositions (3.4-41.0mol% of 4HB) were produced by culturing the engineered E. coli strain expressing xylBC from Caulobacter crescentus, evolved phaC1 from Pseudomonas sp. MBEL 6-19 (phaC1437), and evolved pct from Clostridium propionicum (pct540) in a medium supplemented with sodium 4HB at various concentrations. To produce these polymers without 4HB feeding, the 4HB biosynthetic pathway was additionally constructed by expressing Clostridium kluyveri sucD and 4hbD. The engineered E. coli expressing xylBC, phaC1437, pct540, sucD, and 4hbD successfully produced poly(d-LA-co-GA-co-4HB-co-d-2HB) and poly(d-LA-co-GA-co-4HB) from glucose and xylose. Through modulating the expression levels of the heterologous genes and performing fed-batch cultures, the polymer content and titer could be increased to 65.76wt% and 6.19g/L, respectively, while the monomer fractions in the polymers could be altered as desired. The polymers produced, in particular, the 4HB-rich polymers showed viscous and sticky properties suggesting that they might be used as medical adhesives.

Chain elongation is a microbial process in which an electron donor, such as ethanol, is used to elongate short chain carboxylic acids, such as acetic acid, to medium chain carboxylic acids. This metabolism has been extensively investigated, but the spread and differentiation of chain elongators in the environment remains unexplored. Here, chain elongating communities were enriched from several inocula (3 anaerobic digesters, 2 animal faeces and 1 caproic acid producing environment) using ethanol and acetic acid as substrates at pH 7 and 5.5. This approach showed that (i) the inoculum’s origin determines the pH where native chain elongators can grow; (ii) pH affects caproic acid production, with average caproic acid concentrations of 6.4 ± 1.6 g·L−1 at pH 7, versus 2.3 ± 1.8 g·L−1 at pH 5.5; however (iii) pH does not affect growth rates significantly; (iv) all communities contained a close relative of the known chain elongator Clostridium kluyveri; and (v) low pH selects for communities more enriched in this Clostridium kluyveri-relative (57.6 ± 23.2% at pH 7, 96.9 ± 1.2% at pH 5.5). These observations show that ethanol-consuming chain elongators can be found in several natural and engineered environments, but are not the same everywhere, emphasising the need for careful inoculum selection during process development.

Clostridium kluyveri is an anaerobic microorganism that is well-known for producing butyrate and hexanoate using ethanol and acetate. It is also an important bacterium in the production of Chinese strong flavour baijiu (SFB). To obtain a comprehensive understanding of its metabolism, a curated genome-scale metabolic model (GSMM) of C. kluyveri, including 708 genes, 994 reactions, and 804 metabolites, was constructed and named iCKL708. This model was used to simulate the growth of C. kluyveri on different carbon substrates and the results agreed well with the experimental data. The butyrate, pentanoate, and hexanoate biosynthesis pathways were also elucidated. Flux balance analysis indicated that the ratio of ethanol to acetate, as well as the uptake rate of carbon dioxide, affected hexanoate production. The GSMM iCKL708 described here provides a platform to further our understanding and exploration of the metabolic potential of C. kluyveri.

Bioprocess for conversion of ethanol and short chain fatty acids (SCFAs) into medium chain fatty acids (MCFAs) is termed as microbial chain elongation. MCFAs are saturated monocarboxylic acids with applications as antimicrobials, corrosion inhibitors and are precursors for the production of biodiesel and bioplastics. In the present study, a two-stage mixed culture fermentation was employed for the production of SCFAs and MCFAs from food waste (FW). In the first stage, FW was hydrolyzed into leachate (contains SCFAs) by the addition of anaerobic bacteria and in the second stage SCFAs present in leachate were converted in to MCFAs by the chain elongation bioprocess. The production of MCFAs such as caproic, heptanoic, caprylic and decanoic acids were demonstrated for 91 days in four different experimental conditions, i.e., control (no bacteria), pure culture (Clostridium kluyveri), non-augmentation (only enriched mixed culture), and bio-augmentation (C. kluyveri bio-augmented with enriched mixed culture). Produced SCFAs, MCFAs and biogas were analysed at different time intervals. Bio-augmented culture showed highest SCFAs (8.9 g/l of butyric acid) and MCFAs (8.1 g/l of caproic acid) production than other conditions. Microbial population was characterized by using denaturing gradient gel electrophoresis (DGGE) analysis with the highest MCFAs producing culture. The bacterial diversity observed contained predominantly Clostridia, Sphingobacteriales, Desulfobacteraceae and Bacillus groups in the bioaugmented mixed culture. Results have depicted the potential of employing the bio-augmentation strategy for the valorization of food waste into useful products like SCFAs and MCFAs.

Medium-chain fatty acids (MCFA) are saturated monocarboxylic acids and can be used as antimicrobials, corrosion inhibitors, precursors in biodiesel, and bioplastic production. In the present study, MCFA production was evaluated with acetate and ethanol using the bacteria Clostridium kluyveri. Effects of substrate, electron donor, and methane inhibitor on MCFA production were evaluated. Bacteria successfully converted the ethanol and acetate to butyrate (C4), caproate (C6), and caprylate (C8) by chain elongation process. The highest concentrations of butyrate (4.6 g/l), caproate (3.2 g/l), and caprylate (0.5 g/l) were obtained under methane inhibition conditions than other conditions. The productions of butyrate and caproate were 1.6 and 1.48 times higher under methane inhibition conditions, respectively. Results denoted that the bacteria C. kluyveri can be used for conversion of acetate and ethanol into useful products like butyrate and caproate.

An alternative process for anaerobic wastewater treatment with methane recovery is to elongate the carbon chain of volatile fatty acids (VFAs) with a formation of medium chain carboxylic acids (MCCAs), e.g. n-caproic acid with higher monetary value. A potential electron donor is glycerol as a surplus byproduct from the rapid growth of waste-derived biodiesel industry. In the current approach, an industrial chemical, 1,3-propanediol (1,3-PDO) is produced from crude glycerol along with a formation of other soluble byproducts including ethanol and volatile fatty acids (VFAs), which necessitates a significant amount of energy input for separation and purification. To circumvent the energy sink requirement and upcycle both the wastewater treatment process and the biodiesel industry, it is highly beneficial to produce a valuable secondary product from the byproducts. This pioneer study reports on thermodynamic and physiological insights gained into the co-production of 1,3-PDO and caproate from glycerol. Thermodynamics analysis demonstrated that a higher pH range is more favorable when either glycerol or ethanol acting as an electron donor, whereas a high partial pressure (27% at 1 atm) and a low pH (≤5.5) are advantageous for caproate formation with hydrogen. With the glycerol-to-acetate molar ratio of 4 and pH of 7, the physiological experiments achieved a co-production of 1,3-PDO and caproate. However, the caproate yield was low and found to be kinetic-limited. Caproate formation was significantly increased by the intermediate ethanol addition with the optimal mono-caproate formation obtained at the ethanol-to-acetate molar ratio of 3. A synergistic relationship was evinced through microbial characterization, resulting in Clostridium kluyveri and some bacteria with function of converting glycerol to VFAs. This study demonstrates that sufficient ethanol produced as an intermediate is capable of enhancing caproate formation in a synergistic pathway along with 1,3-PDO. The knowledge gleaned paves new avenues for the biodiesel industry by upcycling the byproduct crude glycerol into 1,3-PDO and caproate.

Anaerobic fermentation-based technologies are used for treating organic residues, and producing high value-added products, such as solvents, gases, and organic acids. Among several organic acids, n-caproic acid can be used as antimicrobial agent, additive in animal feed, flavor additive, and feedstock for chemical and biofuel industries. n-Caproic acid formation occurs through a carboxylic acid chain elongation process, which uses reverse β-oxidation of acetic and/or n-butyric acid, and ethanol or lactic acid as an electron donor. This review addresses important issues in commercial n-caproic acid production: metabolic pathways, kinetics and thermodynamics, substrates, reactors, inhibition of competing biological activities, pH, and acid extraction. Additionally, a mathematical model to describe the reverse β-oxidation kinetics was evaluated from existing literature. Current investigations show a wide range of n-caproic acid production rates (3.0–55.8 g/(L·d)), using different open cultures, fermentation conditions, and methods for inhibiting the methanogenesis. Clostridium kluyveri presence and a dominance of the Clostridium spp. were identified as determinant when ethanol was provided as electron donor. Continuous n-caproic acid extraction through pertraction is a promising technology, which combines selective extraction and enhanced production rates. However, confirming the industrial feasibility of this process requires further investigation.

Microbiome-based anaerobic fermentations are promising platform technologies to convert low grade biomass into chemical building blocks. However, systematic investigations on their scalability are scarce. Here, microbiome-based production of medium-chain carboxylic acids from acetate and ethanol was systematically investigated across scales from serum bottles (110 mL) to standard bioreactors (2.2 L) in batch mode. Microbiome cultivation on serum bottle level for high throughput screening led to a considerable total medium-chain carboxylic acids concentration of 843.2 ± 20 C mM with 77.0 ± 2 mM n-butyrate and 89.2 ± 2 mM n-caproate. Introducing agitation significantly enhanced the maximum product formation rates of n-butyrate (rC4,max) and n-caproate (rC6,max) and was identified as a key parameter for further upscaling. The highest total medium-chain carboxylic acids concentration of 977.8 ± 22.8 C mM with 98.5 ± 2.1 mM n-butyrate and 97.3 ± 2.4 mM n-caproate was reached in the 2.2 L standard bioreactor and was only restricted by end-product inhibition. Further, a carbon recovery of up to 94% was independent of the reactor scale proving the transferability of the microbiome and its functions. Based on cloning and sequencing the most abundant microorganisms were closest related to the model organism for microbial medium-chain carboxylic acid formation, Clostridium kluyveri. The current study demonstrates that time and resource efficient screening of functional microbiomes for relevant cultivation conditions on a small scale can be combined with its subsequent upscaling without performance loss.

Several anaerobic bioconversion technologies produce short chain volatile fatty acids and sometimes ethanol, which can together be elongated to hexanoic acid (C6 acid) by Clostridium kluyveri in a secondary fermentation process. Initiatives are needed to further optimize the process. Therefore, five strategies were tested aiming at elucidating their influence on hexanoic acid production from mixtures of acetic acid, butyric acid and ethanol. pH-regulated bioreactors, maintained at pH 7.5, 6.8 or 6.4 led to maximum C6 acid concentrations of, respectively, 19.4, 18.3 and 13.3 g L−1. At pH 6.8, yeast extract omission resulted in a decrease of the hexanoic acid concentration to 12.0 g L−1 while the addition of an inorganic carbon source, such as bicarbonate, for pH control, increased the C6 acid concentration up to 21.4 g L−1. This research provides guidelines for efficient improved production of hexanoic acid by pure cultures of C. kluyveri, contributing to the state of art.

Clostridium kluyveri was used for chain elongation of C2 C4 fatty acids in stirred tank bioreactors. The influence of different electron acceptors (acetic acid, butyric acid and the mixture of both) on C6 fatty acid production was evaluated in presence of ethanol using similar molar alcohol/acid ratios around 3.5. Bottle batch assays without pH regulation and with only acetic acid as electron acceptor yielded a final C6 fatty acid concentration of 6.8 ± 0.6 g L−1. Then, pH-regulated bioreactors were operated at constant pH of 6.8. Under such conditions, the maximum growth rate was 0.039 h−1 obtained using acetic acid and butyric acid as electron acceptors, whereas the lowest growth rate was 0.010 h−1 with only butyric acid as electron acceptor. The maximum growth rate with acetic acid only, was similar, though slightly lower, as with the mixture of C2 C4 fatty acids. Besides, the maximum productions of hexanoic acid were 11.8 g L−1, 13.1 g L−1 and 21.2 g L−1 using, respectively, acetic acid, butyric acid and the mixture of both acids as electron acceptors. Thus, the use of a mixture of acetic acid and butyric acid in presence of ethanol for chain elongation, at constant pH, proved to be efficient for hexanoic acid production.

Bio-catalytic processes for sustainable production of chemicals and fuels receive increased attention within the concept of circular economy. Strategies to improve these production processes include genetic engineering of bio-catalysts or process technological optimization. Alternatively, synthetic microbial co-cultures can be used to enhance production of chemicals of interest. It remains often unclear however how microbe to microbe interactions affect the overall production process and how this can be further exploited for application. In the present study we explored the microbial interaction in a synthetic co-culture of Clostridium autoethanogenum and Clostridium kluyveri, producing chain elongated products from carbon monoxide. Monocultures of C. autoethanogenum converted CO to acetate and traces of ethanol, while during co-cultivation with C. kluyveri, it shifted its metabolism significantly towards solventogenesis. In C. autoethanogenum, expression of the genes involved in the central carbon- and energy-metabolism remained unchanged during co-cultivation compared to monoculture condition. Therefore the shift in the metabolic flux of C. autoethanogenum appears to be regulated by thermodynamics, and results from the continuous removal of ethanol by C. kluyveri. This trait could be further exploited, driving the metabolism of C. autoethanogenum to solely ethanol formation during co-cultivation, resulting in a high yield of chain elongated products from CO-derived electrons. This research highlights the important role of thermodynamic interactions in (synthetic) mixed microbial communities and shows that this can be exploited to promote desired conversions., The research leading to these results has received funding from the Netherlands Ministry of Education, Culture and Science and from the Netherlands Science Foundation (NWO) under the Gravitation Grant nr. 024.002.002 and Programme ‘Closed Cycles’ with Project nr. ALWGK.2016.029., info:eu-repo/semantics/publishedVersion

Syngas from biomass or steel mills can be fermented into a dilute stream of ethanol and acetic acid, which requires energy intensive distillation for product recovery. This can be circumvented by selective secondary fermentation of the syngas fermentation effluent to caproic acid as easier recoverable platform chemical with Clostridium kluyveri. Here, we explore the impact of biochar and activated carbon on this process. Changes during the fermentation with biochar or activated carbon were monitored, different doses were tested and the recyclability of biochar and activated carbon was assessed. Biochar decreased the lag phase and increased the caproic acid production rate (up to 0.50 g · L - 1 · h - 1 ). Upon recycling for subsequent fermentation, biochar retained this property largely. Activated carbon addition, especially at high dose, could potentially increase the conversion and selectivity towards caproic acid to 14.15 g · L - 1 (control: 11.01 g · L - 1 ) and 92% (control: 84%), respectively.

Medium-chain fatty acids (MCFA, e.g., caproic, heptanoic, caprylic acid) are more valuable than short-chain fatty acids (SCFA, e.g., acetic, propionic, butyric, valeric acid). SCFAs are major products in methane-inhibited mixed-culture anaerobic fermentation. By feeding ethanol to the fermentor, MCFA formation is enhanced through chain elongation. Microorganisms such as Clostridium kluyveri elongate short-chain acids by combining them with alcohol. Very low ethanol concentration reduces chain elongation rates, whereas very high ethanol concentrations inhibit microorganisms. To maximize MCFA production, different ethanol concentrations were investigated in the mixed-culture fermentation of office paper and chicken manure. At 10 g/L ethanol concentration, 10 g/L MCFA was formed. High ethanol concentrations (above 40 g/L) inhibit microorganisms resulting in no chain elongation. For chain elongation, propanol was found to be more inhibitory than ethanol. The data suggest that MCFA production will increase by continuously extracting MCFA and maintaining 5-10 g/L ethanol concentration by periodic addition. Biotechnol. Bioeng. 2016;113: 2597-2604. © 2016 Wiley Periodicals, Inc.

Acetate and ethanol can be converted to caproic acid by microorganisms through reverse β-oxidation. There is limited insight into the versatility of chain elongation in view of different starting substrates, including even- and odd-carbon carboxylates and alcohols other than ethanol. Thermodynamic analyses show that most elongation pathways are energetically feasible. Through incubations of microbial communities with different substrate-pair combinations, we established that ethanol and propanol were both highly suitable for chain elongation. As an electron acceptor, acetate, propionate, and butyrate readily elongated with ethanol, whereas an adaptation period was necessary for formate. Isobutyrate and longer-chained fatty acids above butyrate were not elongated. The microbial communities converged, and consistent enrichment of Clostridium spp. was observed, independent of the supplied alcohol or carboxylate, with a strain related to Clostridium kluyveri dominating the enrichments. Community analysis also showed phylotypes related to Bacteroidaceae and Microbacteriaceae families in all tests that are capable of converting the base substrates to useful intermediates. These organisms were mainly enriched with methanol or formate. Our overall conclusion is thus that multiple substrates can be used for chain elongation and that this process is carried out by highly similar organisms for direct chain elongation irrespective of the substrate.

This paper provides an overview on investigations for a new technology to generate bio-based fuel additives from bio-waste. The investigations are taking place at the composting plant in Darmstadt-Kranichstein (Germany). The aim is to explore the potential of bio-waste as feedstock in producing different bio-based products (or bio-based fuels). For this investigation, a facultative anaerobic process is to be integrated into the normal aerobic waste treatment process for composting. The bio-waste is to be treated in four steps to produce biofuels. The first step is the facultative anaerobic treatment of the waste in a rotting box namely percolate to generate a fatty-acid rich liquid fraction. The Hydrolysis takes place in the rotting box during the waste treatment. The organic compounds are then dissolved and transferred into the waste liquid phase. Browne et al. (2013) describes the hydrolysis as an enzymatically degradation of high solid substrates to soluble products which are further degraded to volatile fatty acids (VFA). This is confirmed by analytical tests done on the liquid fraction. After the percolation, volatile and medium chain fatty acids are found in the liquid phase. Concentrations of fatty acids between 8.0 and 31.5 were detected depending on the nature of the input material. In the second step, a fermentation process will be initiated to produce additional fatty acids. Existing microorganism mass is activated to degrade the organic components that are still remaining in the percolate. After fermentation the quantity of fatty acids in four investigated reactors increased 3-5 times. While fermentation mainly non-polar fatty acids (pentanoic to octanoic acid) are build. Next to the fermentation process, a chain-elongation step is arranged by adding ethanol to the fatty acid rich percolate. While these investigations a chain-elongation of mainly fatty acids with pair numbers of carbon atoms (acetate, butanoic and hexanoic acid) are demonstrated. After these three pre-treatments, the percolate is brought to a refinery to extract the non-polar fatty acids using bio-diesel, which was generated from used kitchen oil at the refinery. The extraction tests in the lab have proved that the efficiency of the liquid-liquid-extraction is directly linked with the chain length and polarity of the fatty acids. By using a non-polar bio-diesel mainly the non-polar fatty acids, like pentanoic to octanoic acid, are extracted. After extraction, the bio-diesel enriched with the fatty acids is esterified. As a result bio-diesel with a lower viscosity than usual is produced. The fatty acids remaining in the percolate after the extraction can be used in another fermentation process to generate biogas.

Microbial production of valerate, a five-carbon carboxylate, can occur from propionate and ethanol through a process called odd-chain elongation. The generation of even-chain compounds in this process lowers product selectivity, forming a key challenge. This study investigated factors determining product selectivity during odd-chain elongation in an odd-chain elongating mixed community and the pure culture Clostridium kluyveri DSM555. Incubations at different ratios of ethanol:propionate showed that increasing ratios (from 0.5 to 7) lowered product specificity, as evidenced by a decrease in the odd:even product ratio from 5.5 to 1.5 for C. kluyveri and from 15 to 0.8 for the mixed community. The consistency of these observations with literature data suggests that control of ethanol:propionate ratio offers a robust tool for process control in odd-chain elongation, while the flexible metabolism can also have implications for efficient use of ethanol during even-chain elongation processes.

Effective sludge treatment with bioenergy production is attracting increasing interests as large quantities of waste activated sludge (WAS) are produced during the wastewater treatment. In this study, a new biotechnical process for converting the WAS alkaline fermentation liquor (WASAFL) into valuable, easy-separated medium chain fatty acids (MCFAs) through chain elongation (CE) was investigated, which may provide a new insight into sludge treatment. In the process, ethanol was served as the electron donor (EDs) and WASAFL were main electron acceptors (EAs). The MCFAs productions were investigated under three different ED to EA ratios (i.e., 1:2, 1:1 and 2:1). The result showed that MCFAs production was increased from 2.88 ± 0.01 to 5.28 ± 0.18 g COD/L with the increase of ED to EA ratio. However, the highest MCFA selectivity was achieved at 72.9% when the ED to EA ratio was 1:1. The decrease in the selectivity at high ED:EA ratio is mainly due to the production of higher alcohol (i.e., n-butanol and n-hexanol). The thermodynamic analysis confirmed all CE processes for MCFAs production from WASAFL were exothermic reactions, with the spontaneity and energy release of the reactions increased with the ethanol level. The microbial community analysis showed that the relative abundances of Clostridium, Oscillibacter, Leptolinea and Exilispira were positively correlated with the MCFAs production. The metagenomic analysis suggested that both the reverse β-oxidization pathway and fatty acid biosynthesis pathway contributed to the CE process in the studied system. The functional enzymes were mainly associated within Clostridium, with Clostridium Kluyveri, Clostridium botulinum and Clostridium magnum being likely the key species responsible for the CE process.

The aim of this study was to develop a bioprocess capable of producing caproic acid using a binary fermentation system consisting of Clostridium kluyveri H068 and Methanogen 166 which could then be applied to the brewing of Chinese strong flavor liquor. We initially explored the mechanism by which the Methanogen 166 strain facilitates caproic acid accumulation, revealing its ability to convert accumulated H2 that is produced by C. kluyveri H068 into methane, thereby eliminating the hydrogen-mediated feedback inhibition that normally constrains C. kluyveri H068 and thus enhancing caproic acid production. In addition, laboratory experiments were conducted to optimize this binary fermentation system, allowing us to determine that the optimum conditions for caproic acid production are a mixed inoculum size of 10% with a C. kluyveri H588/Methanogen 166 inoculation ratio of 2:1 (v/v), a sodium acetate concentration of 20 g/L, a 4% ethanol content (v/v), and a yeast extract concentration of 10 g/L. We further scaled this optimized condition up to use in a 1000 L fermenter and the obtained caproic acid broth was subjected to pit-entry fermentation. Our results demonstrated that this pit-entry fermentation approach was an efficient means of improving the quality of Chinese strong flavor liquor. Electronic supplementary material The online version of this article (10.1186/s13568-018-0705-1) contains supplementary material, which is available to authorized users.

Hexanoic acid (HA), also called caproic acid, can be used as an antimicrobial agent and as a precursor to various chemicals, such as fuels, solvents and fragrances. HA can be produced from ethanol and acetate by the mesophilic anaerobic bacterium Clostridium kluyveri, via two successive elongation steps over butyrate. A high-throughput anaerobic growth curve technique was coupled to a data analysis framework to assess growth kinetics for a range of substrate and product concentrations. Using this method, growth rates and several kinetic parameters were determined for C. kluyveri. A maximum growth rate (µmax) of 0.24 ± 0.01 h−1 was found, with a half-saturation index for acetic acid (KS,AA) of 3.8 ± 0.9 mM. Inhibition by butyric acid occurred at of 124.7 ± 5.7 mM (KI,BA), while the final product, HA, linearly inhibited growth with complete inhibition above 91.3 ± 10.8 mM (KHA of 10.9*10−3 ± 1.3*10−3 mM−1) at pH = 7, indicating that the hexanoate anion also exerts toxicity. These parameters were used to create a dynamic mass-balance model for bioproduction of HA. By coupling data collection and analysis to this modelling framework, we have produced a powerful tool to assess the kinetics of anaerobic micro-organisms, demonstrated here with C. kluyveri, in order further explore the potential of micro-organisms for chemicals production.

An enzyme-based multi-parameter biosensor is developed for monitoring the concentration of formate, d-lactate, and l-lactate in biological samples. The sensor is based on the specific dehydrogenation by an oxidized β-nicotinamide adenine dinucleotide (NAD+)-dependent dehydrogenase (formate dehydrogenase, d-lactic dehydrogenase, and l-lactic dehydrogenase, respectively) in combination with a diaphorase from Clostridium kluyveri (EC 1.8.1.4). The enzymes are immobilized on a platinum working electrode by cross-linking with glutaraldehyde (GA). The principle of the determination scheme in case of l-lactate is as follows: l-lactic dehydrogenase (l-LDH) converts l-lactate into pyruvate by reaction with NAD+. In the presence of hexacyanoferrate(III), the resulting reduced β-nicotinamide adenine dinucleotide (NADH) is then regenerated enzymatically by diaphorase. The electrochemical detection is based on the current generated by oxidation of hexacyanoferrate(II) at an applied potential of +0.3 V vs. an Ag/AgCl reference electrode. The biosensor will be electrochemically characterized in terms of linear working range and sensitivity. Additionally, the successful practical application of the sensor is demonstrated in an extract from maize silage.

This study focused on the optimization of caproic acid production from Clostridium kluyveri H588 and its application in Luzhou-flavor liquor brewing. The critical variables that affected caproic acid production were identified by Plackette-Burman design (ethanol, sodium acetate, yeast extract and initial pH) and further optimized by using a four factor central composite design of response surface methodology. The results demonstrated that the optimum conditions for caproic acid production were determined to be ethanol concentration of 4.046% (v/v), sodium acetate concentration of 0.982% (w/v), yeast extract concentration of 0.145% (w/v) and initial pH of 6.41. Caproic acid production (214.23 mg/100 mL) in the optimized condition was in good agreement with the value predicted by the model equation (219.11 mg/100 mL), thereby assuring its effectiveness. Furthermore, the optimized condition of the lab-scale bioreactor was scaled-up to 5000 L fermentor and the obtained caproic acid broth was subjected to pit-filling fermentation. The result demonstrated this method of pit-filling fermentation was efficient in improving the quality of Luzhou-flavor liquor.

In this study, a dry assay of l -lactate via the enzymatic chromatographic test (ECT) was developed. An l -lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2 −6 U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l -lactate dehydrogenase (1 μl, 0.25 U/μl), and NAD + (2 μl, 1.5 × 10 −5 M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10 mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5 mM with a detection limit of 0.047 mM. This quantitative analysis process for l -lactate was easy to operate with good stability and was proper for the point-of-care testing applications.

Three amperometric biosensors have been developed for the detection of l-malic acid, fumaric acid, and l -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for l-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM−1 (l-malate biosensor) and 0.4 μA mM−1 (fumarate biosensor). The l-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM−1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.

Caproate is a valuable industrial product and chemical precursor. In this study, batch tests were conducted to investigate the fermentative caproate production through chain elongation from acetate and ethanol. The effect of acetate/ethanol ratio and initial ethanol concentration on caproate production was examined. When substrate concentration was controlled at 100 mM total carbon, hydrogen was used as an additional electron donor. The highest caproate concentration of 3.11 g/L was obtained at an ethanol/acetate ratio of 7:3. No additional electron donor was needed upon an ethanol/acetate ratio ≥7:3. Caproate production increased with the increase of carbon source until ethanol concentration over 700 mM, which inhibited the fermentation process. The highest caproate concentration of 8.42 g/L was achieved from high ethanol strength wastewater with an ethanol/acetate ratio of 10:1 (550 mM total carbon). Results obtained in this study can pave the way towards efficient chain elongation from ethanol-rich wastewater.

Microbial electrosynthesis is a highly promising application of microbial electrochemical technologies for the sustainable production of organic compounds. At the same time a multitude of questions need to be answered and challenges to be met. Central for its further development is using appropriate electroactive microorganisms and their efficient extracellular electron transfer (EET) as well as wiring of the metabolism to EET. Among others, Clostridia are believed to represent electroactive microbes being highly promising for microbial electrosynthesis. We investigated the potential steps and challenges for the bio-electrochemical fermentation (electro-fermentation) of mid-chain organic acids using Clostridium kluyveri. Starting from a metabolic model the potential limitations of the metabolism as well as beneficial scenarios for electrochemical stimulation were identified and experimentally investigated. C. kluyveri was shown to not be able to exchange electrons with an electrode directly. Therefore, exogenous mediators (2-hydroxy-1,4-naphthoquinone, potassium ferrocyanide, neutral red, methyl viologen, methylene blue, and the macrocyclic cobalt hexaamine [Co(trans-diammac)]3+) were tested for their toxicity and electro-fermentations were performed in 1L bioreactors covering 38 biotic and 8 abiotic runs. When using C. kluyveri and mediators, maximum absolute current densities higher than the abiotic controls were detected for all runs. At the same time, no significant impact on the cell metabolism (product formation, carbon recovery, growth rate) was found. From this observation, we deduce general potential limitations of electro-fermentations with C. kluyveri and discuss strategies to successfully overcome them.

We used metabolic modeling to computationally investigate the potential of bacterial coculture system designs for CO conversion to the platform chemical butyrate. By taking advantage of the native capabilities of wild-type strains, we developed two anaerobic coculture designs by combining Clostridium autoethanogenum for CO-to-acetate conversion with bacterial strains that offer high acetate-to-butyrate conversion capabilities: the environmental bacterium the human gut bacteriumEubacterium rectale. When grown in continuous stirred tank reactor on a 70/0/30 CO/H(2)/N(2) gas mixture, the C. autoethanogenum-C Kluyveri co-culture was predicted to offer no mprovement in butyrate volumetric productivity compared to an engineered C. autoethanogenum monoculture despite utilizing vinyl acetate as a secondary carbon source for C. kluyveri growth enhancement. A coculture consisting of C. autoethanogenum and C. kluyveri engineered in silico to eliminate hexanoate synthesis was predicted to enhance both butyrate productivity and titer. The C. autoethanogenum-E. rectale coculture offered similar improvements in butyrate productivity without the need for metabolic engineering when glucose was provided as a secondary carbon source to enhance E. rectale growth. A bubble column model developed to assess the potential for large-scale butyrate production of the C. autoethanogenum-E. rectale design predicted that a 40/30/30 CO/H(2)/N(2) gas mixture and a 5 m column length would be preferred to enhance C. autoethanogenum growth and counteract CO inhibitory effects on E. rectale.

Valeric acid and its ester derivatives are chemical compounds with a high industrial interest. Here we report a new approach to produce them from crude glycerol, by combining propionic acid fermentation with chain elongation. Propionic acid was produced by Propionibacterium acidipropionici (8.49 ± 1.40 g·L−1). In the subsequent mixed population chain elongation, valeric acid was the dominant product (5.3 ± 0.69 g·L−1) of the chain elongation process. Residual glycerol negatively impacted the selectivity of mixed culture chain elongation towards valeric acid, whereas this was unaffected when Clostridium kluyveri was used as bio-catalyst. Valeric acid could be selectively isolated and upgraded to ethyl valerate by using dodecane as extractant and medium for esterification, whereas shorter-chain carboxylic acids could be recovered by using a 10 wt% solution of trioctylphosphine oxide (TOPO) in dodecane. Overall, our work shows that the combined fermentation, electrochemistry and homogeneous catalysis enables fine chemical production from side streams.

Carboxydotrophic bacteria (CTB) have received attention due to their ability to synthesize commodity chemicals from producer gas and synthesis gas (syngas). CTB have an important advantage of a high product selectivity compared to chemical catalysts. However, the product spectrum of wild-type CTB is narrow. Our objective was to investigate whether a strategy of combining two wild-type bacterial strains into a single, continuously fed bioprocessing step would be promising to broaden the product spectrum. Here, we have operated a syngas-fermentation process with Clostridium ljungdahlii and Clostridium kluyveri with in-line product extraction through gas stripping and product condensing within the syngas recirculation line. The main products from C. ljungdahlii fermentation at a pH of 6.0 were ethanol and acetate at net volumetric production rates of 65.5 and 431 mmol C·L-1·d-1, respectively. An estimated 2/3 of total ethanol produced was utilized by C. kluyveri to chain elongate with the reverse β-oxidation pathway, resulting in n-butyrate and n-caproate at net rates of 129 and 70 mmol C·L-1·d-1, respectively. C. ljungdahlii likely reduced the produced carboxylates to their corresponding alcohols with the reductive power from syngas. This resulted in the longer-chain alcohols n-butanol, n-hexanol, and n-octanol at net volumetric production rates of 39.2, 31.7, and 0.045 mmol C·L-1·d-1, respectively. The continuous production of the longer-chain alcohols occurred only within a narrow pH spectrum of 5.7-6.4 due to the pH discrepancy between the two strains. Regardless whether other wild-type strains could overcome this pH discrepancy, the specificity (mol carbon in product per mol carbon in all other liquid products) for each longer-chain alcohol may never be high in a single bioprocessing step. This, because two bioprocesses compete for intermediates (i.e., carboxylates): (1) chain elongation; and (2) biological reduction. This innate competition resulted in a mixture of n-butanol and n-hexanol with traces of n-octanol.

Dehydrogenases are an important target for the development of cancer therapeutics. Dehydrogenases either produce or consume NAD(P)H, which is fluorescent but at a wavelength where many compounds found in chemical libraries are also fluorescent. By coupling dehydrogenases to diaphorase, which utilizes NAD(P)H to produce the fluorescent molecule resorufin from resazurin, the assay can be red-shifted into a spectral region that reduces interference from compound libraries. Dehydrogenases that produce NAD(P)H, such as isocitrate dehydrogenase 1 (IDH1), can be read in kinetic mode. Dehydrogenases that consume NAD(P)H, such as mutant IDH1 R132H, can be read in endpoint mode. Here, we report protocols for robust and miniaturized 1,536-well assays for WT IDH1 and IDH1 R132H coupled to diaphorase, and the counterassays used to further detect compound interference with the coupling reagents. This coupling technique is applicable to dehydrogenases that either produce or consume NAD(P)H, and the examples provided here can act as guidelines for the development of high-throughput screens against this enzyme class.

Synthesis gas, a mixture of CO, H2, and CO2, is a promising renewable feedstock for bio-based production of organic chemicals. Production of medium-chain fatty acids can be performed via chain elongation, utilizing acetate and ethanol as main substrates. Acetate and ethanol are main products of syngas fermentation by acetogens. Therefore, syngas can be indirectly used as a substrate for the chain elongation process., ERC Grant (Project 323009) and the Gravitation Grant (Project 024.002.002) of the Netherlands Ministry of Education, Culture and Science, and the Netherlands Science Foundation (NWO)

Gasification of organic wastes coupled to syngas fermentation allows the recovery of carbon in the form of commodity chemicals, such as carboxylates and biofuels. Acetogenic bacteria ferment syngas to mainly two-carbon compounds, although a few strains can also synthesize four-, and six-carbon molecules. In general, longer carbon chain products have a higher biotechnological (and commercial) value due to their higher energy content and their lower water solubility. However, de-novo synthesis of medium-chain products from syngas is quite uncommon in acetogenic bacteria. An alternative to de-novo synthesis is bioproduction of short-chain products (C2 and C4), and their subsequent elongation to C4, C6, or C8 through reversed β-oxidation metabolism. This two-step synergistic approach has been successfully applied for the production of up to C8 compounds, although the accumulation of alcohols in these mixed cultures remained below detection limits. The present work investigates the production of higher alcohols from syngas by open mixed cultures (OMC). A syngas-fermenting community was enriched from sludge of an anaerobic digester for a period of 109 days in a lab-scale reactor. At the end of this period, stable production of ethanol and butanol was obtained. C6 compounds were only transiently produced at the beginning of the enrichment phase, during which Clostridium kluyveri, a bacterium able to carry out carbon chain elongation, was detected in the community. Further experiments showed pH as a critical parameter to maintain chain elongation activity in the co-culture. Production of C6 compounds was recovered by preventing fermentation pH to decrease below pH 4.5–5. Finally, experiments showed maximal production of C6 compounds (0.8 g/L) and alcohols (1.7 g/L of ethanol, 1.1 g/L of butanol, and 0.6 g/L of hexanol) at pH 4.8. In conclusion, low fermentation pH is critical for the production of alcohols, although detrimental to C. kluyveri. Fine control of fermentation pH to final values around 4.8 could allow sustained production of higher alcohols The authors would like to thank the Spanish Ministry (BestEnergy, CTQ2011-23632 and Bescarbox, CTM2013-43454-R for its financial support in this study. LEQUIA and EcoAqua have been recognized as consolidated research groups by the Catalan Government (2014-SGR-1168 and 2014-SGR-0484). RG gratefully acknowledges support from Ghent University BOF postdoctoral fellowship (BOF15/PDO/068)

The molecular basis of butyrate production in Porphyromonas gingivalis has not been fully elucidated, even though butyrate, a short chain fatty acid (SCFA), can exert both beneficial and harmful effects on a mammalian host. A database search showed that the amino acid sequence of PGN_0723 protein was 50.6% identical with CoA-dependent succinate semialdehyde dehydrogenase (SSADH) in Clostridium kluyveri. By contrast, the protein has limited identity (19.1%) with CoA-independent SSADH in Escherichia coli. Compared with the wild type, growth speed, and final turbidity were lower in the PGN_0723 deletion strain that was constructed by replacing the PGN_0723 gene with an erythromycin resistance cassette. Gas chromatography mass spectrometry revealed the supernatant concentrations of the SCFAs butyrate, isobutyrate, and isovalerate, but not propionate, in the PGN_0723 deletion strain were also lower than those in the wild type. The wild-type phenotype was restored in a complemented strain. We cloned the PGN_0723 gene, purified the recombinant protein, and computationally constructed its three-dimensional model. A colorimetric assay and liquid chromatography-tandem mass spectrometry analysis demonstrated that the recombinant PGN_0723 produces succinate semialdehyde, which is an intermediate in the P. gingivalis butyrate synthesis pathway, not from succinate but from succinyl-CoA in the presence of NAD(P)H via a ping-pong bi-bi mechanism. Asn110Ala and Cys239Ala mutations resulted in a significant loss of the CoA-dependent PGN_0723 enzymatic activity. The study provides new insights into butyrate production, which constitutes a virulence factor in P. gingivalis.

During the late 1930s, Barker isolated an anaerobic bacterium which he called Clostridium kluyveri. This organism is able to grow on a mixture of acetate and ethanol and produces n-butyrate and n-caproate. From this organism, Barker and Stadtman obtained cell-free extracts which could carry the reactions leading to the C4 and C6 acids. Ethanol is oxidized to acetic acid in two steps, respectively, catalyzed by alcohol dehydrogenase and an acetaldehyde dehydrogenase depending on the presence of coenzyme A

The ruminal microbiome rapidly converts plant biomass to short-chain fatty acids (SCFA) that nourish the ruminant animal host. Because of its high species diversity, functional redundancy, and ease of extraruminal cultivation, this mixed microbial community is a particularly accomplished practitioner of the carboxylate platform for producing fuels and chemical precursors. Unlike reactor microbiomes derived from anaerobic digesters or sediments, the ruminal community naturally produces high concentrations of SCFA, with only modest methane production owing to the absence of both proton-reducing acetogens and aceticlastic methanogens. The extraruminal fermentation can be improved by addition of ethanol or lactate product streams, particularly in concert with reverse β-oxidizing bacteria (e.g., Clostridium kluyveri or Megasphaera elsdenii) that facilitate production of valeric and caproic acids. Application of fundamental principles of thermodynamics allows identification of optimal conditions for SCFA chain elongation, as well as discovery of novel synthetic capabilities (e.g., medium-chain alcohol and alkane production) by this mixed culture system.

Caproate always appears during fermentative H(2) production but its formation was not well explained. It possibly results from the secondary fermentation of ethanol and acetate or butyrate by some special species like Clostridium kluyveri. This study attempts to elucidate caproate formation during the fermentation H(2) production by using C. kluyveri as an example and evaluating several possible pathways of caproate formation. A detailed energetic analysis of the empirical data of an H(2)-producing reactor demonstrated that caproate can be formed from two substrates, either ethanol and acetate or ethanol and butyrate. The analysis showed that at least 5 mol ethanol per mole reaction was essential to support caproate formation under the experimental condition. The analysis also indicated that the secondary fermentation by C. kluyveri might be another pathway to spontaneously produce H(2), butyrate, and acetate in addition to the butyrate-acetate pathway. Co-production of caproate and H(2) from ethanol was thermodynamically feasible and contributed to at least 10-20% of total H(2) production in the reactor studied. It is also clarified that caproate formation is hydrogenogenic rather than hydrogenotrophic.

Phosphotransacetylase (EC 2.3.1.8 ) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA): CH 3 COOPO 3 2− + CoASH ⇆ CH 3 COSCoA + HPO 4 2− . The role of arginine residues was investigated for the phosphotransacetylase from Methanosarcina thermophila . Kinetic analysis of a suite of variants indicated that Arg 87 and Arg 133 interact with the substrate CoA. Arg 87 variants were reduced in the ability to discriminate between CoA and the CoA analog 3′-dephospho-CoA, indicating that Arg 87 forms a salt bridge with the 3′-phosphate of CoA. Arg 133 is postulated to interact with the 5′-phosphate of CoA. Large decreases in k cat and k cat / K m for all of the Arg 87 and Arg 133 variants indicated that these residues are also important, although not essential, for catalysis. Large decreases in k cat and k cat / K m were also observed for the variants in which lysine replaced Arg 87 and Arg 133, suggesting that the bidentate interaction of these residues with CoA or their greater bulk is important for optimal activity. Desulfo-CoA is a strong competitive inhibitor of the enzyme, suggesting that the sulfhydryl group of CoA is important for the optimization of CoA-binding energy but not for tight substrate binding. Chemical modification of the wild-type enzyme by 2,3-butanedione and substrate protection by CoA indicated that at least one reactive arginine is in the active site and is important for activity. The inhibition pattern of the R87Q variant indicated that Arg 87 is modified, which contributes to the inactivation; however, at least one additional active-site arginine is modified leading to enzyme inactivation, albeit at a lower rate.

The oxidation of NADH using NADH oxidase from Bacillus licheniformis or diaphorase from Clostridium kluyveri, was found to produce superoxide anion (O2−). The generated O2− reduced water-soluble tetrazolium salt WST-1 to WST-1 formazan. The formation of WST-1 formazan was monitored as a change in the absorbance at 438 nm. The formation of WST-1 formazan was found to be proportional to the NADH concentration. As a result, a linear curve was obtained within the range of 0.5 μM–50 μM NADH concentration. The concentration of NADH was determined by chemiluminescence using lucigenin specific for O2− instead of WST-1. Application of NADH oxidase from Bacillus licheniformis was found to be 400 times stronger chemiluminescence than that of diaphorase with a lower detection limit of 15 nM. The present method was applied to determine ethanol combined with yeast alcohol dehydrogenase. The ethanol concentrations of various kinds of alcoholic beverages obtained by the present method were compared with those obtained using F-kit method. A good linear correlation was observed between them (r=0.9997) and the slope (1.005) was very close to unity, suggesting that the present method could be applied for the determination of ethanol in practical samples.

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway.

In the presence of NADH- reductases (dihydrolipoamide: NAD oxidoreductase E. C.l.8.1.4 from pig heart or from Clostridium kluyveri; frequently also addressed as “diaphorases”) and NADH lucigenin strongly increases ethylene production from a-ketomethylthiobutyrate (KMB) as an indicator for strong oxidants of the OH-radical type. These reactions are further stimulated in the presence of Fe3+ ions. With these NADH- “diaphorases”, the structurally similar poison, paraquat, in the absence or presence of Fe3+ has no effect. With ferredoxin-NADP reductase (E. C.1.18.1.2.), however, paraquat reacts quasi identical to lucigenin. Superoxide dismutase, catalase, free radical- or OH - scavengers such as mannitol, propylgallate, DABCO, and desferal inhibit the reaction whereas EDTA (in the presence or absence of added Fe3+) is stimulatory. From these data we conclude that the superoxide - indicator LUC is redox-active after unspecific coupling to several almost ubiquitory NAD(P)H- reductases catalyzing monovalent oxygen reduction. Lucigenin thus should no longer be used as a “specific” superoxide indicator. This report is in agreement with very recent results by Liochev and Fridovich (Arch. Biochem. Biophys. 337, 115 [1997]) and Vasquez-Vivar et al. (FEBS Lett. 403, 127 (1997).

Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.

Different microbial pathways can elongate the carbon chains of molecules in open cultures of microbial populations (i.e. reactor microbiomes) under anaerobic conditions. Here, we discuss three such pathways: 1. homoacetogenesis to combine two carbon dioxide molecules into acetate; 2. succinate formation to elongate glycerol with one carbon from carbon dioxide; and 3. reverse β oxidation to elongate short-chain carboxylates with two carbons into medium-chain carboxylates, leading to more energy-dense and insoluble products (e.g. easier to separate from solution). The ability to use reactor microbiomes to treat complex substrates can simultaneously address two pressing issues: 1. providing proper waste management; and 2. producing renewable chemicals and fuels., The authors thank Wolfgang Bucket (MPI Marburg) for assistance with Figure 1. C.M.S. and L.T.A. were supported by the U. S. Army Research Laboratory and the U. S. Army Research Office under contract/grant number W911NF-12-1-0555. H.R. was supported for this work by the Cornell University Agricultural Experiment Station federal formula funds, Project No. NYC-123452 received from the National Institutes for Food and Agriculture (NIFA), U.S. Department of Agriculture. K.R. was supported by the European Research Council Starter Grant Electrotalk and the Multidisciplinary Research Partnership Ghent Bio-Economy. A.J.M.S. was supported by the Chemical Sciences division of the Netherlands Science Foundation (CW-TOP 700.55.343) and the European Research Council (ERC grant 323009).