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ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri. In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used.IMPORTANCEThough dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum, C. ljungdahlii, and Clostridium kluyveri. We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Abstract Background Dilute ethanol streams generated during fermentation of biomass or syngas can be used as feedstocks for the production of higher value products. In this study, we describe a novel synthetic microbial co-culture that can effectively upgrade dilute ethanol streams to odd-chain carboxylic acids (OCCAs), specifically valerate and heptanoate. The co-culture consists of two strict anaerobic microorganisms: Anaerotignum neopropionicum, a propionigenic bacterium that ferments ethanol, and Clostridium kluyveri, well-known for its chain-elongating metabolism. In this co-culture, A. neopropionicum grows on ethanol and CO2 producing propionate and acetate, which are then utilised by C. kluyveri for chain elongation with ethanol as the electron donor. Results A co-culture of A. neopropionicum and C. kluyveri was established in serum bottles with 50 mM ethanol, leading to the production of valerate (5.4 ± 0.1 mM) as main product of ethanol-driven chain elongation. In a continuous bioreactor supplied with 3.1 g ethanol L−1 d−1, the co-culture exhibited high ethanol conversion (96.6%) and produced 25% (mol/mol) valerate, with a steady-state concentration of 8.5 mM and a rate of 5.7 mmol L−1 d−1. In addition, up to 6.5 mM heptanoate was produced at a rate of 2.9 mmol L−1 d−1. Batch experiments were also conducted to study the individual growth of the two strains on ethanol. A. neopropionicum showed the highest growth rate when cultured with 50 mM ethanol (μ max = 0.103 ± 0.003 h−1) and tolerated ethanol concentrations of up to 300 mM. Cultivation experiments with C. kluyveri showed that propionate and acetate were used simultaneously for chain elongation. However, growth on propionate alone (50 mM and 100 mM) led to a 1.8-fold reduction in growth rate compared to growth on acetate. Our results also revealed sub-optimal substrate use by C. kluyveri during odd-chain elongation, where excessive ethanol was oxidised to acetate. Conclusions This study highlights the potential of synthetic co-cultivation in chain elongation processes to target the production of OCCAs. Furthermore, our findings shed light on to the metabolism of odd-chain elongation by C. kluyveri.

Background: Dilute ethanol streams generated during fermentation of biomass or syngas can be used as feedstocks for the production of higher value products. In this study, we describe a novel synthetic microbial co-culture that can effectively upgrade dilute ethanol streams to odd-chain carboxylic acids (OCCAs), specifically valerate and heptanoate. The co-culture consists of two strict anaerobic microorganisms: Anaerotignum neopropionicum, a propionigenic bacterium that ferments ethanol, and Clostridium kluyveri, well-known for its chain-elongating metabolism. In this co-culture, A. neopropionicum grows on ethanol and CO2 producing propionate and acetate, which are then utilised by C. kluyveri for chain elongation with ethanol as the electron donor. Results: A co-culture of A. neopropionicum and C. kluyveri was established in serum bottles with 50 mM ethanol, leading to the production of valerate (5.4 ± 0.1 mM) as main product of ethanol-driven chain elongation. In a continuous bioreactor supplied with 3.1 g ethanol L−1 d−1, the co-culture exhibited high ethanol conversion (96.6%) and produced 25% (mol/mol) valerate, with a steady-state concentration of 8.5 mM and a rate of 5.7 mmol L−1 d−1. In addition, up to 6.5 mM heptanoate was produced at a rate of 2.9 mmol L−1 d−1. Batch experiments were also conducted to study the individual growth of the two strains on ethanol. A. neopropionicum showed the highest growth rate when cultured with 50 mM ethanol (μmax = 0.103 ± 0.003 h−1) and tolerated ethanol concentrations of up to 300 mM. Cultivation experiments with C. kluyveri showed that propionate and acetate were used simultaneously for chain elongation. However, growth on propionate alone (50 mM and 100 mM) led to a 1.8-fold reduction in growth rate compared to growth on acetate. Our results also revealed sub-optimal substrate use by C. kluyveri during odd-chain elongation, where excessive ethanol was oxidised to acetate. Conclusions: This study highlights the potential of synthetic co-cultivation in chain elongation processes to target the production of OCCAs. Furthermore, our findings shed light on to the metabolism of odd-chain elongation by C. kluyveri. [ABSTRACT FROM AUTHOR]

Anaerobic soil disinfestation (ASD) consists of the application of labile organic materials to soil, flooding, and covering the soil surface with plastic film. Anaerobic soil disinfestation is a widely used ecofriendly alternative to chemical fumigation for eliminating soil-borne plant pathogens. However, the exact mode of action of ASD has not been elucidated. In particular, the potential role of anaerobic soil bacteria in disinfestation is unclear. In this study, we isolated a predominant bacterium designated as strain E801 from cocopeat medium after laboratory-scale ASD with ethanol as the carbon source. The strain was closely related with Clostridium kluyveri, and fermentatively produced butyrate and caproate from ethanol and acetate. Interestingly, the culture supernatant of strain E801 strongly suppressed the growth of Fusarium oxysporum f. sp. lycopersici (Fol) in a pH-dependent manner. Among the volatile fatty acids produced by E801, only caproate showed significant growth suppression at pHs below 5.5. In addition, caproate eliminated Fol conidia completely at pHs 5.5 and 5.0 and suppressed Fol growth even at a low temperature (15 °C). Furthermore, cocopeat medium amended with caproate eliminated Fol conidia completely within 6 days. These results suggest that caproate is one of the key disinfestation factors in ethanol-based ASD and that the direct application of caproate to soil could be a promising strategy for rapid and stable soil disinfestation. [ABSTRACT FROM AUTHOR]

Chain elongation is a relevant bioprocess in support of a circular economy as it can use a variety of organic feedstocks for production of valuable short and medium chain carboxylates, such as butyrate (C4), caproate (C6), and caprylate (C8). Alcohols, including the biofuel, butanol (C4), can also be generated in chain elongation but the bioreactor conditions that favor butanol production are mainly unknown. In this study we investigated production of butanol (and its precursor butyrate) during ethanol and acetate chain elongation. We used semi-batch bioreactors (0.16 L serum bottles) fed with a range of ethanol concentrations (100–800 mM C), a constant concentration of acetate (50 mM C), and an initial total gas pressure of ∼112 kPa. We showed that the butanol concentration was positively correlated with the ethanol concentration provided (up to 400 mM C ethanol) and to chain elongation activity, which produced H2 and further increased the total gas pressure. In bioreactors fed with 400 mM C ethanol and 50 mM C acetate, a concentration of 114.96 ± 9.26 mM C butanol (∼2.13 g L−1) was achieved after five semi-batch cycles at a total pressure of ∼170 kPa and H2 partial pressure of ∼67 kPa. Bioreactors with 400 mM C ethanol and 50 mM C acetate also yielded a butanol to butyrate molar ratio of 1:1. At the beginning of cycle 8, the total gas pressure was intentionally decreased to ∼112 kPa to test the dependency of butanol production on total pressure and H2 partial pressure. The reduction in total pressure decreased the molar ratio of butanol to butyrate to 1:2 and jolted H2 production out of an apparent stall. Clostridium kluyveri (previously shown to produce butyrate and butanol) and Alistipes (previously linked with butyrate production) were abundant amplicon sequence variants in the bioreactors during the experimental phases, suggesting the microbiome was resilient against changes in bioreactor conditions. The results from this study clearly demonstrate the potential of ethanol and acetate-based chain elongation to yield butanol as a major product. This study also supports the dependency of butanol production on limiting acetate and on high total gas and H2 partial pressures.

[Display omitted] • C. kluyveri thrived in batch & continuous systems with three ethanol:acetate ratios. • 12.9 g/L (92.9 % selectivity) caproate were reached in batch mode with a 3.5:1 ratio. • Higher ratios favored batch mode selectivity over continuous mode at ratios ≥ 3.5:1. • Pure-strain continuous bioreactor worked without contamination for over 23 days. • An increase in EEO was observed as the ethanol:acetate molar ratio increased. Recently, there has been notable interest in researching and industrially producing medium-chain carboxylic acids (MCCAs) like n -caproate and n -caprylate via chain elongation process. This study presents a comprehensive assessment of the behavior and MCCA production profiles of Clostridium kluyveri in batch and continuous modes, at different ethanol:acetate molar ratios (1.5:1, 3.5:1 and 5.5:1). The highest n -caproate concentration, 12.9 ± 0.67 g/L (92.9 ± 1.39 % MCCA selectivity), was achieved in batch mode at a 3.5:1 ratio. Interestingly, higher ratios favored batch mode selectivity over continuous mode when this was equal or higher to 3.5:1. Steady state operation yielded the highest n -caproate (9.5 ± 0.13 g/L) and n -caprylate (0.35 ± 0.020 g/L) concentrations at the 3.5:1 ratio. Increased ethanol:acetate ratios led to a higher excessive ethanol oxidation (EEO) in both operational modes, potentially limiting n -caproate production and selectivity, especially at the 5.5:1 ratio. Overall, this study reports the efficient MCCA production of both batch and continuous modes by C. kluyveri. [ABSTRACT FROM AUTHOR]

The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism's population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture's health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used., Importance: Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, "state-of-the-art" labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures., Competing Interests: The authors declare no conflict of interest.

Caproate (hexanoate) and other medium-chain fatty acids are valuable platform chemicals produced by processes utilizing petroleum or plant oil. Clostridium kluyveri, growing on short chain alcohols (notably ethanol) and carboxylic acids (such as acetate) is noted for its ability to perform chain elongation to produce 4- to 8-carbon carboxylates. C. kluyveri has been studied in monoculture and coculture conditions, which lead to relatively modest carboxylate titers after long fermentation times. To assess the biosynthetic potential of C. kluyveri for caproate production from sugars through coculture fermentations, in the absence of monoculture data in the literature suitable for our coculture experiments, we first explored C. kluyveri monocultures. Some monocultures achieved caproate titers of 150 to over 200 mM in 40–50 h with a production rate of 7.9 mM/h. Based on that data, we then explored two novel, syntrophic coculture partners for producing caproate from sugars: Clostridium acetobutylicum and Clostridium saccharolyticum. Neither species has been cocultured with C. kluyveri before, and both demonstrate promising results. Our experiments of C. kluyveri monocultures and C. kluyveri—C. saccharolyticum cocultures demonstrate exceptionally high caproate titers (145–200 mM), fast production rates (3.25–8.1 mM/h), and short fermentation times (18–45 h). These results represent the most caproate produced by a C. kluyveri coculture in the shortest known fermentation time. We also explored the possibility of heterologous cell fusion between the coculture pairs similar to the results seen previously in our group with C. acetobutylicum and Clostridium ljungdahlii. Fusion events were observed only in the C. acetobutylicum—C. kluyveri coculture pair, and we offer an explanation for the lack of fusion between C. saccharolyticum and C. kluyveri. This work supports the promise of coculture biotechnology for sustainable production of caproate and other platform chemicals.

Anaerobic soil disinfestation (ASD) consists of the application of labile organic materials to soil, flooding, and covering the soil surface with plastic film. Anaerobic soil disinfestation is a widely used ecofriendly alternative to chemical fumigation for eliminating soil-borne plant pathogens. However, the exact mode of action of ASD has not been elucidated. In particular, the potential role of anaerobic soil bacteria in disinfestation is unclear. In this study, we isolated a predominant bacterium designated as strain E801 from cocopeat medium after laboratory-scale ASD with ethanol as the carbon source. The strain was closely related with Clostridium kluyveri, and fermentatively produced butyrate and caproate from ethanol and acetate. Interestingly, the culture supernatant of strain E801 strongly suppressed the growth of Fusarium oxysporum f. sp. lycopersici (Fol) in a pH-dependent manner. Among the volatile fatty acids produced by E801, only caproate showed significant growth suppression at pHs below 5.5. In addition, caproate eliminated Fol conidia completely at pHs 5.5 and 5.0 and suppressed Fol growth even at a low temperature (15 °C). Furthermore, cocopeat medium amended with caproate eliminated Fol conidia completely within 6 days. These results suggest that caproate is one of the key disinfestation factors in ethanol-based ASD and that the direct application of caproate to soil could be a promising strategy for rapid and stable soil disinfestation.

Caproic acid can be used as spices, preservatives, animal feed additives, and biofuels. At the same time, caproic acid plays an important role in Chinese Baijiu. It is the precursor substance for the synthesis of ethyl caproate, which directly affects the quality of Chinese Baijiu. Caproic acid-producing bacteria are the main microorganisms that synthesize caproic acid in Chinese Baijiu, and the most common strain is Clostridium kluyveri. Caproic acid-producing bacteria synthesize n-caproic acid through reverse β-oxidation to extend the carboxylic acid chain. This method mainly uses ethanol and lactic acid as substrates. Ethanol and lactic acid are converted into acetyl-CoA, and acetyl-CoA undergoes a series of condensation, dehydrogenation, dehydration, and reduction to extend the carboxylic acid chain. This review addresses the important issues of caproic acid-producing bacteria in the brewing process of Baijiu: the common caproic acid-producing bacteria that have been reported metabolic pathways, factors affecting acid production, biological competition pathways, and the effect of mixed bacteria fermentation on acid production. It is hoped that this will provide new ideas for the study of caproic acid-producing bacteria in Chinese Baijiu. [ABSTRACT FROM AUTHOR]

Caproic acid can be used as spices, preservatives, animal feed additives, and biofuels. At the same time, caproic acid plays an important role in Chinese Baijiu. It is the precursor substance for the synthesis of ethyl caproate, which directly affects the quality of Chinese Baijiu. Caproic acid-producing bacteria are the main microorganisms that synthesize caproic acid in Chinese Baijiu, and the most common strain is Clostridium kluyveri. Caproic acid-producing bacteria synthesize n-caproic acid through reverse β-oxidation to extend the carboxylic acid chain. This method mainly uses ethanol and lactic acid as substrates. Ethanol and lactic acid are converted into acetyl-CoA, and acetyl-CoA undergoes a series of condensation, dehydrogenation, dehydration, and reduction to extend the carboxylic acid chain. This review addresses the important issues of caproic acid-producing bacteria in the brewing process of Baijiu: the common caproic acid-producing bacteria that have been reported metabolic pathways, factors affecting acid production, biological competition pathways, and the effect of mixed bacteria fermentation on acid production. It is hoped that this will provide new ideas for the study of caproic acid-producing bacteria in Chinese Baijiu.

Microbial fermentation of synthesis gas (syngas) is becoming more attractive for sustainable production of commodity chemicals. To date, syngas fermentation focuses mainly on the use of Clostridium species for the production of small organic molecules such as ethanol and acetate. The co-cultivation of syngas-fermenting microorganisms with chain-elongating bacteria can expand the range of possible products, allowing, for instance, the production of medium-chain fatty acids (MCFA) and alcohols from syngas. To explore these possibilities, we report herein a genome-scale, constraint-based metabolic model to describe growth of a co-culture of Clostridium autoethanogenum and Clostridium kluyveri on syngas for the production of valuable compounds. Community flux balance analysis was used to gain insight into the metabolism of the two strains and their interactions, and to reveal potential strategies enabling production of butyrate and hexanoate.The model suggests that one strategy to optimize the production of medium-chain fatty-acids from syngas would be the addition of succinate. According to the prediction, addition of succinate would increase the pool of crotonyl-CoA and the ethanol/acetate uptake ratio in C. kluyveri, resulting in a flux of up to 60% of electrons into hexanoate. Another potential way to further optimize butyrate and hexanoate production would be an increase of C. autoethanogenum ethanol production. Blocking either acetaldehyde dehydrogenase or formate dehydrogenase (ferredoxin) activity or formate transport, in the C. autoethanogenum metabolic model could potentially lead to an up to 150% increase in ethanol production.

[Display omitted] • C. kluyveri bioaugmentation is proposed to enhance caproic acid fermentation. • Caproic acid concentration and selectivity reached 12.89 g/L and 55.08 %. • Microbial community structure was shifted by bioaugmentation. • Bacteria involved in degrading straw and producing caproic acid were enriched. Producing caproic acid via carboxylate platform is an environmentally-friendly approach for treating lignocellulosic agricultural waste. However, its implementation is still challenged by low product yields and selectivity. A microbiome named cellulolytic acid-producing microbiome (DCB), proficient in producing cellulolytic acid, was successfully acquired and shows promise for producing high-level caproic acid. In this study, a bioaugmentation method utilizing Clostridium kluyveri is proposed to enhance caproic acid yield of DCB using rice straw. With exogenous ethanol, bioaugmentation with Clostridium kluyveri significantly improved the caproic acid concentration and selectivity by 7 times and 4.5 times, achieving 12.9 g/L and 55.1 %, respectively. The addition of Clostridium kluyveri introduced reverse β-oxidation pathway, a more efficient caproic acid production pathway. Meanwhile, bioaugmentation enriched the bacteria proficient in degrading straw and producing short-chain fatty acids, providing more substrates for caproic acid production. This study provides potential bioaugmentation strategies for optimizing caproic acid yield from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

• C. kluyveri managed to grow in a medium with different levels of available CO 2. • 11.0 g/L C 6 was reached from acetate and ethanol in a bioreactor with CO 2 supply. • There is a direct correlation between CO 2 concentration and C. kluyveri 's growth. • CO 2 uptake by C. kluyveri at bioreactor level was demonstrated. Chain elongation technology allows medium-chain fatty acids (MCFAs) production as an alternative to fossil resources. Clostridium kluyveri generates n -caproate primarily from ethanol and acetate, presumably requiring CO 2 for growth. Here, the impact of CO 2 on C. kluyveri was explored. Bottle studies revealed the bacterium's adaptability to low CO 2 levels, even in conditions with minimal dissolved NaHCO 3 (0.0003 M) and unfavorable pH (below 6) under 1 bar CO 2. Bioreactor investigations demonstrated a direct correlation between CO 2 availability and bacterial growth. The highest n -caproate production (11.0 g/L) with 90.1 % selectivity was achieved in a bioreactor with continuous CO 2 supply at 3 mL/min. Additional bottle experiments pressurized with 1 bar CO 2 and varying ethanol:acetate ratios (1:1, 2:1, 4:1) also confirmed CO 2 consumption by C. kluyveri. However, increasing the ethanol:acetate ratio did not enhance n -caproate selectivity, likely due to overly acidic pH conditions. These findings provide insights into chain-elongators responses under diverse conditions. [ABSTRACT FROM AUTHOR]

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. • C. kluyveri was proven to be able to produce unusually high amount of hexanoic acid. • Maintaining a constant pH of 6.8 enhances hexanoic acid production by C. kluyveri. • C. kluyveri grows better with a mixture of ethanol and acetic and butyric acids. • Ethanol, acetic and butyric acids yielded as much as 21.2 g L−1 hexanoic acid. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Sufficient FVW degradation and more acetate formation was gained by optimizing ISR. • High caproate yield and selectivity were achieved by regulating E/A ratio and pH. • The mixed culture caproate fermentation was dominated by C. kluyveri. • Caproate was mainly generated in the middle and late stages of C. kluyveri growth. Abstract 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. [ABSTRACT FROM AUTHOR]

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. Unlabelled Image • Boosted caproate production by cell immobilization on wheat straw • Shortened lag phase from 104 to 15 h after immobilization for chain elongation • The highest caproate production of 10.1 g/L was achieved with immobilized cells. • The immobilized cells as biofilm showed good stability and productivity. [ABSTRACT FROM AUTHOR]

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[Display omitted] • Corn-stalk derived biochar was used in dark fermentation and chain elongation. • Corn-stalk derived biochar showed the promotion effect in dark fermentation. • Dark fermentation metabolites can produce caproate without extra electron donor. • Corn-stalk derived biochar increased the caproate production in chain elongation. This study proposes a new model in which ethanol and acetate produced by dark fermentation are processed by Clostridium kluyveri for chain elongation to produce caproate with an addition of biochar prepared from cornstalk residues after acid pretreatment and enzymatic hydrolysis (AERBC) in the dark fermentation and chain elongation processes. The results show a 6–25% increase in hydrogen production in dark fermentation with adding AERBC, and the maximum concentration of caproate in the new model reached 1740 mg/L, 61% higher than that in the control group. In addition, caproate was obtained by dark fermentation, using liquid metabolites as substrates with an initial pH range of 6.5–7.5. Finally, the electron balance and electron transfer efficiency in the new model were analyzed, and the role of AERBC in dark fermentation and chain elongation was investigated. This study provides a new reference for the use of dark-fermented liquid metabolites and cornstalk residue. [ABSTRACT FROM AUTHOR]

Background: Lignocellulosic biomass plays a crucial role in creating a circular bioeconomy and minimizing environmental impact. Enset biomass is a byproduct of traditional Ethiopian Enset food processing that is thrown away in huge quantities. This study aimed to produce caproate from Enset fiber using Neocallimastix cameroonii strain G341 and Clostridium kluyveri DSM 555 in one-pot two-step fermentation., Results: The process started by growing N. cameroonii on Enset fiber as a carbon source for 7 days. Subsequently, the fungal culture was inoculated with active C. kluyveri preculture and further incubated. The results showed that N. cameroonii grew on 0.25 g untreated Enset fiber as the sole carbon source and produced 1.16 mmol acetate, 0.51 mmol hydrogen, and 1.34 mmol formate. In addition, lactate, succinate, and ethanol were detected in small amounts, 0.17 mmol, 0.08 mmol, and 0.7 mmol, respectively. After inoculating with C. kluyveri, 0.3 mmol of caproate and 0.48 mmol of butyrate were produced, and hydrogen production also increased to 0.95 mmol compared to sole N. cameroonii fermentation. Moreover, after the culture was supplemented with 2.18 mmol of ethanol during C. kluyveri inoculation, caproate, and hydrogen production was further increased to 1.2 and 1.36 mmol, respectively, and the consumption of acetate also increased., Conclusion: A novel microbial cell factory was developed to convert untreated lignocellulosic Enset fiber into the medium chain carboxylic acid caproate and H 2 by a co-culture of the anaerobic fungi N. cameroonii and C. kluyveri. This opens a new value chain for Enset farmers, as the process requires only locally available raw materials and low-price fermenters. As the caproate production was mainly limited by the available ethanol, the addition of locally produced ethanol-containing fermentation broth ("beer") would further increase the titer., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Chinese strong-flavor liquor (CSFL), accounting for more than 70% of both Chinese liquor production and sales, was produced by complex fermentation with pit mud. Clostridium kluyveri, an important species coexisted with other microorganisms in fermentation pit mud (FPM), could produce caproic acid, which was subsequently converted to the key CSFL flavor substance ethyl caproate. In this study, we present the first complete genome sequence of C. kluyveri isolated from FPM. Clostridium kluyveri JZZ contains one circular chromosome and one circular plasmid with length of 4,454,353 and 58,581 bp, respectively. 4158 protein-coding genes were predicted and 2792 genes could be assigned with COG categories. It possesses the pathway predicted for biosynthesis of caproic acid with ethanol. Compared to other two C. kluyveri genomes, JZZ consists of longer chromosome with multiple gene rearrangements, and contains more genes involved in defense mechanisms, as well as DNA replication, recombination, and repair. Meanwhile, JZZ contains fewer genes involved in secondary metabolites biosynthesis, transport, and catabolism, including genes encoding Polyketide Synthases/Non-ribosomal Peptide Synthetases. Additionally, JZZ possesses 960 unique genes with relatively aggregating in defense mechanisms and transcription. Our study will be available for further research about C. kluyveri isolated from FPM, and will also facilitate the genetic engineering to increase biofuel production and improve fragrance flavor of CSFL. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The caproic acid-producing bacterial consortium (CAPBC) J30, isolated from Chinese Luzhou-flavored liquor distillery's pit mud, produces caproic acid as a metabolite. Constructed a 16S rDNA gene clone library and analyzed amplified ribosomal DNA restriction analysis, Clostridium genus was found predominating in CAPBC J30 and C. kluyveri was the chief contributor to its caproic acid-producing ability Then based on the Daqu extraction medium, effects of different carbon sources, nitrogen sources and growth factors on the caproic acid-producing ability were investigated, among which six key factors were chosen and optimized by Taguchi design. The estimated optimal medium was the Daqu extraction medium added 10 g⋅-1 sodium acetate, ethanol 15 ml⋅-1, liquor tailing 15 ml⋅-1, urea 3g⋅1-1, yellow mud extraction 1 ml⋅-1, and (NH4)2S04 0.5 g⋅1-1. After cultivated in this optimized medium for 15 days at 37°C, the caproic acid yield was up to 5024.11 mg⋅-1. This optimal medium will help to produce high caproic acid of CAPBC J30 to circumvent bottlenecks encountered in the high effective and adaptable ingredients in the field of Chinses Luzhou-dwortd liquor manufacture. [ABSTRACT FROM AUTHOR]

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.

[Display omitted] • Caproate production increased by 170.52% in bioaugmented electro-fermentation system. • Synergy of A. woodii and C. kluyveri was observed in electro-fermentation system. • Colonization of C. kluyveri on the biocathode was promoted by the electric field. • Electron transfer capacity of biocathode was promoted by the electric field. As a chain elongation (CE) model strain, Clostridium kluyveri has been used in the studies of bioaugmentation of caproate production. However, its application in the novel electro-fermentation CE system for bioaugmentation is still unclear. In this study, the CE performances, with or without bioaugmentation and in conventional or electro-fermentation systems were compared. And the mechanism of electrochemical-bioaugmentation by constructing a co-culture of Acetobacterium woodii and Clostridium kluyveri were further verified. Results demonstrated that the bioaugmentation treatments have better CE performance, especially in electro-fermentation system, with a highest caproate concentration of 4.68 g·L-1. Mechanism analysis revealed that C. kluyveri responded to the electric field and emerged synergy with the acetogens, which was proved by the increases of C. kluyveri colonization and the acetogens abundance in biofilm and supported by the co-culture experiment. This study provides a novel insight of microbial synergy mechanism of C. kluyveri during CE bioaugmentation in electro-fermentation system. [ABSTRACT FROM AUTHOR]

The open culture system exhibits several advantages in bioconversion of complex substrates into caproate, yet it is urgent to develop an advanced approach to overcome the low fermentative efficiency. In this study, three reinforcement techniques were employed to reinforce the chain elongation in the open culture system, including bioaugmentation, pretreatment of anaerobic sludge (AS) and addition of cell carrier. Results showed that the caproate production was improved to 48.9 mmol/L using bioaugmentation technology. With removal of methanogen by alkaline-thermal pretreatment, the caproate production increased to 94.8 mmol/L. The highest caproate production of 130.5 mmol/L was obtained with addition of cell carrier, which was explained by the formation of biofilm. The genus Clostridium_sensu_stricto capable of medium chain fatty acids production dominated the microbiome of the AS. This study provides an insight that efficient caproate production can be achieved in the open culture system by reinforcing the pathway of chain elongation. [Display omitted] • Caproate production was improved in the open culture system. • Acetate competition was reduced via eliminating acetoclastic methanogen. • The biofilm on the cell carrier reinforced the pathway of chain elongation. • The genus Clostridium_sensu_stricto dominated the microbiome of anaerobic sludge. [ABSTRACT FROM AUTHOR]

Chain elongation is a relevant bioprocess in support of a circular economy as it can use a variety of organic feedstocks for production of valuable short and medium chain carboxylates, such as butyrate (C4), caproate (C6), and caprylate (C8). Alcohols, including the biofuel, butanol (C4), can also be generated in chain elongation but the bioreactor conditions that favor butanol production are mainly unknown. In this study we investigated production of butanol (and its precursor butyrate) during ethanol and acetate chain elongation. We used semi-batch bioreactors (0.16 L serum bottles) fed with a range of ethanol concentrations (100-800 mM C), a constant concentration of acetate (50 mM C), and an initial total gas pressure of ∼112 kPa. We showed that the butanol concentration was positively correlated with the ethanol concentration provided (up to 400 mM C ethanol) and to chain elongation activity, which produced H 2 and further increased the total gas pressure. In bioreactors fed with 400 mM C ethanol and 50 mM C acetate, a concentration of 114.96 ± 9.26 mM C butanol (∼2.13 g L -1 ) was achieved after five semi-batch cycles at a total pressure of ∼170 kPa and H 2 partial pressure of ∼67 kPa. Bioreactors with 400 mM C ethanol and 50 mM C acetate also yielded a butanol to butyrate molar ratio of 1:1. At the beginning of cycle 8, the total gas pressure was intentionally decreased to ∼112 kPa to test the dependency of butanol production on total pressure and H 2 partial pressure. The reduction in total pressure decreased the molar ratio of butanol to butyrate to 1:2 and jolted H 2 production out of an apparent stall. Clostridium kluyveri (previously shown to produce butyrate and butanol) and Alistipes (previously linked with butyrate production) were abundant amplicon sequence variants in the bioreactors during the experimental phases, suggesting the microbiome was resilient against changes in bioreactor conditions. The results from this study clearly demonstrate the potential of ethanol and acetate-based chain elongation to yield butanol as a major product. This study also supports the dependency of butanol production on limiting acetate and on high total gas and H 2 partial pressures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Robles, Sundar, Mohana Rangan and Delgado.)

Nitrogenase is the only known biological enzyme capable of reducing N 2 to bioavailable NH 3 . Most nitrogenases use Mo as a metallocofactor, while alternative cofactors V and Fe are also viable. Both geological and bioinformatic evidence suggest an ancient origin of Mo-based nitrogenase in the Archean, despite the low concentration of dissolved Mo in the Archean oceans. This apparent paradox would be resolvable if mineral-bound Mo were bioavailable for nitrogen fixation by ancient diazotrophs. In this study, the bioavailability of mineral-bound Mo, V, and Fe was determined by incubating an obligately anaerobic diazotroph Clostridium kluyveri with Mo-, V-, and Fe-bearing minerals (molybdenite, cavansite, and ferrihydrite, respectively) and basalt under diazotrophic conditions. The results showed that C. kluyveri utilized mineral-associated metals to express nitrogenase genes and fix nitrogen, as measured by the reverse transcription quantitative polymerase chain reaction and acetylene reduction assay, respectively. C. kluyveri secreted chelating molecules to extract metals from the minerals. As a result of microbial weathering, mineral surface chemistry significantly changed, likely due to surface coating by microbial exudates for metal extraction. These results provide important support for the ancient origin of Mo-based nitrogenase, with profound implications for coevolution of the biosphere and geosphere.

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 b-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. [ABSTRACT FROM AUTHOR]

Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H 2 . The genes encoding butyrate-producing enzymes, including electron-bifurcating butyryl-CoA dehydrogenase/electron transfer flavoprotein complex and NADH-dependent reduced ferredoxin:NADP + oxidoreductase, form a cluster, which is preceded by a gene annotated as the transcriptional regulator Rex. Northern blotting and RT-PCR experiments indicated that the gene cluster forms a large transcriptional unit that possibly includes several small transcriptional units. The deduced Rex protein contains a winged helix DNA-binding domain and a Rossmann fold potentially interacting with NAD(H). Bioinformatics analysis revealed that Rex can bind the promoter regions of numerous genes, which are involved in carbon and energy metabolism, including NADH oxidation, hydrogen production, ATP synthesis, butyrate formation, and succinate metabolism. Rex may regulate the transcription of genes encoding certain transcriptional regulators and transporters. Electrophoretic mobility shift and isothermal titration calorimetry assays revealed that Rex specifically formed protein-DNA complexes with the promoter regions of target genes, which could be inhibited by NADH but restored by an excess amount of NAD + . These results suggest that Rex plays a key role in the carbon and energy metabolism of C. kluyveri as a global transcriptional regulator in response to the cellular NADH/NAD + ratio. [ABSTRACT FROM AUTHOR]

Background: 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. Results: Here, we report the establishment of a synthetic co-culture consisting of Clostridium autoethanogenum and Clostridium kluyveri. Together, these bacteria are capable of converting CO and syngas to a mixture of C4 and C6 fatty acids and their respective alcohols. The co-culture is able to grow using solely CO or syngas as a substrate, and presence of acetate significantly stimulated production rates. The co-culture produced butyrate and caproate at a rate of 8.5 ± 1.1 and 2.5 ± 0.63 mmol/l/day, respectively. Butanol and hexanol were produced at a rate of 3.5 ± 0.69 and 2.0 ± 0.46 mmol/l/day, respectively. The pH was found to be a major factor during cultivation, influencing the growth performance of the separate strains and caproate toxicity. Conclusion: This co-culture poses an alternative way to produce medium-chain fatty acids and higher alcohols from carbon monoxide or syngas and the process can be regarded as an integration of syngas fermentation and chain elongation in one growth vessel. [ABSTRACT FROM AUTHOR]

Chinese strong-aroma type liquor (CSAL) is a popular distilled alcoholic beverage in China. It is produced by a complex fermentation process that is conducted in pits in the ground. Ethyl caproate is a key flavor compound in CSAL and is thought to originate from caproic acid produced by Clostridia inhabiting the fermentation pit mud. However, the particular species of Clostridium associated with this production are poorly understood and problematic to quantify by culturing. In this study, a total of 28 closest relatives including 15 Clostridia and 8 Bacilli species in pit muds from three CSAL distilleries, were detected by culture-dependent and -independent methods. Among them, Clostridium kluyveri was identified as the main producer of caproic acid. One representative strain C . kluyveri N6 could produce caproic, butyric and octanoic acids and their corresponding ethyl esters, contributing significantly to CSAL flavor. A real time quantitative PCR assay of C. kluyveri in pit muds developed showed that a concentration of 1.79 × 10 7 16S rRNA gene copies/g pit mud in LZ-old pit was approximately six times higher than that in HLM and YH pits and sixty times higher than that in LZ-new pit respectively. This method can be used to improve the management of pit mud microbiology and its impact on CSAL quality. [ABSTRACT FROM AUTHOR]