Efficient caproic acid production from lignocellulosic biomass by bio-augmented mixed microorganisms.

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 ß-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.

Carbon dioxide as key player in chain elongation and growth of Clostridium kluyveri: Insights from batch and bioreactor studies.

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 CO2 for growth. Here, the impact of CO2 on C. kluyveri was explored. Bottle studies revealed the bacterium's adaptability to low CO2 levels, even in conditions with minimal dissolved NaHCO3 (0.0003 M) and unfavorable pH (below 6) under 1 bar CO2. Bioreactor investigations demonstrated a direct correlation between CO2 availability and bacterial growth. The highest n-caproate production (11.0 g/L) with 90.1 % selectivity was achieved in a bioreactor with continuous CO2 supply at 3 mL/min. Additional bottle experiments pressurized with 1 bar CO2 and varying ethanol:acetate ratios (1:1, 2:1, 4:1) also confirmed CO2 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.

Caproate production from Enset fiber in one-pot two-step fermentation using anaerobic fungi (Neocallimastix cameroonii strain G341) and Clostridium kluyveri DSM 555.

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 H2 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.

Improved sequential production of hydrogen and caproate by addition of biochar prepared from cornstalk residues.

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.

Enhanced ethanol-driven carboxylate chain elongation by Pt@C in simulated sequencing batch reactors: Process and mechanism.

Carboxylate chain elongation can create value-added bioproducts from the organic waste. The effects of Pt@C on chain elongation and associated mechanisms were investigated in simulated sequencing batch reactors. 5.0 g/L of Pt@C greatly increased the synthesis of caproate, with an average yield of 21.5 g COD/L, which was 207.4% higher than the trial without Pt@C. Integrated metagenomic and metaproteomic analyses were used to reveal the mechanism of Pt@C-enhanced chain elongation. Pt@C enriched chain elongators by increasing the relative abundance of dominant species by 115.5%. The expression of functional genes related to chain elongation was promoted in the Pt@C trial. This study also demonstrates that Pt@C may promote overall chain elongation metabolism by enhancing CO2 uptake of Clostridium kluyveri. The study provides insights into the fundamental mechanisms of how chain elongation can perform CO2 metabolism and how it can be enhanced by Pt@C to upgrade bioproducts from organic waste streams.

Clostridium kluyveri enhances caproate production by synergistically cooperating with acetogens in mixed microbial community of electro-fermentation system.

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.

Efficient production of n-caproate from syngas by a co-culture of Clostridium aceticum and Clostridium kluyveri.

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.

Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry.

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO2 -gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.

Monitoring co-cultures of Clostridium carboxidivorans and Clostridium kluyveri by fluorescence in situ hybridization with specific 23S rRNA oligonucleotide probes.

The development of co-cultures of clostridial strains which combine different physiological traits represents a promising strategy to achieve the environmentally friendly production of biofuels and chemicals. For the optimization of such co-cultures it is essential to monitor their composition and stability throughout fermentation. FISH is a quick and sensitive method for the specific labeling and quantification of cells within microbial communities. This technique is neither limited by the anaerobic fermenter environment nor by the need of prior genetic modification of strains. In this study, two specific 23S rRNA oligonucleotide probes, ClosKluy and ClosCarb, were designed for the monitoring of C. kluyveri and C. carboxidivorans, respectively. After the optimization of hybridization conditions for both probes, which was achieved at 30% (v/v) formamide, a high specificity was observed with epifluorescence microscopy using cells from different pure reference strains. The discriminating properties of the ClosKluy and ClosCarb probes was verified with samples from heterotrophic co-cultures in anaerobic flasks as well as autotrophic stirred-tank bioreactor co-cultures of C. kluyveri and C. carboxidivorans. Besides being suited to monitor defined co-cultures of these two species, the new specific FISH oligonucleotide probes for C. kluyveri and C. carboxidivorans additionally have potential to be applied in environmental studies.

The occurrence and ecology of microbial chain elongation of carboxylates in soils.

Chain elongation is a growth-dependent anaerobic metabolism that combines acetate and ethanol into butyrate, hexanoate, and octanoate. While the model microorganism for chain elongation, Clostridium kluyveri, was isolated from a saturated soil sample in the 1940s, chain elongation has remained unexplored in soil environments. During soil fermentative events, simple carboxylates and alcohols can transiently accumulate up to low mM concentrations, suggesting in situ possibility of microbial chain elongation. Here, we examined the occurrence and microbial ecology of chain elongation in four soil types in microcosms and enrichments amended with chain elongation substrates. All soils showed evidence of chain elongation activity with several days of incubation at high (100 mM) and environmentally relevant (2.5 mM) concentrations of acetate and ethanol. Three soils showed substantial activity in soil microcosms with high substrate concentrations, converting 58% or more of the added carbon as acetate and ethanol to butyrate, butanol, and hexanoate. Semi-batch enrichment yielded hexanoate and octanoate as the most elongated products and microbial communities predominated by C. kluyveri and other Firmicutes genera not known to undergo chain elongation. Collectively, these results strongly suggest a niche for chain elongation in anaerobic soils that should not be overlooked in soil microbial ecology studies.

Biochar and activated carbon enhance ethanol conversion and selectivity to caproic acid by Clostridium kluyveri.

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.

Ethanol:propionate ratio drives product selectivity in odd-chain elongation with Clostridium kluyveri and mixed communities.

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.

Enrichment Versus Bioaugmentation-Microbiological Production of Caproate from Mixed Carbon Sources by Mixed Bacterial Culture and Clostridium kluyveri.

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.

Effect of pH, yeast extract and inorganic carbon on chain elongation for hexanoic acid production.

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.

Influence of electron acceptors on hexanoic acid production by Clostridium kluyveri.

Clostridium kluyveri was used for chain elongation of C2C4 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 ±â€¯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 C2C4 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.

Performance and microbial characterization of two-stage caproate fermentation from fruit and vegetable waste via anaerobic microbial consortia.

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.

Medium chain carboxylic acids production from waste biomass: Current advances and perspectives.

Alternative chemicals to diverse fossil-fuel-based products is urgently needed to mitigate the adverse impacts of fossil fuel depletion on human development. To this end, researchers have focused on the production of biochemical from readily available and affordable waste biomass. This is consistent with current guidelines for sustainable development and provides great advantages related to economy and environment. The search for suitable biochemical products is in progress worldwide. Therefore, this review recommends a biochemical (i.e., medium chain carboxylic acids (MCCAs)) utilizing an emerging biotechnological production platform called the chain elongation (CE) process. This work covers comprehensive introduction of the CE mechanism, functional microbes, available feedstock types and corresponding utilization strategies, major methods to enhance the performance of MCCAs production, and the challenges that need to be addressed for practical application. This work is expected to provide a thorough understanding of the CE technology, to guide and inspire researchers to solve existing problems in depth, and motivate large-scale MCCAs production.

Immobilization of Clostridium kluyveri on wheat straw to alleviate ammonia inhibition during chain elongation for n-caproate production.

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.

Design, Optimization and Verification of 16S rRNA Oligonucleotide Probes of Fluorescence in-situ Hybridization for Targeting Clostridium spp. and Clostridium kluyveri.

Fluorescence in-situ hybridization (FISH) is a common and popular method used to investigate microbial communities in natural and engineered environments. In this study, two specific 16S rRNA-targeted oligonucleotide probes, CLZ and KCLZ, were designed and verified to quantify the genus Clostridium and the species Clostridium kluyveri. The optimal concentration of hybridization buffer solution for both probes was 30% (w/v). The specificity of the designed probes was high due to the use of pellets from pure reference strains. Feasibility was tested using samples of Chinese liquor from the famed Luzhou manufacturing cellar. The effectiveness of detecting target cells appears to vary widely in different environments. In pit mud, the detection effectiveness of the target cell by probes CLZ and KCLZ was 49.11% and 32.14%, respectively. Quantitative analysis by FISH technique of microbes in pit mud and fermented grains showed consistency with the results detected by qPCR and PCR-DGGE techniques, which showed that the probes CLZ and KCLZ were suitable to analyze the biomass of Clostridium spp. and C. kluyveri during liquor fermentation. Therefore, this study provides a method for quantitative analysis of Clostridium spp. and C. kluyveri and monitoring their community dynamics in microecosystems.

Complete Genome Sequence of Clostridium kluyveri JZZ Applied in Chinese Strong-Flavor Liquor Production.

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.