Caproic Acid Production Research Articles

Novel synthetic co-culture of Acetobacterium woodii and Clostridium drakei using CO2 and in situ generated H2 for the production of caproic acid via lactic acid.

Acetobacterium woodii is known to produce mainly acetate from CO2 and H2, but the production of higher value chemicals is desired for the bioeconomy. Using chain-elongating bacteria, synthetic co-cultures have the potential to produce longer-chained products such as caproic acid. In this study, we present first results for a successful autotrophic co-cultivation of A.woodii mutants and a Clostridium drakei wild-type strain in a stirred-tank bioreactor for the production of caproic acid from CO2 and H2 via the intermediate lactic acid. For autotrophic lactate production, a recombinant A. woodii strain with a deleted Lct-dehydrogenase complex, which is encoded by the lctBCD genes, and an inserted D-lactate dehydrogenase (LdhD) originating from Leuconostoc mesenteroides, was used. Hydrogen for the process was supplied using an All-in-One electrode for in situ water electrolysis. Lactate concentrations as high as 0.5g L-1 were achieved with the AiO-electrode, whereas 8.1gL-1 lactate were produced with direct H2 sparging in a stirred-tank bioreactor. Hydrogen limitation was identified in the AiO process. However, with cathode surface area enlargement or numbering-up of the electrode and on-demand hydrogen generation, this process has great potential for a true carbon-negative production of value chemicals from CO2.

High-rate conversion of lactic acid-rich streams to caproic acid in a fermentative granular system

Lactic acid-driven chain elongation enables upgrading low-value organic streams into caproic acid. Recently, volumetric production rates over 0.5 g L-1 h-1have been reported for carbohydrate-rich streams in expanded granular sludge bed (EGSB) reactors. However, many target streams contain mixtures of carbohydrates and lactic acid, and little is known about their impact on product profile and microbial ecology, or the importance of carbohydrates as substrate to achieve high rates. This manuscript investigated varying glucose-to-lactate ratios and observed that decreasing glucose-content eliminated odd-chain by-products, while glucose omission required acetic acid addition to support lactic acid conversion. Decreasing the glucose-content fed resulted in decreasing amounts of granular biomass, with the disappearance of granules when no glucose was fed. Lowering the HRT to 0.3 days while feeding only lactic and acetic acid likely triggered re-granulation, enabling the highest lactic acid-driven caproic acid production rates reported thus far at 16.4 ± 1.7 g L-1 d-1.

Substrate loading and nutrient composition steer caproic acid production and biofilm aggregation in high-rate granular reactors

Carbohydrate-rich waste-streams form attractive feedstocks for the production of caproic acid via microbial chain elongation. However, process upscaling and implementation is hampered by low volumetric production rates. Biomass retention with granular fermentation technologies enables high-rate production of caproic acid at scale, but feedstock characteristics enabling microbial granulation under chain-elongating conditions are unknown. Here, we characterised the feedstock requirements for caproic acid production in high-rate expanded granular sludge bed reactors by investigating the impact of substrate concentration and nutrient composition on caproic acid production and granular biofilm aggregation. First, thin stillage was used as model feedstock to demonstrate that low substrate concentrations (11–49 g COD L-1) do not affect product profile, maintaining a caproic acid selectivity of 47 ± 3%, and improve granular biofilm aggregation. High substrate concentrations (55–88 g COD L-1) reduced caproic acid selectivity in favour of butyric acid, potentially due to concomitant product toxicity, and negatively affected granulation. Second, the impact of nutrient composition on production and granulation was investigated by developing a synthetic medium. This demonstrated that complex nutrients (here supplied as tryptone) were required to steer the microbial community from producing mainly acetic acid, butyric acid and iso-butyric acid to the target caproic acid. However, granules were observed in the system regardless of the addition of tryptone. Overall, these results provide crucial insight to identify suitable feedstocks for granular caproic acid producing technologies, opening the door to high-rate production of caproic acid from various underexplored carbohydrate-rich streams.

Production of caproic acid from acetate and ethanol through microbial chain elongation: A techno-economic assessment

Over the past years, microbial chain elongation has received increased attention as a way to produce high value products. This study investigates the economic feasibility of converting ethanol and acetate into caproic acid, using pertraction-based continuous product removal. Feedstock cost, CAPEX and electricity consumption were identified as being the most important cost factors. It was found that the system performance achieved at pilot scale (productivity of 2.5 g/L·d, COD conversion efficiency of 50%) is insufficient to make the process competitive with current market prices. Higher productivity levels could not be achieved experimentally, as when additional inputs were fed to the system these remained largely unconverted. This suggests that the main constraint lies at the fermentation side.

Exploring the response patterns of strong-flavor baijiu brewing microecosystem to fortified Daqu under different pit ages

The fermentation of strong-flavor baijiu depends largely on the evolution of the microbial community that originated from Daqu and pit mud (PM). Applying fortified Daqu (FD) has been proven to be an effective strategy to improve the quality and yield of baijiu. However, it is unclear what the effects of FD on the liquor brewing microecosystem under different pit ages because of the temporal heterogeneity of the PM community. Taking 2-year (new) and 40-year (aged) pits as the objects, the influence of FD on the metabolic profile, physicochemical parameters, and community diversity in Zaopei and PM was investigated by polyphase detecting approaches. Present results showed that the metabolic profiles of Zaopei were significantly improved by FD, whereas those of PM were mainly dependent on pit age. Aspergillus, Caproiciproducens, and Methanosarcina were more abundant in the aged pit, while Kazachstania, Lactobacillus, and Sphingomonas dominated in the new pit, whether in Zaopei or in PM. The interaction relationships among the communities were also altered by FD, and the co-occurrence network, especially the increased links between archaea and bacteria in the new pit. Notably, this interaction in the aged pit distinctly affected the hexanoic acid content based on the Mantel test. The results of PICRUSt2 analysis inferred that FD perhaps improved the interspecies hydrogen transfer in the new pit and increased the carbon flow of hexanoic acid production during chain elongation in the aged pit. These results provide new insights into the production of high-quality strong-flavor baijiu and the aging of PM.

Characterization of a Novel Acetogen Clostridium sp. JS66 for Production of Acids and Alcohols: Focusing on Hexanoic Acid Production from Syngas

Characterization of a Novel Acetogen Clostridium sp. JS66 for Production of Acids and Alcohols: Focusing on Hexanoic Acid Production from Syngas

Development of an enhanced chain elongation process for caproic acid production from waste-derived lactic acid and butyric acid

Medium chain carboxylic acids (MCCA), such as caproic acid, are high-value chemicals with many industrial applications. The development of a biological mixed culture chain elongation (CE) process, using waste-derived lactic acid offers an opportunity to sustainably produce MCCA, such as caproic acid. However, the nature of the biological conversions in mixed communities is still poorly understood. In particular, inhibition of methanogens and MCCA competitors for the development of a specialised inoculum and optimisation of lactic acid conversion and selectivity toward caproic acid are required. Here, the impacts of physical and chemical treatments of the inoculum (anaerobic granular sludge) on the efficiency of CE were evaluated. Additionally, the effects of lactic acid/butyric acid concentrations and ratio (r LA/BA) on selectivity and concentration of caproic acid were investigated. Blended and acidified (pH 3) granular sludge yielded the highest caproic acid concentrations. Moreover, using pH 3 acidified and blended granular sludge, substrate concentrations of 250 mM (r LA/BA = 1.5:1) and 300 mM (r LA/BA = 1:1) were optimal for efficient caproic acid accumulation with a selectivity ≥90 and a mole of lactic to mole of butyric acid consumption (C r LA/BA) of 2:1. However, a similar outcome was achieved when using an enriched caproic acid producing culture with a lower substrate concentration of 200 mM (r LA/BA = 1:1). We report a lactic acid to butyric acid threshold concentration, below and above which the selectivity toward caproic acid is reduced. With these optimum substrate concentrations and ratios, the feasibility for efficient caproic acid synthesis using lactic acid as electron donor and butyric acid as electron acceptor was established. Caproic acid synthesis was completely inhibited when the substrate concentrations were increased to 400 mM (r LA/BA = 1:1). Finally, species affiliated with Ruminococcaceae were likely involved in the caproic acid synthesis. These findings have strong potential for process-design applications in continuous reactor systems, thus allowing for continuous, efficient and sustainable caproic acid production from biomass-derived lactic acid and butyric acid.

A Downstream Processing Cascade for Separation of Caproic and Caprylic Acid from Maize Silage-Based Fermentation Broth.

Production of caproic and caprylic acid through anaerobic fermentation of crops or residual and waste biomass has been regarded as an alternative to the conventional ways, where plant oils and animal fats are mostly used. The downstream processing of the fermentation broth is a particular challenge since the broth has a highly complex composition and low concentrations of the target products. In this study, the proof-of-principle for a separation cascade for caproic (C6) and caprylic acid (C8) produced in a maize silage-based fermentation process was demonstrated. For clarification of the fermentation broth, a filter press and a ceramic ultrafiltration membrane was used to remove coarse solids and to separate suspended particles and macromolecules from the fermentation broth, respectively. With both techniques, the dry matter content was reduced from 6.8 to 2.3% and a particle-free product solution was obtained. Subsequently, the carboxylic acids were extracted with oleyl alcohol by liquid-liquid extraction with an extraction efficiency of 85% for C6 and 97% for C8. Over the whole cascade, 58% of caproic acid and 66% of caprylic acid were recovered from the fermentation broth into the extract. Among all separation steps, solid-liquid separation with the filter press caused the major part of the product loss of 21% of each carboxylic acid. By using separation equipment with a better solid separation efficiency such as decanter centrifuges or belt filter presses this loss could be minimized.

Impact of Dietary Isoflavone Supplementation on the Fecal Microbiota and Its Metabolites in Postmenopausal Women.

Isoflavones are metabolized by components of the gut microbiota and can also modulate their composition and/or activity. This study aimed to analyze the modifications of the fecal microbial populations and their metabolites in menopausal women under dietary treatment with soy isoflavones for one month. Based on the level of urinary equol, the women had been stratified previously as equol-producers (n = 3) or as equol non-producers (n = 5). The composition of the fecal microbiota was assessed by high-throughput sequencing of 16S rRNA gene amplicons and the changes in fatty acid excretion in feces were analyzed by gas chromatography. A greater proportion of sequence reads of the genus Slackia was detected after isoflavone supplementation. Sequences of members of the family Lachnospiraceae and the genus Pseudoflavonifractor were significantly increased in samples from equol-producing women. Multivariable analysis showed that, after isoflavone treatment, the fecal microbial communities of equol producers were more like each other. Isoflavone supplementation increased the production of caproic acid, suggesting differential microbial activity, leading to a high fecal excretion of this compound. However, differences between equol producers and non-producers were not scored. These results may contribute to characterizing the modulating effect of isoflavones on the gut microbiota, which could lead to unravelling of their beneficial health effects.

Co-digestion of cheese whey with sewage sludge for caproic acid production: Role of microbiome and polyhydroxyalkanoates potential production

The main aim of this work was to evaluate the efficiency of producing caproic acid and other volatile fatty acids using a co-digestion between cheese whey and sewage sludge in a continuous reactor. The effect of two different feeding regimes (one and two per day) and three hydraulic retention times (HRT) (15, 10 and 6 days) on the organic acids production were studied. The optimal conditions for the process were 10 days HRT, 2 feeding cycles per day, reaching a maximum degree of acidification of 44%. Under these conditions, the most abundant organic acid was caproic acid. The analysis of the microbial community dynamics in the reactor during the HRT changes revealed a microbiome enriched in organisms involved in caproic acid production. Additionally, the production of polyhydroxyalkanoates using the organic acids stream as feeding was verified in a fed-batch experiment obtaining a copolymer formed by hydroxybutyrate, hydroxyvalerate and hydroxyhexanoate.

Profiling the influence of physicochemical parameters on the microbial community and flavor substances of zaopei

Strong-flavor baijiu is a traditional distilled alcoholic beverage with a long history in China. The fermented grains play a pivotal role in the production of baijiu. The purpose of this study was to evaluate and compare the microbiota and flavor substances present in fermented zaopei (ZP) from pits of different ages. High-throughput sequencing, headspace solid-phase microextraction gas chromatography-mass spectrometry, principal component analysis, community composition analysis, and redundancy analysis were used to analyze and evaluate the impact of environmental factors on microbial communities and flavor substances. Six genera of bacteria (e.g., Caproiciproducens, Syntrophaceticus, Sedimentibacter, Hydrogenispora, Pelotomaculum and Bacillus) and seven genera of fungi (Cladosporium, Debaryomyces, Dipodascus, Auxarthron, Cephalotrichum, unclassified Stachybotryaceae, unclassified Microascaceae and Cephalotrichum) notably affected the production of hexanoic acid (an important flavor compound). Moisture and alcohol content also had considerable effects on the production of the flavor compounds such as ethyl lactate, hexanoic acid, and ethyl hexanoate. The profiles of volatile compounds present in ZP were different between the aged and new pits; these profiles were mainly reflected in the concentration and types of alcohols, aldehydes, esters, and aromatic compounds. This paper provides a comprehensive overview of the physicochemical parameters, flavor substances, and microbial population distribution of ZP. Characterization of various ZP samples help to elucidate the fermentation mechanisms and offer a theoretical reference to control and enhance the quality of Baijiu. © 2021 Society of Chemical Industry.

Evaluation of hidden H2-consuming pathways using metabolic flux-based analysis for a fermentative side-stream dynamic membrane bioreactor using untreated seed sludge

This study investigates the performance and hidden hydrogen consuming metabolic pathways of a fermentative side stream dynamic membrane (DM) bioreactor using flux balance analysis (FBA). The bioreactor was inoculated with untreated methanogenic seed sludge. It was found that fouling rate aggravated with increasing COD concentration (10–30 g/L) and was positively correlated to it rather than to the applied solid flux on the DM module. Due to increased fouling rate the hydraulic retention time (HRT) could not be reduced less than 0.82 ± 0.02 d. An increase in the organic loading rate (OLR) led to an increase in H2 yield from 0.01 to 0.76 mol H2/mol of sucrose. FBA revealed that homoacetogenesis was the main H2-consuming pathway at lower OLRs (corresponding to 10 and 15 g COD/L), while for the OLR corresponding to 30 g COD/L, homoacetogens were suppressed. More importantly, caproic acid production pathway was identified for the first time as another H2-consuming pathway at high OLR which was not significant at lower OLRs during fermentative dynamic membrane bioreactor operations.

Metagenome and analysis of metabolic potential of the microbial community in pit mud used for Chinese strong-flavor liquor production

Complex microbiomes of pit mud (PM) play significant roles in imbuing flavors and qualities of Chinese strong-flavor liquor (CSFL) during fermentation. However, understanding both of the taxonomic and functional diversity of the whole microorganisms in PM still remain a major challenge. Here, PM microbiomes were investigated based on metagenomic sequencing, assembly and binning. Metagenomic data revealed that Euryarchaeota was the predominant phylum, followed by Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. For further functional exploration, 703 metagenome-assembled genomes (MAGs), including 304 novel strains, 197 novel species, and 94 novel genera were reconstructed. Three primary groups of Firmicutes (n = 406), Euryarchaeota (n = 130) and Bacteroidetes (n = 74), particularly genus of them Syntrophomonas, Thermacetogenium and Clostridium, methanogens (Methanobacterium, Methanoculleus, and Methanosarcina), Proteiniphilum and Prevotella, contained most of metabolic potential genes. Additionally, Chloroflexi was firstly reported to have potential to be involved in the caproic acid (CA) production. Bacteroidetes could be the key phylum to synthesize terpenes, and Armatimonadetes, Firmicutes, Ignavibacteriae and Verrucomicrobia may possess the same metabolic potential as well. Overall, this study will significantly improve our understanding of the diverse PM microbiome and help guide the future exploration of microbial resources for modifying PM fermentation processes.

Effect of reactor operating conditions on carboxylate production and chain elongation from co-fermented sludge and food waste

Abstract Nowadays, fermentation of organic wastes for the production of carboxylic acids as precursors of higher-value products has attracted significant attention. In this paper, sewage sludge and food waste were co-fermented to produce carboxylic acids and study the subsequent chain elongation process. The Copenhagen waste stream scenario was taken as a case study. Firstly, design of experiments was used to investigate the overall carboxylic acids and hexanoic acid production in batch, as a function of the co-fermentation ratio, substrate to co-culture ratio and initial pH. Optimal operating conditions for hexanoic acid were obtained with SS/FW 6.61, S/Xo 6.73 and initial pH 6.83. Statistical optimization increased the overall carboxylic acid titer by 41%, while co-fermentation allowed to increase hexanoate annual production up to 77%. Furthermore, a continuous fermentation experiment was performed to study the effect of reactor operating conditions. The overall carboxylates titer was 2 times higher, which also favored chain elongation compared to batch mode. An increasing loading rate did not affect the overall carboxylate titer, however the hexanoic acid titer increased by 44%. A maximum titer of 4.9 g/l of hexanoic acid was produced, achieving a productivity of 2.46 g/l/d of hexanoic acid with a retention time of 2 d and no external electron donor addition. This would correspond to 610 t/y of hexanoic acid and 350 t/y of other carboxylic acids that could be produced, based on the waste availability in Copenhagen.

Effect of ethanol or lactic acid on volatile fatty acid profile and microbial community in short-term sequentially transfers by ruminal fermented with wheat straw in vitro

At present, anaerobic fermentation with waste has become a new utilization direction for the production of VFA, and it has advantages for chemical platforms in replacing non-renewable resources that use petrochemicals as raw materials. In this experiment, the pH value, gas production, VFA production, bacterial and fungal community structure of rumen fermented wheat straw with ethanol or lactic acid in vitro within 8 transfers (3-day interval) were investigated. Ethanol significantly increased the pH value (P < 0.05), while ethanol and lactic acid significantly decreased the gas production (P < 0.05). Ethanol and lactic acid addition increased the production of valeric acid and caproic acid (P < 0.01). The fermentation of ethanol and lactic acid addition showed marked differences in bacterial composition. Clostridium and Ruminococcus genera were the main bacteria producing caporic acid in ethanol culture, while Megasphaera genus was the main bacteria producing valeric acid in lactic acid culture. The relative abundance of Wallemia genus was positively correlated with valeric acid yield (P < 0.05). This result provides reference data that wheat straw co-cultured with ethanol or lactic acid in vitro increases valeric acid and caproic acid production, and bacteria plays more important role than fungi.

Performance of native open cultures (winery effluents, ruminal fluid, anaerobic sludge and digestate) for medium-chain carboxylic acid production using ethanol and acetate

The production of medium-chain carboxylic acids (MCCA) was studied by using five different native microbiomes (ruminal fluid, endogenous red and white winery effluents, acidogenic digestate and anaerobic granular sludge). The presence of Clostridium was found to be essential in an efficient endogenous open culture inocula. The endogenous winery effluent microbiota produced the highest hexanoic acid concentration, followed by anaerobic sludge inoculum. Similarly, the highest volumetric and specific rates of hexanoic acid production (263 g hexanoic acid/L/d and 13 g/g of total solids/d) were observed for the inoculum harvested from the red wine winery effluent. However, the microbiota present in the ruminal fluid inoculum was found to be more versatile, allowing the production of longer-chain carboxylic acids such as heptanoic and octanoic acids. The fermentative digestate inoculum produced the lowest concentration of hexanoic acid at the lowest rate.

Caproic acid production from lactate using Megasphaera hexanoica

Contribution to the International Chain Elongation Conference 2020 | ICEC 2020. An abstract can be found in the right column.

The effect of substrate concentration on granular fermentation for caproic acid production

Contribution to the International Chain Elongation Conference 2020 | ICEC 2020. An abstract can be found in the right column.

Development of an enhanced chain elongation process for caproic acid production from waste-derived lactic acid and butyric acid

Contribution to the International Chain Elongation Conference 2020 | ICEC 2020. An abstract can be found in the right column.

Effect of ultrasonic pretreatment on chain elongation of saccharified residue from food waste by anaerobic fermentation

Converting biowaste into value-added products has raised the researchers' interests. In this study, bioconversion was applied to produce chain acids from food waste by anaerobic fermentation. To improve the caproic acid production, different pretreatments (i.e., ultrasonic, hydrothermal, and alkaline-thermal) were used for investigating their effects on the acidogenic production and microbial communities. The results showed that ultrasonic and hydrothermal pretreatments (207.8 and 210.1mg COD/g VS, respectively) were very efficient for enhancing the caproic acid production, compared to the alkaline-thermal pretreated samples and control samples (72.6 and 97.5mg COD/g VS, respectively). The ultrasonic pretreatment was beneficial for reducing volatile fatty acids (VFAs) during the caproic acid production, resulting in converting more lactic acid to caproic acid by adding the hydrothermal pretreatment. The microbial community analysis showed that the acidogenic bacteria Caproiciproducens dominated the fermentation in this bioconversion process of food waste into chain acids. The Caproiciproducens mainly degraded the proteins and carbohydrates from the saccharified residues of food waste to produce caproic acids through chain elongation procedure. The investigation and optimized method may help develop the bioconversion technology for producing VFAs products from food wastes.