Caproic Acid Production Research Articles

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.

A kinetic study on carboxylic acids production using bovine slaughterhouse wastewater: a promising substrate for resource recovery in biotechnological processes.

Carboxylic acids (CA) are considered high added-value compounds, and their production from wastes has gained economic and environmental notoriety. However, the CA production and kinetic modeling using some agro-industrial wastewaters, such as bovine slaughterhouse wastewater (SHW), are not well reported in the literature. Therefore, the objective of this work was to evaluate the CA production potential using SHW as a substrate under acidogenic conditions and to apply mathematical models to estimate the kinetic parameters of particulate organic matter hydrolysis, soluble organic matter consumption, and CA production. Tests were carried out in quadruplicate batch reactors with a 250-mL reaction volume, with brewery sludge as inoculum and using chloroform (0.05%, v/v) for methanogenesis inhibition. The obtained yield was 0.55g acids gCODA-1, corresponding to 0.76 gCOD gCODA-1. The production of caproic acid without the addition of electron donors was achieved. Mathematical models that describe exponential growth, such as the first-order exponential model, cone model, and Fitzhugh model, were the most suitable to describe the production kinetics of CA. Finally, SHW seems to be a promising substrate to be investigated in the carboxylic platform.

Caproic Acid Formation by Carbon Chain Elongation During Fermentative Hydrogen Production of Cassava Wastewater

In this work, cassava wastewater was used to evaluate hydrogen production and the subsequent production of caproic acid via acetate carbon chain elongation, adding ethanol as electron donor. Batch reactors were operated with an initial chemical oxygen demand of 10 and 20 g O2/L (A10 and A20, respectively), under self-fermentative operation at 40 ± 1 °C. The initial pH was set to 5.5 to inhibit methanogens growth. Hydrogen production yields were 1.01 and 1.98 mol H2/mol glucose for A10 and A20, respectively. In both cases, the main metabolites were ethanol and acetic, butyric, propionic, and caproic acids during the fermentative step; when adding ethanol for the chain elongation the pH was adjusted to 7.0 using a 2-M NaOH solution. The maximum total caproic acid concentration obtained after ethanol addition was 4.2 and 7.2 g/L for A10 and A20, respectively; of which 84% and 90% resulted from the carbon chain elongation step, respectively.

Dark fermentative hydrogen production from hydrolyzed sugar beet pulp improved by iron addition

This study evaluated the impact of three different iron compounds (Fe2O3, FeSO4, FeCl3) on hydrogen production via mesophilic dark fermentation (DF) of hydrolyzed sugar beet pulp (SBP). In batch tests, the maximum hydrogen yield of over 200 dm3H2/kgVS was achieved with the addition of 0.1 gFe2O3/dm3, which was twice greater than the control. In semi-continuous experiments, the highest hydrogen production of 52.11 dm3H2/kgVS combined with 19.4 dm3CH4/kgVS methane yield was obtained at a dose of 1 gFe2O3/dm3. Acetic, lactic and caproic acids were the main metabolic products of DF. Microbiological studies showed some balance between hydrogen producing microorganisms from the order Clostridiales and lactic acid producers (LAB) affiliated with the orders Lactobacillales and Coriobacteriales. Moreover, the presence of methanogens affiliated to the genera Methanobrevibacter and Methanosphaera was also documented. An interesting finding was the appearance of rare bacteria from the genus Caproiciproducens, which was responsible for increased caproic acid production.

Production of volatile fatty acids through co-digestion of sewage sludge and external organic waste: Effect of substrate proportions and long-term operation

Volatile fatty acids (VFAs) are intermediates of anaerobic digestion with high value and wide range of usage. Co-digestion of sewage sludge and external organic waste (OW) for VFA production can help achieve both resource recovery and ensure sustainable and innovative waste management. In view of this, the effect of substrate proportions on VFA production from co-digestion of primary sewage sludge and OW is studied. Long-term operation in a semi-continuous reactor was performed to assess the resilience of such a system and the VFA-rich effluent was tested for its ability to be used as carbon source for denitrification. Co-digestion was initially carried out in batch reactors with OW proportion of 0%, 25%, 50%, 75%, 100% in terms of COD and scaled up in a semi-continuous reactor operation with 50% OW. In the short-term operation in the batch mode, acetic acid dominated, however, increasing OW fraction resulted in increased valeric and caproic acid production. Moreover, in the long-term semi-continuous operation, caproic acid dominated, accounting for ≈55% of VFAs. The VFA-rich effluent from the semi-continuous reactor achieved the highest denitrification rate as a carbon source when compared with acetic acid and methanol. The results demonstrate that co-fermentation can increase VFA yield and shift products from acetic acid to caproic acid in long-term operation and the VFAs can be used within wastewater treatment plants to close the loop.

Characterization of Microbial Diversity and Community Structure in Fermentation Pit Mud of Different Ages for Production of Strong-Aroma Baijiu.

In the traditional fermentation process of strong-aroma Baijiu, a fermentation pit mud (FPM) provides many genera of microorganisms for fermentation. However, the functional microorganisms that have an important effect on the quality of Baijiu and their changes with the age of fermentation pit (FP) are poorly understood. Herein, the Roche 454 pyrosequencing technique and a phospholipid fatty-acid analysis were employed to reveal the structure and diversity of prokaryotic communities in FPM samples that have been aged for 5, 30, and 100 years. The results revealed an increase in total prokaryotic biomass with an FP age; however, Shannon’s diversity index decreased significantly (p < 0.01). These results suggested that a unique microbial community structure evolved with uninterrupted use of the FP. The number of functional microorganisms, which could produce the flavor compounds of strong-aroma Baijiu, increased with the FP age. Among them, Clostridium and Ruminococcaceae are microorganisms that directly produce caproic acid. The increase of their relative abundance in the FPM might have improved the quality of strong-aroma Baijiu. Syntrophomonas, Methanobacterium, and Methanocorpusculum might also be beneficial to caproic acid production. They are not directly involved but provide possible environmental factors for caproic acid production. Overall, our study results indicated that an uninterrupted use of the FP shapes the particular microbial community structure in the FPM. This research provides scientific support for the concept that the aged FP yields a high-quality Baijiu.

Genome-Scale Metabolic Network Reconstruction and In Silico Analysis of Hexanoic acid Producing Megasphaera elsdenii.

Hexanoic acid and its derivatives have been recently recognized as value-added materials and can be synthesized by several microbes. Of them, Megasphaera elsdenii has been considered as an interesting hexanoic acid producer because of its capability to utilize a variety of carbons sources. However, the cellular metabolism and physiology of M. elsdenii still remain uncharacterized. Therefore, in order to better understand hexanoic acid synthetic metabolism in M. elsdenii, we newly reconstructed its genome-scale metabolic model, iME375, which accounts for 375 genes, 521 reactions, and 443 metabolites. A constraint-based analysis was then employed to evaluate cell growth under various conditions. Subsequently, a flux ratio analysis was conducted to understand the mechanism of bifurcated hexanoic acid synthetic pathways, including the typical fatty acid synthetic pathway via acetyl-CoA and the TCA cycle in a counterclockwise direction through succinate. The resultant metabolic states showed that the highest hexanoic acid production could be achieved when the balanced fractional contribution via acetyl-CoA and succinate in reductive TCA cycle was formed in various cell growth rates. The highest hexanoic acid production was maintained in the most perturbed flux ratio, as phosphoenolpyruvate carboxykinase (pck) enables the bifurcated pathway to form consistent fluxes. Finally, organic acid consuming simulations suggested that succinate can increase both biomass formation and hexanoic acid production.

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.

Impact of feedstocks and downstream processing technologies on the economics of caproic acid production in fermentation by Megasphaera elsdenii T81

Caproic acid (CA) was produced by Megasphaera elsdenii T81 with Jerusalem artichoke tubers (JA) as a feedstock. More CA was produced under the medium with the acid hydrolysate of JA than the comparative medium with a carbon composition similar to that of JA. CA was produced up to 13.0 g/L and 0.52 g/L/h with extractive fermentation using a mixed solvent of alamine 336 in oleyl alcohol at 37 °C. The JA cost to produce 1 ton of CA is only 505 USD, which is much lower than that required for purchasing sucrose (860 USD) in CA production. As a result of the analysis performed using SuperPro Designer, including the cost of distillation to obtain pure CA, the estimated production cost for CA from dry JA is 1869 USD/ton CA at the production scale of 2000 ton/year, which is lower than the current market price for petroleum-derived CA (~2500 USD/ton).

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.

Preliminary Study on Caproic Acid Production by Compound Bacterial Agent of Luzhou-Flavor Liquor Pit Mud

Pit mud plays an important role in the fermentation of Luzhou-flavor liquor, It is a common problem for Luzhou-flavor liquors when the mud is aging and the fragrance is not prominent.Choose high quality mud, Inoculating the mash extract as a mixed bacterial solution in sodium acetate medium, Anaerobic culture method to increase the activity of anaerobic microorganisms, Increase hexanoic acid production; Characterization of hexanoic acid by color reaction of copper sulfate diethyl ether, Determination of hexanoic acid by gas chromatography, Its output is in the range of 1.5g/Lto 3.6 g/L, This is the production of mud maintenance liquid, Technical support is provided to prevent aging of the mud and improve the fragrance of the main body.

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 ± 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.

Caproic acid production from acid whey via open culture fermentation – Evaluation of the role of electron donors and downstream processing

The objective of this study was to investigate the potential of supplementing ethanol and lactic acid as electron donors in reverse β-oxidation for short chain carboxylic acids chain elongation during anaerobic fermentation of acid whey. Best results were achieved when lactic acid was added at concentration of 300 mM. It resulted in medium chain carboxylic acids (MCCAs) concentration of 5.0 g/L. In the trials with ethanol addition, the overall yield was 20% lower. Subsequently liquid-liquid extraction with ionic liquids (ILs) was investigated as a potential purification method of caproic acid. The most promising, with respect to recovery of caproic acid, was piperazinium IL [C1C1C10Ppz][NTF2], however, the selectivity was only 0.39. Less effective [C1C1C6Ppz][NTF2] recovered 85.9% of caproic acid while reaching a higher selectivity of 0.53. Technoeconomic model revealed that to meet the conservative value of $2.25 per kg of caproic acid, the downstream processing should not exceed $0.65 per kg.

Improvement of caproic acid production in a Clostridium kluyveri H068 and Methanogen 166 co-culture fermentation system

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

New coculture system of Clostridium spp. and Megasphaera hexanoica using submerged hollow-fiber membrane bioreactors for caproic acid production

In this study, a coculture bioprocess was developed with Clostridium strains producing butyric acid and Megasphaera hexanoica producing caproic acid from the butyric acid. The two bacterial strains were each cultivated in two submerged hollow-fiber membrane bioreactors (s-HF/MBRs), separately. Each fermentation broth was filtered through the membrane modules, and the filtered broth was either interchanged on another reactor or obtained sequentially through. Using s-HF/MBRs, the caproic acid concentration increased to 10.08 g L−1, with the fastest productivity of 0.69 g L−1 h−1, which higher than that previously reported.

Bioaugmentation with Clostridium tyrobutyricum to improve butyric acid production through direct rice straw bioconversion

One-pot bioconversion is an economically attractive biorefinery strategy to reduce enzyme consumption. Direct conversion of lignocellulosic biomass for butyric acid production is still challenging because of competition among microorganisms. In a consolidated hydrolysis/fermentation bioprocessing (CBP) the microbial structure may eventually prefer the production of caproic acid rather than butyric acid production. This paper presents a new bioaugmentation approach for high butyric acid production from rice straw. By dosing 0.03 g/L of Clostridium tyrobutyricum ATCC 25755 in the CBP, an increase of 226% higher butyric acid was yielded. The selectivity and concentration also increased to 60.7% and 18.05 g/L, respectively. DNA-sequencing confirmed the shift of bacterial community in the augmented CBP. Butyric acid producer was enriched in the bioaugmented bacterial community and the bacteria related to long chain acids production was degenerated. The findings may be useful in future research and process design to enhance productivity of desired bio-products.

Short chain and medium chain fatty acids production using food waste under non-augmented and bio-augmented conditions

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.

Temperature-Phased Conversion of Acid Whey Waste Into Medium-Chain Carboxylic Acids via Lactic Acid: No External e-Donor

Summary Acid whey is a Greek-yogurt waste stream and can be a resource to produce biofuel precursors. Our objective was to convert acid whey into medium-chain carboxylic acid (MCCA) oil with the open-culture carboxylate platform. Here, we developed a temperature-phased bioprocess with different anaerobic reactor microbiomes, performing thermophilic lactic acid production and mesophilic chain elongation, to produce MCCAs (C6–C9) from acid whey via lactic acid as an intermediate. For the lactic acid-producing bioreactor, we achieved a volumetric lactic acid production rate of 1,230 mmol C L −1 day −1 (1.54 g L −1 hr −1 ). For the chain-elongating bioreactor, we achieved a volumetric MCCA production rate of 111 mmol C L −1 day −1 and a volumetric n -caproic acid (C6) production rate of 81 mmol C L −1 day −1 (0.07 g L −1 hr −1 ). We converted a real waste stream into mainly MCCAs without the external addition of an electron donor.

The effect of methanogenesis inhibition, inoculum and substrate concentration on hydrogen and carboxylic acids production from cassava wastewater.

Manipueira is a carbohydrate-rich agro-industrial waste from cassava processing. It is considered well suitable for biotechnological processes, such as hydrogen and carboxylic acids production, due to the high content of easily degradable organic matter. However, the proper methanogenesis inhibition method, inoculum type, and organic loads are factors still limiting the processes. The objective in this work was to evaluate the effects of such factors on byproducts production in anaerobic reactors. Batch experiments were conducted with 2.3-L flasks during two operational phases. In the first phase (P1), inhibition of methanogens in the sludge was evaluated using acetylene (1% v/v of headspace) and heat treatment (120°C, 1atm for 30min). In the second phase (P2), three inoculum types obtained from common anaerobic sludges (bovine rumen and sludges from municipal and textile industrial wastewater treatment plants) were individually assayed. P2 aimed to identify the best inoculum, based on hydrogen production ability, which was tested for three initial concentrations of manipueira in terms of chemical oxygen demand (COD) (10, 20 and 40g O2/L). Results of P1 indicated that either acetylene or heat treatment efficiently inhibited methanogenesis, with no methane production. However, the maximum H2 production potential by applying heat treatment (~563mL) was more than twice compared with that by acetylene treatment (~257mL); and butyrate was the main carboxylic acid by-product (~3g/L). In P2 experiments after sludge heat treatment, the highest hydrogen yield (1.66±0.07mol H2/mol glucose) and caproic acid production (~2g/L) were observed at 20g O2/L of manipueira COD, when bovine rumen was the inoculum. The primary metabolic degradation products in all P2 experiments were ethanol, acetic, butyric, propionic and caproic acids. The finding of caproic acid detection indicated that the applied conditions in manipueira anaerobic degradation favored carbon chain elongation over methanogenesis.

Optimization of hexanoic acid production in recombinant Escherichia coli by precise flux rebalancing

The aim of this study is to demonstrate that rebalancing of metabolic fluxes at acetyl-CoA branch node can substantially improve the titer and productivity of hexanoic acid in recombinant Escherichia coli strains. First, a hexanoic acid-producing E. coli strain was constructed by expressing genes encoding β-ketothiolase (BktB) from Cupriavidus necator and acetyl-CoA transferase (ACT) from Megasphaera sp. MH in a butyric acid producer strain. Next, metabolic flux was optimized at the acetyl-CoA branch node by fine-tuning the expression level of the gene for acetyl-CoA acetyltransferase (AtoB). Four synthetic 5′-untranslated regions were designed for atoB using UTR Designer to modulate the expression level of the gene. Notably, the productivity of the optimized strain (14.7 mg/L/h) was the highest among recombinant E. coli strains in literature when using a similar inoculum size for fermentation. These results show that fine-tuning the expression level of atoB is critical for production of hexanoic acid.