Caproate Formation Research Articles

Significant enhancement by casamino acids of caproate production via chain elongation

In this study, the effects of casamino acids, as the main component in yeast extract, on the bioproduction of caproate were evaluated. A dosage of 5 g/L casamino acids addition significantly promoted caproate production and shortened the lag phase for caproate production by 7.6-folds compared with the control. Both the net carbon and electron conversion efficiencies were enhanced. The addition of casamino acids shaped a dissimilar microbial community from that of the control and that of the inoculum. The caproate-producing Clostridium and Caproiciproducens, the proteolytic Proteiniphilum and Brassicibacter, and the saccharide-utilizing Hydrogenoanaerobacteriu were enriched, and their abundance were distinguished between with and without the addition of casamino acids as fermentation progressed. Further metagenomics analysis recovered genome bins of Clostridium kluyveri, Clostridium cochlearium, Caprobacter fermentans and Proteiniphilum acetatigenes, representing the majority of the functional bacteria with casamino acids addition. The complete reverse β-oxidation and amino acids (serine/glycine) metabolic pathway for caproate production and butyrate formation, respectively, were fully recovered. Furthermore, all the genes involved were highly expressed and metabolically active in meta-transcriptomics analysis. The finding proved that casamino acids was able to enhance caproate formation by promoting ethanol utilization and butyrate formation. In general, yeast extract could not only support microbial growth but also contribute to the formation of medium chain fatty acids, therefore promoting the efficiency of chain elongation process.

Effect of Oxygen Contamination on Propionate and Caproate Formation in Anaerobic Fermentation

Mixed microbial cultures have become a preferred choice of biocatalyst for chain elongation systems due to their ability to convert complex substrates into medium-chain carboxylates. However, the complexity of the effects of process parameters on the microbial metabolic networks is a drawback that makes the task of optimizing product selectivity challenging. Here, we studied the effects of small air contaminations on the microbial community dynamics and the product formation in anaerobic bioreactors fed with lactate, acetate and H2/CO2. Two stirred tank reactors and two bubble column reactors were operated with H2/CO2 gas recirculation for 139 and 116 days, respectively, at pH 6.0 and 32°C with a hydraulic retention time of 14 days. One reactor of each type had periods with air contamination (between 97 ± 28 and 474 ± 33 mL O2 L−1 d−1, lasting from 4 to 32 days), while the control reactors were kept anoxic. During air contamination, production of n-caproate and CH4 was strongly inhibited, whereas no clear effect on n-butyrate production was observed. In a period with detectable O2 concentrations that went up to 18%, facultative anaerobes of the genus Rummeliibacillus became predominant and only n-butyrate was produced. However, at low air contamination rates and with O2 below the detection level, Coriobacteriia and Actinobacteria gained a competitive advantage over Clostridia and Methanobacteria, and propionate production rates increased to 0.8–1.8 mmol L−1 d−1 depending on the reactor (control reactors 0.1–0.8 mmol L−1 d−1). Moreover, i-butyrate production was observed, but only when Methanobacteria abundances were low and, consequently, H2 availability was high. After air contamination stopped completely, production of n-caproate and CH4 recovered, with n-caproate production rates of 1.4–1.8 mmol L−1 d−1 (control 0.7–2.1 mmol L−1 d−1). The results underline the importance of keeping strictly anaerobic conditions in fermenters when consistent n-caproate production is the goal. Beyond that, micro-aeration should be further tested as a controllable process parameter to shape the reactor microbiome. When odd-chain carboxylates are desired, further studies can develop strategies for their targeted production by applying micro-aerobic conditions.

Fermentation of organic wastes and CO2 + H2 off-gas by microbiotas provides short-chain fatty acids and ethanol for n-caproate production

• Caproate was produced in a two-stage process. • In the first stage SCFAs were produced from three complex substrates. • In the second stage CO 2 + H 2 promoted the production of ethanol. • Caproate was produced from SCFAs and ethanol previously accumulated. • A CO 2 -enriched atmosphere improved the caproate production up to 6 g/L. This study describes a two-stage process for the valorization of organic wastes and CO 2 /H 2 off-gas for the production of short-chain fatty acids (SCFAs) and ethanol, as precursors for n -caproate formation. Three complex organic wastes with previously identified acetogenic members were used to inoculate batch reactors: 1) fibers and 2) leachates from a bagasse fermentation process, and 3) an anaerobic granular sludge from a reactor treating brewery effluents. In the first stage, the reactors loaded with a CO 2 /H 2 [25:75, v/v] gas mixture produced SCFAs at similar concentrations between 4,274 and 5,381 mg/L. In this stage, fiber reactors had the highest H 2 consumption. In the second stage, reactors consumed all available H 2 and accumulated solvents as well as low concentrations of n -caproate. In particular, sludge reactors produced 3,528 mg/L of solvents with more than 50% ethanol content, and leachate reactors produced 617 mg/L of n -caproate. By the end of the second stage, in a CO 2 -enriched atmosphere, leachate reactors produced 6,157 mg/L of n -caproate at the expenses of the SCFAs and solvents. Fiber and leachate microbiotas shared autotrophic members and SCFA-elongating bacteria while sludge microbiota differed in composition. This study demonstrated that the use of CO 2 /H 2 off-gas shifted the composition of SCFAs to a mixture with ethanol which promoted its condensation into n -caproate. A CO 2 -enriched atmosphere improved significantly the final production of n -caproate. The results of this study can be used to integrate the n -caproate production, and possibly other medium-chain fatty acids, with biorefinery schemes in which SCFAs and CO 2 /H 2 off-gases are produced.

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.

Thermodynamic and physiological study of caproate and 1,3-propanediol co-production through glycerol fermentation and fatty acids chain elongation

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

Isobutyrate biosynthesis via methanol chain elongation: converting organic wastes to platform chemicals

AbstractBACKGROUNDIsobutyrate is a platform chemical that is currently produced from a non‐renewable fossil‐based feedstock. This study aimed at developing a renewable isobutyrate production process by using methanol chain elongation, a novel bioprocess that uses organic waste as primary feedstocks and an undefined reactor microbiome as the catalyst.RESULTSA continuous anaerobic bioreactor experiment was first performed using a synthetic medium containing acetate, butyrate and methanol, all of which are common derivatives from organic residues. Continuous isobutyrate (2.0 g L−1 day−1) and caproate formation (0.2 g L−1 day−1) from methanol, acetate and butyrate were demonstrated. A batch test to synthesise isobutyrate from a real organic waste, i.e. acidified supermarket waste (ASW), was performed. Up to 6.2 g L−1 isobutyrate was formed which accounted for 63% of all identified products.CONCLUSIONIn this study, a proof‐of‐principle for isobutyrate production from organic waste via methanol chain elongation was demonstrated. The continuous accumulation or supply of butyrate, the suppression of methanogenic activity and methanol addition were shown to be of use to provide conditions for isobutyrate formation. © 2016 Society of Chemical Industry

Methanol as an alternative electron donor in chain elongation for butyrate and caproate formation

Chain elongation is an emerging mixed culture biotechnology converting acetate into valuable biochemicals by using ethanol as an external electron donor. In this study we proposed to test another potential electron donor, methanol, in chain elongation. Methanol can be produced through the thermochemical conversion of lignocellulosic biowaste. Use of methanol in chain elongation integrates the lignocellulosic feedstocks and the thermochemical platform technologies into chain elongation. After such integration, the feedstocks for chain elongation are solely from 2nd generation biomass resources. A proof-of-principle study of chain elongation using methanol and acetate was performed in both a batch and a continuous experiment. In the batch experiment, butyrate (191 mMC) and caproate (3 mMC) production from methanol and acetate was observed. A mixed culture microbiome taken from a previous chain elongation reactor fed with ethanol was responsible for the observed organic acid production. The continuous experiment was performed in an upflow anaerobic bioreactor (UAB). The hydraulic retention time (HRT) was 36 h and the operational period lasted for 45 days. In the continuous experiment, butyrate production (Rate > 30 mMC/day) was observed; the caproate concentration was below the detection limit during the entire continuous operational period. In both experiments, methanol and acetate were both substrates contributing to the butyrate production. To the authors' current knowledge, this study is the first attempt at a mixed culture fermentation utilising methanol and acetate for biochemical production. Further research should focus on elevating the butyrate production rate and concentration in the continuous operation of methanol chain elongation, which may stimulate caproate formation.

Changes in hydrogenase genetic diversity and proteomic patterns in mixed-culture dark fermentation of mono-, di- and tri-saccharides

Dark fermentation using mixed cultures is a promising biotechnology for producing hydrogen (H 2) from renewable organic waste at a low cost. The impact of the characteristics of carbohydrates was evaluated on H 2 production and the associated changes in clostridial populations. A series of H 2-producing batch experiments was performed from mono-, di- to tri-saccharides (i.e. fructose, glucose, sucrose, maltose, cellobiose, maltotriose). Both chain length and alpha- or beta-linkage of carbohydrates impacted H 2 production performance as well as the patterns of hydrogenases. The H 2 yield, ranging from 1.38 to 1.84 mol-H 2/mol-hexose, decreased with the increasing chain length of the carbohydrates, showing a negative effect of the hydrolysis step on H 2 production efficiency. Changes in H 2 yield were associated with a specialization of clostridial species, which used different metabolic routes. The rise in H 2 production was associated with butyrate and acetate increases while H 2 consumption was related to caproate formation. Both clostridial [FeFe]- and [NiFe]-hydrogenases were identified in cellobiose cultures by a proteomic approach. This is the first study that combines genetic and proteomic analyses focused on H 2-producing bacteria under various conditions and it opens very interesting perspectives to better understand and optimize H 2 production using mixed cultures.

Biological formation of caproate and caprylate from acetate: fuel and chemical production from low grade biomass

This research introduces an alternative mixed culture fermentation technology for anaerobic digestion to recover valuable products from low grade biomass. In this mixed culture fermentation, organic waste streams are converted to caproate and caprylate as precursors for biodiesel or chemicals. It was found that acetate, as the main intermediate of anaerobic digestion, can be elongated to medium chain fatty acids with six and eight carbon atoms. Mixed microbial communities were able to produce 8.17 g l−1 caproate and 0.32 g l−1 caprylate under methanogenesis-suppressed conditions in a stable batch reactor run. The highest production rate was 25.6 mM C caproate per day with a product yield of 0.6 mol C per mol C. This elongation process occurred with both ethanol and hydrogen as electron donors, demonstrating the flexibility of the process. Microbial characterization revealed that the microbial populations were stable and dominated by relatives of Clostridium kluyveri.

Caproate formation in mixed-culture fermentative hydrogen production

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

Comparative performance of two upflow anaerobic biohydrogen-producing reactors seeded with different sludges

Comparisons were made between the performance of two upflow anaerobic biohydrogen-producing reactors. One reactor (R1) was seeded with methanogenic sludge, but was operated at low pH levels, whereas another (R2) was seeded with heat-pretreated sludge. Experiment results showed that H 2 partial pressure in biogas, H 2 production rate and H 2 yield were pH-dependent in the two reactors. The optimum pH for H 2 partial pressure was observed at pH 3.10 for R1 and 4.00 for R2, respectively. The optimum pH for maximum H 2 production rate and H 2 yield was 4.00 for both R1 and R2. Acetate, propionate, butyrate, i-butyrate, valerate, caproate and ethanol were main aqueous products and their distribution was also significantly influenced by pH. For R1, caproate was dominant at pH < 4.00 and the butyrate-type fermentation predominated at pH 4.5–6.0. For R2, butyrate was dominant at pH > 3.60 , while the ethanol-type fermentation was important at pH < 3.60 . With a thermodynamic analysis, possible metabolic pathways for the formation of caproate and valerate in both reactors were suggested.

The role of pH in the fermentative H 2 production from an acidogenic granule-based reactor

The role of pH in the fermentative H 2 production from an upflow acidogenic granule-based reactor was investigated in this study. Experimental results show that all H 2 partial pressure, H 2 production rate and H 2 yield were pH-dependent, in the range of 2.8 × 10 4–5.2 × 10 4 Pa, 61–145 ml-H 2 l −1 h −1 and 0.68–1.61 mol-H 2 mol-glucose −1, respectively. The maximum H 2 partial pressure was observed at pH 3.4, while both maximum H 2 production rate and H 2 yield were found at pH 4.2. Acetate, propionate, butyrate, i-butyrate, valerate, caporate and ethanol were present in the effluent of this UASB reactor, and their distribution was also pH-dependent. As pH was decreased from 4.2 to a lower level of 3.4 or increased to a higher level of 6.3, the fermentative type of this H 2-producing reactor would shift from butyrate-type to caporate- or ethanol-type. Thermodynamic analysis was performed to explore the possible metabolic pathways of caproate and valerate formation. The metabolic pathway of caproate formation was pH-dependent, while that of valerate formation was pH-independent. A neural network model was designed, trained and validated. It was able to successfully describe the daily variations of H 2 partial pressure and H 2 yield of the reactor, and to predict its steady state performance at various pHs.

Biological hydrogen production in a UASB reactor with granules. II: Reactor performance in 3-year operation

The experiment was conducted to evaluate the performance of an upflow anaerobic sludge blanket (UASB) with granules for H(2) production from a sucrose-rich synthetic wastewater at various substrate concentrations (5.33-28.07 g-COD/L) and hydraulic retention times (HRTs) (3-30 h) for over 3 years. The kinetics of H(2) production was evaluated, and the sludge yield and endogenous decay coefficient of the H(2)-producing granules were estimated to be 0.334 g-VSS/g-COD and 0.004/h, respectively. Based on Gibbs free energy calculations, the formation thermodynamics of caproate, an important aqueous product, were analyzed. Experimental results show that the H(2) partial pressure in biogas decreased with increasing substrate concentration, but was not sensitive to the variation of HRT in a range of 6-22 h. The H(2) production rate increased with increasing substrate concentration, but decreased with increasing HRT. The H(2) yield was in the range of 0.49-1.44 mol-H(2)/mol-glucose. Acetate, butyrate, caporate, and ethanol were the main aqueous products in the reactor, and their concentrations were dependent on both substrate concentration and HRT. An elevated substrate concentration resulted in a shift of fermentation from butyrate- to caporate-type in the reactor and the formation of caproate was dependent on the H(2) partial pressure. The 3-year experimental results demonstrate that H(2) could be produced continuously and stably from the acidogenic-granule-based UASB reactor.

Short chain flavour ester synthesis by a new esterase from Bacillus licheniformis

The newly characterized esterase from Bacillus licheniformis was used for ethyl ester synthesis in n-heptane by direct esterification with fatty acids of different chain lengths. The highest reaction rates and yields were obtained with caproic (C6) and caprylic (C8) acids reflecting the enzyme specificity for mid-chain length fatty acids. The rate of ethyl caproate formation increased linearly with the enzyme concentration in the range 0–40 mg/ml. The enzyme displayed maximum activity with 0.1% (v/v) addition of water. The initial reaction rate was maximal at 67°C but the highest yield (95%) was obtained at 45°C. Kinetic parameters were determined for caproic acid: K M of 38.4 mM and V M of 8.3 μmol/min/ml (at 0.25 M ethanol), and for ethanol K M of 12.3 mM and V M of 1.3 μmol/min/ml (at 0.25 M caproic acid). The highest activities and yields were observed for solvents having log P≥3.2. The enzyme was used three times without activity loss.

Formation of interim by-products in methanogenic degradation of butyrate

The formation of interim by-products during the methanogenic degradation of butyrate was monitored and analyzed in this study. Two series of experiments were conducted at various butyrate concentrations and under the influence of increased partial pressure of hydrogen ( P H 2 ). In all batches, acetate was found most abundant in the mixed liquor, accounting for over 52–83% of carbon in the original butyrate. This confirms that the degradation of butyrate was through acetate by β -oxidation, and the rate of butyrotrophic acetogenesis was considerably higher than that of acetotrophic methanogenesis. Assuming the degradation followed the Monod equation, the maximum-rate constant μ max was found in the range of 3.4–6.0 mg (mg VSS d) −1 and the half-rate concentration K s was 700–1150 mg l −1 . Other interim by-products included hydrogen (up to 10 −3.8 atm), propionate (up to 25 mg l −1 ), i -butyrate (up to 780 mg l −1 ), and several higher-molecular-weight carboxylic acids (up to 21 mg l −1 ). All these by-products became fully degraded eventually after reaching the maximum levels, except propionate which remained at the peak concentration. This suggests that the butyrate-degrading sludge was incapable of degrading propionate. Addition of formate to the feed solution increased the P H 2 , which enhanced the formation of propionate but did not affect butyrate degradation. The effect of P H 2 on propionate was in accordance with the chemical energy analysis. The chemical energy analysis also suggests that the formation of caproate, one of the higher-molecular-weight acids, was independent of the increase of P H 2 .

Homogeneous catalytic hydrogenation of sorbic acid with pentacyanocobaltate II

AbstractThe homogeneous hydrogenation of sodium sorbate, with pentacyanocobaltate II used as the catalyst, was followed manometrically at room temperature and atmospheric hydrogen pressure. One mole of hydrogen was absorbed by one mole of sorbate. UV and IR analyses demonstrated the reduction of sorbate to hexenoate. Gas liquid chromatography (GLC) of hexenoate indicated 82, 17, and 1% yields of 2‐,3‐, and 4‐hexenoates, respectively, with traces of sorbate. Increasing either the period of hydrogenation or the ratio of catalyst to sorbate, or both, did not cause the formation of caproate or change the ratio of the three isomers. Homogeneous catalytic hydrogenation in methanolic solutions showed higher selectivity since 2‐hexenoate was present in 96% yield. To standardize GLC columns, hexenoic acid isomers were synthesized by the Knoevenagel condensation.Trans‐ 2‐ hexenoic andtrans‐ 3‐ hexe‐ noic acids were prepared in 75 and 85% yields by a condensation ofn‐butanal and malonic acid in the presence of pyridine and triethanolamine, respectively.Trans‐ i‐ h.exea.oic acid was prepared in 70% yield by condensing diethyl malo‐nate with crotyl bromide. Physical properties, including UV and IR spectra, were determined for the purified synthetic acids and methyl esters. Although the absolute specificity of attack at the 4,5‐double bond of sorbic acid was not confirmed, the high degree of selectivity obtained does encourage further study of homogeneous catalysis on higher fatty acids.