Fermentative Acid Production Research Articles

Effect of D-limonene on volatile fatty acids production from anaerobic fermentation of waste activated sludge under pH regulation: performance and mechanisms

D-limonene extracted from citrus peels possesses an inhibitory effect on methanogenic archaea. This study is aimed to bridge the research gap on the influence of D-limonene on volatile fatty acids (VFA) production from waste activated sludge (WAS) and to address the low VFA yield in standalone anaerobic fermentation of WAS. When the initial pH was not controlled, 1.00 g/g TSS D-limonene resulted in a VFA accumulation of 1175.45 ± 101.36 mg/L (174.45 ± 8.13 mgCOD/gVS). When the initial pH was controlled at 10 and the D-limonene concentration was 0.50 g/g TSS, the VFA accumulation reached 2707.44 ± 183.65 mg/L (445.51 ± 17.10 mgCOD/gVS). The pH-regulated D-limonene treatment enhanced solubilization and acidification, slightly inhibited hydrolysis, and significantly suppressed methanogenesis. D-limonene under alkaline conditions can increase the relative abundance of Clostridium_sensu_stricto, significantly enhancing acidification. Moreover, it markedly inhibited methanogenesis by particularly reducing the relative abundance of Methanothrix that was responsible for acetate consumption, thus favoring the accumulation of VFA. The research reveals the potential mechanism of pH regulation and D-limonene on anaerobic fermentation acid production, providing a theoretical basis for improving the acid production performance of the anaerobic fermentation of WAS.

Expression of a Recombinant Cholesterol Esterase from Mustela putorius furo in Pichia pastoris and Its Applicability for γ-Oryzanol Hydrolysis.

Ferulic acid (FA) exhibits antioxidant and anti-inflammatory properties, making it valuable for numerous industrial applications. Traditionally, FA is produced by the alkaline hydrolysis of γ-oryzanol, which is typically associated with wastewater generation. Recently, an increasing demand of natural FA necessitates its green production via enzymatic hydrolysis of γ-oryzanol, a mixture comprising triterpene alcohol ferulates and phytosteryl ferulates. Thus far, γ-oryzanol can be hydrolyzed by only four commercial cholesterol esterases with low yields. Herein, we report a recombinant cholesterol esterase from Mustela putorius furo (MPFCE) for the enzymatic hydrolysis of γ-oryzanol. The enzyme yielded 25.5% FA, which is the highest reported through enzymatic means thus far. The hydrolysis profile revealed that the enhanced yield primarily resulted from the near-complete hydrolysis of phytosteryl ferulates, together with slight hydrolysis of triterpene alcohol ferulates. MPFCE serves as a potential candidate for the enzymatic production of FA through targeted hydrolysis of γ-oryzanol.

A growth-based screening strategy for engineering the catalytic activity of an oxygen-sensitive formate dehydrogenase.

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli. This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (EcFDH-H). Using an EcFDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active EcFDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using EcFDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 EcFDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO2 and N2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications.

Effects of acetic acid fermentation product and its components on defense signaling in rice.

Acetic acid fermentation product made from isomalto-oligosaccharide as the main raw material is composed of isomalto-oligosaccharide and acetic acid. In this paper, we have shown that the fermentation product enhanced the expression of disease resistance genes in rice, and that its main functional component was acetic acid. It has been reported so far that acetic acid enhances the jasmonic acid signaling pathway, while the role of isomalto-oligosaccharide in plant defense signaling remains unclear. In this study, we demonstrated the possibility that isomalto-oligosaccharide shifted part of the jasmonic acid signaling pathway, which is enhanced by acetic acid, to the salicylic acid signaling pathway, which is the other major defense pathway. Furthermore, glucose, a constituent monosaccharide of isomalto-oligosaccharide, and a disaccharide maltose had little effect on the signaling pathway, but a trisaccharide maltotriose tended to have a similar effect to isomalto-oligosaccharide on the defense signaling pathway.

Study on the effect of alkali on the production of volatile fatty acids from rhamnolipid‐alkali‐thermal pretreated waste activated sludge

AbstractThis article systematically explores the strengthening effect of NaOH dosage on the fermentation acid production of Waste Activated Sludge (WAS) by the rhamnolipids‐alkali‐thermal combined pretreatment. Analyzed the dissolution characteristics of organic matter in sludge, changes in total volatile fatty acids (VFAs) content and components, and microbial community structure. Research has shown that increasing the amount of NaOH can promote the dissolution of sludge extracellular polymeric substances (EPS) and cell lysis. When NaOH dose was 60 mg/g VS, the dissolution of SCOD was the largest, reaching 5427.78 mg/L. It provides high‐quality substrate for anaerobic flora through the decomposition of refractory humus‐like substances and macromolecular substances, thus improving the production of VFAs. When NaOH dose was 80 mg/g VS, the maximum VFAs yield was 1663.76 mg C/L, and the fermentation type was butyric acid. The increase of NaOH dose promotes the effective enrichment of bacteria, and the dominant bacteria are Proteobacteria, Firmicutes, and Bacteroides at the gate level, with different abundance. The fermentation time plays a decisive role in the evolution of microbial population, and the increase of NaOH dose promotes the enrichment of acid‐producing bacteria, thus improving the production of VFAs.

Bipolar membrane electrodialysis for efficient production of ferulic acid in alcohol/water mixed solvent

In this study, bipolar membrane electrodialysis (BMED) was proposed to produce ferulic acid, as a kind of poorly water-soluble organic acids, in the alcohol/water mixed solvent system. Among the mixed solvents of EtOH/H2O, n-PrOH/H2O, and i-PrOH/H2O, high solubilities of both ferulic acid and sodium ferulate were achieved in the solvent of 50 v/v% n-PrOH/H2O. Meanwhile, the ionization of the produced ferulic acid was relatively low in the above system due to a feasible pKa of ferulic acid, resulting in a high purity of the product. Furthermore, a low current density of 10 mA/cm2 was recommended for the BMED process due to the stable membrane performance (no obvious membrane fouling observed) and low energy consumption. Moreover, the ionization of ferulic acid can be further lowered due to the solvation effects at a high feed concentration (0.5 mol/L, nearly saturated concentration). The low ionization of ferulic acid means that the migration of H+ ions instead of Na+ ions through cation exchange membrane can be suppressed efficiently, resulting in a high concentration of the product. Under the optimized parameters, the purity of ferulic acid, the current efficiency and energy consumption were 99.5%, 99.4%, and 0.30 kWh/kg ferulic acid, respectively. Also, the process economy estimation shows a low total process cost of 0.49 $/kg ferulic acid. Therefore, the BMED process exhibits an excellent economic competitiveness for efficient production of ferulic acid.

Bacterial diversity of stingless bee honey in Yunnan, China: isolation and genome sequencing of a novel acid-resistant Lactobacillus pentosus (SYBC-MI) with probiotic and L. tryptophan producing potential via millet fermentation.

Stingless bee (Hymenoptera, Apidae, and Trigona) honey is a remarkable "miracle liquid" with a wide range of medical benefits for conditions including gastroenteritis, cataracts, and wound healing. Our study aimed to isolate, identify, and characterize acid-resistant Lactobacillus spp. from sour honey distributed in Yunnan, China. To assess the safety of an entirely novel Lactobacillus pentosus strain, S4 (OM618128), based on probiotic property evaluation and whole-genome sequencing analysis. A 16S rRNA gene high-throughput sequencing analysis showed that Lactobacillus was abundant at the genus level in sour honey. Seven Lactobacillus strains (viz. S1-7) were isolated from sour honey using a multiple-anaerobic culture enrichment method. One potential acid-resistant isolate, Lactobacillus sp. S4, was obtained after screening the seven Lactobacillus isolates, and it had the highest lactic acid production (17.62g/L), followed by Lactobacillus sp. S3 (17.07g/L). Phylogenetic and comparative analyses of conserved sequence regions have shown that all seven strains are phylogenetically located in the Lactobacillus pentosus sub-cluster. In L. pentosus SYBC-MI, there is a circular chromosome (3288615 bps) and 11,466 bps plasmids. GC content is 44.03%. The number of predicted genes is 3,129, with 16 rRNAs and 74 tRNAs present. During the fermentation of foxtail millet by seven Lactobacillus pentosus (S1-7) strains isolated from sour honey, a potential tryptophan accumulating isolate, Lactobacillus pentosus S4, was obtained, which could reach a maximum tryptophan content of 238.43mgL-1 that is 1.80 times the initial tryptophan content in the fermentation broth. This strain has strong acid tolerance, salt tolerance, and fermentation acid production abilities. This strain degrades nitrite at a rate of over 99%, and it has high probiotic potential as well. This project has established a solid foundation for further exploring the excellent lactic acid bacteria in sour honey. It is also investigating the key taxa and their role in the environment. According to the results of our studies, these LAB isolates provide a lot of potential for use in the future, as a source of probiotics for human, animals, and starter cultures for food applications.

Nitrogen fertilization enhances growth and development of Cacopsylla chinensis by modifying production of ferulic acid and amino acids in pears

Nitrogen fertilization enhances growth and development of Cacopsylla chinensis by modifying production of ferulic acid and amino acids in pears

Production of ferulic acid from dried bamboo shoots for the biotransformation into vanillin using a novel microbe Enterobacter aerogenes

Production of ferulic acid from dried bamboo shoots for the biotransformation into vanillin using a novel microbe Enterobacter aerogenes

Structural insights into the oligomeric effects on catalytic activity of a decameric feruloyl esterase and its application in ferulic acid production

Oligomeric feruloyl esterase (FAE) has great application prospect in industry due to its potentially high stability and fine-tuned activity. However, the relationship between catalytic capability and oligomeric structure remains undetermined. Here we identified and characterized a novel, cold-adapted FAE (BtFae) derived from Bacteroides thetaiotaomicron. Structural studies unraveled that BtFae adopts a barrel-like decameric architecture unique in esterase families. By disrupting the interface, the monomeric variant exhibited significantly reduced catalytic activity and stability toward methyl ferulate, potentially due to its impact on the flexibility of the catalytic triad. Additionally, our results also showed that the monomerization of BtFae severely decreased the ferulic acid release from de-starched wheat bran and insoluble wheat arabinoxylan by 75 % and 80 %, respectively. Collectively, this study revealed novel connections between oligomerization and FAE catalytic function, which will benefit for further protein engineering of FAEs at the quaternary structure level for improved industrial applications.

A Rehmannia glutinosa caffeic acid O-methyltransferase functional identification: Reconstitution of the ferulic acid biosynthetic pathway in Saccharomyces cerevisiae using Rehmannia glutinosa enzymes.

Rehmannia glutinosa produces many pharmacological natural components, including ferulic acid (FA) which is also an important precursor of some medicinal ingredients, so it is very significant to explore FA biosynthesis for enhancing the production of FA and its derivations. This study aimed to determine and reconstitute the R. glutinosa FA biosynthetic pathway from phenylalanine (Phe) metabolism in Saccharomyces cerevisiae as a safe host for the biosynthesis of plant-derived products. Although plant caffeic acid O-methyltransferases (COMTs) are thought to be a vital catalytic enzyme in FA biosynthesis pathways, to date, none of the RgCOMTs in R. glutinosa has been characterized. This study identified an RgCOMT and revealed its protein enzymatic activity for FA production in vitro. The RgCOMT overexpression in R. glutinosa significantly increased FA yield, suggesting that its molecular function is involved in FA biosynthesis. Heterologous expression of the RgCOMT and reported R. glutinosa genes, RgPAL2 (encoding phenylalanine ammonia-lyase [PAL] protein), RgC4H (cinnamate 4-hydroxylase [C4H]), and RgC3H (p-coumarate-3-hydroxylase [C3H]), in S. cerevisiae confirmed their catalytic abilities in the reaction steps for the FA biosynthesis. Importantly, in this study, these genes were introduced into S. cerevisiae and coexpressed to reconstitute the R. glutinosa FA biosynthetic pathway from Phe metabolism, thus obtaining an engineered strain that produced an FA titer of 148.34mgL-1 . This study identified the functional activity of RgCOMT and clarified the R. glutinosa FA biosynthesis pathway in S. cerevisiae, paving the way for the efficient production of FA and its derivatives.

커피발효물의 발효특성 및 항비만 효과

This study was conducted to investigate the fermentation characteristics and anti-obesity effects of acetic acid fermentation products of coffee wine. The live cell counts, soluble solids, pH and total acidity of the acetic acid unfermented coffee wine (AUFCW; day 0, before fermentation) were 6.35 log CFU/mL, 8.10 °Brix, 3.88, and 1.29%, respectively, while the acetic acid fermented coffee wine (AFCW; day 15, after fermentation) were 4.40 log CFU/mL, 8.57 °Brix, 3.07, and 7.45%, respectively. Pancreatic lipase inhibitory activity tended to increase as the acetic acid fermentation period increased. The anti-obesity effects of AFCW on 3T3-L1 cells, which was induced by MDI, were evaluated based on the lipid accumulation rate, leptin expression, and fat production-related gene expression (PPAR-γ and SREBP-1c) at the mRNA level. In the case of AFCW, the lipid accumulation rate and leptin expression were decreased to 69.37% and 50.20% at a concentration of 200 μg/mL, respectively, and the expression levels of PPAR-γ and SREBP-1c at the mRNA level were decreased to 79.89% and 48.81%, respectively. These results indicate that anti-obesity effect of acetic acid fermentation products could be increased by acetic acid fermentation of coffee wine.

Extracellular Expression of Feruloyl Esterase and Xylanase in Escherichia coli for Ferulic Acid Production from Agricultural Residues.

There is still a large amount of ferulic acid (FA), an outstanding antioxidant, present in agricultural residues. Enzymatic hydrolysis has been regarded as the most effective way to release FA. This present study therefore selected feruloyl esterase (FAE) and xylanase (XYN) from the metagenomes of a cow rumen and a camel rumen, respectively, for their recombinant expression in Escherichia coli BL21 and further application in releasing FA. After screening the candidate signal peptides, the optimal one for each enzyme, which were selected as SP1 and SP4, respectively, was integrated into the vectors pET22b(+) and pETDuet-1. Among the generated E. coli strains SP1-F, SP4-X, and SP1-F-SP4-X that could express extracellular enzymes either separately or simultaneously, the latter one performed the best in relation to degrading the biomass and releasing FA. Under the optimized culture and induction conditions, the strain SP1-F-SP4-X released 90% of FA from 10% of de-starched wheat bran and produced 314.1 mg/L FA, which was deemed to be the highest obtained value to the best of our knowledge. This result could pave a way for the re-utilization of agricultural residues and enhancing their add-value.

Feruloyl Esterase (LaFae) from Lactobacillus acidophilus: Structural Insights and Functional Characterization for Application in Ferulic Acid Production.

Ferulic acid and related hydroxycinnamic acids, used as antioxidants and preservatives in the food, cosmetic, pharmaceutical and biotechnology industries, are among the most abundant phenolic compounds present in plant biomass. Identification of novel compounds that can produce ferulic acid and hydroxycinnamic acids, that are safe and can be mass-produced, is critical for the sustainability of these industries. In this study, we aimed to obtain and characterize a feruloyl esterase (LaFae) from Lactobacillus acidophilus. Our results demonstrated that LaFae reacts with ethyl ferulate and can be used to effectively produce ferulic acid from wheat bran, rice bran and corn stalks. In addition, xylanase supplementation was found to enhance LaFae enzymatic hydrolysis, thereby augmenting ferulic acid production. To further investigate the active site configuration of LaFae, crystal structures of unliganded and ethyl ferulate-bound LaFae were determined at 2.3 and 2.19 Å resolutions, respectively. Structural analysis shows that a Phe34 residue, located at the active site entrance, acts as a gatekeeper residue and controls substrate binding. Mutating this Phe34 to Ala produced an approximately 1.6-fold increase in LaFae activity against p-nitrophenyl butyrate. Our results highlight the considerable application potential of LaFae to produce ferulic acid from plant biomass and agricultural by-products.

Ferulic acid production from wheat bran by integration of enzymatic pretreatment and a cold-adapted carboxylesterase catalysis

High-value chemical production from natural lignocellulose transformation is a reliable waste utilization approach. A gene encoding cold-adapted carboxylesterase in Arthrobacter soli Em07 was identified. The gene was cloned and expressed in Escherichia coli to obtain a carboxylesterase enzyme with a molecular weight of 37.2 KDa. The activity of the enzyme was determined using α-naphthyl acetate as substrate. Results showed that the optimum enzyme activity of carboxylesterase was at 10 °C and pH 7.0. It was also found that the enzyme could degrade 20 mg enzymatic pretreated de-starched wheat bran (DSWB) to produce 235.8 μg of ferulic acid under the same conditions, which was 5.6 times more than the control. Compared to the chemical strategy, enzymatic pretreatment is advantageous because it is environmentally friendly, and the by-products can be easily treated. Therefore, this strategy provides an effective method for high-value utilization of biomass waste in agriculture and industry.

Efficient production of ferulic acid and p-coumaric acid from reed straws via combined enzymatic hydrolysis and hydrothermal pretreatment

The present study aimed to evaluate an available process for production of ferulic acid (FA) and p-coumaric acid (pCA) that could be potentially used as functional agents in food processing. The hydrothermal pretreatment combined with enzymatic hydrolysis (HP-CEH) was explored for efficient production of phenolic acids from reed straws. The results indicated that HP-CEH significantly increased production of phenolic acids. The recovery yield of ferulic acid and p-coumaric acid was respectively found to be 0.69 and 0.81 mg/g dry reed straw when performed by HP-CEH for 16 h. The degradation of hemicellulose, lignocellulose and lignin, and disruption of cell walls or cellulose fibers contributed to phenolic acids production from reed straws. Moreover, the scavenging activity of hydroxyl radical and DPPH radical of the phenolic acids extracted by HP-CEH was respectively obtained to be 77.32% and 86.77%.

Beyond purified dietary fibre supplements: Compositional variation between cell wall fibre from different plants influences human faecal microbiota activity and growth in vitro.

Dietary fibre is a major energy source for the human gut microbiota, but it is unclear to what extent the fibre source and complexity affect microbial growth and metabolite production. Cell wall material and pectin were extracted from five different dicotyledon plant sources, apples, beet leaves, beetroots, carrots and kale, and compositional analysis revealed differences in the monosaccharide composition. Human faecal batch incubations were conducted with 14 different substrates, including the plant extracts, wheat bran and commercially available carbohydrates. Microbial activity was determined for up to 72 h by measuring gas and fermentation acid production, total bacteria (by qPCR) and microbial community composition by 16S rRNA amplicon sequencing. The more complex substrates gave rise to more microbiota variation compared with the pectins. The comparison of different plant organs showed that the leaves (beet leaf and kale) and roots (carrot and beetroot) did not give rise to similar bacterial communities. Rather, the compositional features of the plants, such as high arabinan levels in beet and high galactan levels in carrot, appear to be major predictors of bacterial enrichment on the substrates. Thus, in-depth knowledge on dietary fibre composition should aid the design of diets focused on optimizing the microbiota.

High efficiency production of ferulic acid using feruloyl esterase and xylanase in deep eutectic solvents

The mixed enzymatic system showed the best performance in the ChCl-Gly DES aqueous solution (a volume ratio of 1 : 2).

Concurrent production of ferulic acid and glucose from wheat bran by catalysis of a putative bifunctional enzyme

The aim of this work is to study a bifunctional endoglucanase/carboxylesterase in Sphingobacterium soilsilvae Em02 and express it in soluble form in engineered Escherichia coli. The molecular weight of the recombinant protein of the bifunctional enzyme was 41 KDa. This research also determined the enzymatic activities of the bifunctional enzymes using microcrystalline cellulose and p-nitrophenyl butyrate as substrates and found 40 °C as the optimum temperature for their enzymatic activities. The optimal pH in dual function was 6.0 for endoglucanase and 7.0 for carboxylesterase. The bifunctional enzyme also exhibited enzymatic activities on the natural biomass by generating up to 3.94 mg of glucose and 49.4 μg of ferulic acid from 20 mg of destarched wheat bran. This indicates the broad application prospects of the bifunctional enzyme in agriculture and industry.

Effects of rhamnolipid pretreatment on DOM dissolution characteristics and anaerobic fermentation acid production of waste activated sludge

ABSTRACT In this paper, the change characteristics of DOM (dissolved organic matter) and acid production characteristics of anaerobic fermentation in waste activated sludge(WAS) pretreated with rhamnolipid (RL) were studied. The results showed that RL at the dose of 80 mg/gTS could significantly promote the disintegrating of EPS (extracellular polymers) and cell wall in WAS, and a large number of proteins and carbohydrates were dissolved. Three dimensional fluorescence parallel factor analysis showed that the addition of RL enhanced the dissolution and biodegradability of humus-like substances. LC-OCD (Liquid chromatography – organic carbon detection) analysis showed that RL promotes the dissolution of biodegradable components such as Biopolymer, Building Blocks and LMW Neutrals, and ensures the increase of VFA (volatile fatty acids) production in the process of anaerobic fermentation. Under the RL dose of 80 mg/gTS, the maximum VFA production of WAS was obtained at 108 h of anaerobic fermentation, which was 2699.39 mg/L. Acetic acid and propionic acid were the main components in the WAS fermentation broth pretreated by RL. The concentration of butyric acid increased with the increase of RL dose. The RL dose can significantly affect the composition of VFA in WAS fermentation broth.