13C-labelled Glucose Reveals Shifts In Fermentation Pathway During Cathodic Electro-Fermentation With Mixed Microbial Culture.

Cathodic Electro Fermentation (CEF) is an innovative approach to manage the spectrum of products deriving from anaerobic fermentation. Herein, mixed microbial culture fermentation using a ternary mixture containing labelled 13C glucose and non-labelled acetate and ethanol was studied to identify the role of polarization on the metabolic pathways of glucose fermentation. CEF at an applied potential of -700 mV (vs. SHE, Standard Hydrogen Electrode) enhanced the production yield of acetate, propionate, and butyrate (0.90 ± 0.10, 0.22 ± 0.03, and 0.34 ± 0.05 mol/mol; respectively) compared to control tests performed at open circuit potential (OCP) (0.54 ± 0.09, 0.15 ± 0.04 and, 0.21 ± 0.001 mol/mol, respectively). Results indicate that CEF affected the 13C labelled fermented product levels and their fractional 13C enrichments, allowing to establish metabolic pathway models. This work demonstrates that, under cathodic polarization, the abundance of both fully 13C labelled propionate and butyrate isotopomers increased compared to control tests. The effect of CEF is mainly due to intermediates initially produced from the glucose metabolic transformation in the presence of non-labelled acetate and ethanol as external substrates. These findings represent a significant advancement in current knowledge of CEF, which offers a promising tool to control mixed cultures bioprocesses.

Environmental pH and compound structure affect the activity of short-chain carboxylic acids against planktonic growth, biofilm formation, and eradication of the food pathogen Salmonella enterica.

Short-chain carboxylic acids (SCCAs) that are naturally produced by microbial fermentation play an essential role in delaying microbial spoilage. SCCAs are structurally diverse, but only a few of them are routinely used in food biopreservation. This study investigated the effects of environmental pH and intrinsic properties of 21 structurally different SCCAs on the antimicrobial and antibiofilm activity against Salmonella enterica. Inhibition of SCCA toward planktonic and biofilm growth of S. enterica was higher in an acidic environment (pH 4.5) that is common in fermented products, and for SCCA that possessed both a high acid dissociation strength (pKa) (>4.0) and a positive hydrophobicity [octanol/water partition coefficient (log Kow)]. Crotonic and caproic acids were identified as SCCAs with potential as biopreservatives even at near-neutral pH. SCCA with hydrophilic groups such as lactic acid did not inhibit S. enterica at concentrations up to 50 mM, while SCCA with benzene or methyl groups or a double bond prevented S. enterica growth and biofilm formation. Stimulation of biofilm formation was observed for formic, acetic, and propionic acid close to the minimum inhibitory concentration to reduce 50% of cell density (MIC50) of planktonic cells, and for citric and isocitric acid with an MIC50 of ≥50 mM. The presence of low concentrations of formic and propionic acids during biofilm formation conferred protection during eradication possibly due to a pre-adaptation effect, yet two consecutive acid treatments were successful in eradicating biofilms if the first acid treatment was two- to threefold of the MIC50.IMPORTANCEThis study provides a systematic comparison on the antimicrobial and antibiofilm activity of more than 20 structurally different SCCAs against a common food pathogen. We tested the antimicrobial activity at controlled pH and identified the structure-dependent antimicrobial effects of SCCA without the confounding influence of acidification. The combined effect of pKa and log Kow was identified as an important feature that should be considered when deciding for a specific SCCA in the application as antimicrobial. Our results imply that additional phenomena such as the use of SCCA as substrate and cellular pre-adaption effects have to be taken into consideration. We finally present a two-step treatment as an efficient approach to eradicate biofilms, which can be applied for the disinfection of contact surfaces and manufacturing equipment. Results obtained here can serve as guidelines for application of SCCA to avoid the growth of food pathogens and/or to develop biopreserved food systems.

Short-chain fatty and carboxylic acid changes associated with fecal microbiota transplant communally influence microglial inflammation.

The intestinal microbiota has been proposed to influence human mental health and cognition through the gut-brain axis. Individuals experiencing recurrent Clostridioides difficile infection (rCDI) frequently report depressive symptoms, which are improved after fecal microbiota transplantation (FMT); however, mechanisms underlying this association are poorly understood. Short-chain fatty acids and carboxylic acids (SCCA) produced by the intestinal microbiota cross the blood brain barrier and have been proposed to contribute to gut-brain communication. We hypothesized that changes in serum SCCA measured before and after successful FMT for rCDI influences the inflammatory response of microglia, the resident immune cells of the central nervous system. Serum SCCA were quantified using gas chromatography-mass spectroscopy from 38 patients who participated in a randomized trial comparing oral capsule-vs colonoscopy-delivered FMT for rCDI, and quality of life was assessed by SF-36 at baseline, 4, and 12 weeks after FMT treatment. Successful FMT was associated with improvements in mental and physical health, as well as significant changes in a number of circulating SCCA, including increased butyrate, 2-methylbutyrate, valerate, and isovalerate, and decreased 2-hydroxybutyrate. Primary cultured microglia were treated with SCCA and the response to a pro-inflammatory stimulus was measured. Treatment with a combination of SCCA based on the post-FMT serum profile, but not single SCCA species, resulted in significantly reduced inflammatory response including reduced cytokine release, reduced nitric oxide release, and accumulation of intracellular lipid droplets. This suggests that both levels and diversity of SCCA may be an important contributor to gut-brain communication.

Assay of short-chain carboxylic acids in plasma and urine using gas chromatography after extractive alkylation.

After extractive alkylation combined with plasma deproteinization, we used gas chromatography to assay for short-chain carboxylic acids from formic acid to valeric acid in plasma and urine. It was possible to provide highly sensitive analysis with 0.1-3.4 µg/mL as the limit of detection for plasma and 0.6-8.0 µg/mL for urine, with a correlation coefficient of 1.000 for the linear regression calibration curves. For plasma, deproteinization using ultrafiltration before extractive alkylation resulted in a higher sensitivity for acetic, propionic, butyric, and valeric acids compared with the method without deproteinization. The concentrations of formic acid and acetic acid were determined to be 6 µg/mL and 10 µg/mL, respectively, in the tested plasma, and 22 µg/mL and 32 µg/mL, respectively, in the tested urine. Concentrations from propionic acid to valeric acid were ≤ 1.3 µg/mL. In addition, high concentrations of sulfate, phosphate, hydrogen carbonate, ammonium, and/or sodium ions did not remarkably inhibit the derivatization of carboxylic acids, although hydrogen carbonate ions significantly inhibited that of formic acid.

Improving production of lactic acid and volatile fatty acids from dairy cattle manure and corn straw silage: Effects of mixing ratios and temperature.

Anaerobic co-fermentation (AcoF) of dairy cattle manure (DCM) and corn straw silage (CSS) for producing lactic acid (LA) and volatile fatty acids (VFAs) was investigated. Batch experiments were conducted at seven different DCM/CSS ratios and at mesophilic and thermophilic temperatures. Results indicated that the highest concentration of LA was 17.50 ± 0.70 g/L at DCM:CSS ratio of 1:3 and thermophilic temperature, while VFAs was 18.23 ± 2.45 g/L at mono-CSS fermentation and mesophilic temperature. High solubilization of thermophilic conditions contributed to LA accumulation in AcoF process. Presence of the CSS increased the relative abundance of Lactobacillus for LA production at thermophilic. Meanwhile, the abundance of Bifidobacterium was increased when CSS was added at mesophilic, which could conduce to VFAs production. This study provides a new route for enhancing the biotransformation of DCM and CSS into short-chain fatty acids, potentially bringing economic benefits to agricultural waste treatment.

Potential of anaerobic co-fermentation in wastewater treatments plants: A review.

Fermentation (not anaerobic digestion) is an emerging biotechnology to transform waste into easily assimilable organic compounds such as volatile fatty acids, lactic acid and alcohols. Co-fermentation, the simultaneous fermentation of two or more waste, is an opportunity for wastewater treatment plants (WWTPs) to increase the yields of sludge mono-fermentation. Most publications have studied waste activated sludge co-fermentation with food waste or agri-industrial waste. Mixing ratio, pH and temperature are the most studied variables. The highest fermentation yields have been generally achieved in mixtures dominated by the most biodegradable substrate at circumneutral pH and mesophilic conditions. Nonetheless, most experiments have been performed in batch assays which results are driven by the capabilities of the starting microbial community and do not allow evaluating the microbial acclimation that occurs under continuous conditions. Temperature, pH, hydraulic retention time and organic load are variables that can be controlled to optimise the performance of continuous co-fermenters (i.e., favour waste hydrolysis and fermentation and limit the proliferation of methanogens). This review also discusses the integration of co-fermentation with other biotechnologies in WWTPs. Overall, this review presents a comprehensive and critical review of the achievements on co-fermentation research and lays the foundation for future research.

Improvement of Extractive Alkylation Gas Chromatography of Short-chain Carboxylic Acids in Aqueous Solution.

In the analysis of short-chain carboxylic acids such as formic acid and acetic acid in aqueous solution using extractive alkylation gas chromatography, tetrahexylammonium bromide (THAB) as a phase-transfer catalyst (PTC) causes a high intensity and broad peaks in the gas chromatogram, and interfere with the detection of carboxylic acid derivatives. By an easy treatment of the extractive alkylation solution with perchloric acid and n-hexane, it is possible to remove more than 95% of THAB, and to provide good gas chromatogram with a little admixture of carboxylic acid derivatives. The desensitization was 16% at the maximum, the contamination of the glass insert in gas chromatograph and liquid phase in column by THAB was minimized, and trouble in continuous measurement could be avoided.

Short chain carboxylic acids production and dynamicity of microbial communities from co-digestion of swine manure and corn silage.

Short chain carboxylic acids (SCCAs) have attracted much attention due to their wide application and benefit of high economy. This study investigated the influence of organic load rates (OLRs) on short-chain carboxylic acids (SCCAs) production and microbial communities for co-digestion of swine manure (SM) and corn silage (CS) during four different OLRs stages in a semi-continuous mode. The results showed that relatively stable SCCAs concentration of 10.5-13.6 g COD/L and SCCAs yield of 0.42 mg COD/mg VS was achieved at OLR of 3.0 g VS/L·d. The maximum concentration of 19.1 g COD/L was achieved at 3.5 g VS/L·d. Volatile fatty acids (VFAs) (include acetic, n-butyric and caproic acids) accounted for approximately 80% of SCCAs. Hydrolysis bacteria (HB) including Clostridium, Terriporobacter, Intestinibacter, and Turiibacter decreased with the increase of OLR, while acidogenic bacteria (AB) including Acetobacter, Lactobacillus, Aeriscardovia, and Pseudomonas increased, resulting in insufficient degradation of CS.

Acidogenic fermentation of food waste for volatile fatty acid production with co-generation of biohydrogen.

Fermentation experiments were designed to elucidate the functional role of the redox microenvironment on volatile fatty acid (VFA, short chain carboxylic acid) production and co-generation of biohydrogen (H2). Higher VFA productivity was observed at pH 10 operation (6.3g/l) followed by pH 9, pH 6, pH 5, pH 7, pH 8 and pH 11 (3.5 g/l). High degree of acidification, good system buffering capacity along with co-generation of higher H2 production from food waste was also noticed at alkaline condition. Experiments illustrated the role of initial pH on carboxylic acids synthesis. Alkaline redox conditions assist solubilization of carbohydrates, protein and fats and also suppress the growth of methanogens. Among the carboxylic acids, acetate fraction was higher at alkaline condition than corresponding neutral or acidic operations. Integrated process of VFA production from waste with co-generation of H2 can be considered as a green and sustainable platform for value-addition.