Volatile Fatty Acids Effective as Antibacterial Agents against Three Enteric Bacteria during Mesophilic Anaerobic Incubation.

The antibacterial effects of a selection of volatile fatty acids (acetic, propionic, butyric, valeric, and caproic acids) relevant to anaerobic digestion were investigated at 1, 2 and 4 g/L. The antibacterial effects were characterised by the dynamics of Enterococcus faecalis NCTC 00775, Escherichia coli JCM 1649 and Klebsiella pneumoniae A17. Mesophilic anaerobic incubation to determine the minimum bactericidal concentration (MBC) and median lethal concentration of the VFAs was carried out in Luria Bertani broth at 37 °C for 48 h. Samples collected at times 0, 3, 6, 24 and 48 h were used to monitor bacterial kinetics and pH. VFAs at 4 g/L demonstrated the highest bactericidal effect (p < 0.05), while 1 g/L supported bacterial growth. The VFA cocktail was the most effective, while propionic acid was the least effective. Enterococcus faecalis NCTC 00775 was the most resistant strain with the VFAs MBC of 4 g/L, while Klebsiella pneumoniae A17 was the least resistant with the VFAs MBC of 2 g/L. Allowing a 48 h incubation period led to more log decline in the bacterial numbers compared to earlier times. The VFA cocktail, valeric, and caproic acids at 4 g/L achieved elimination of the three bacteria strains, with over 7 log10 decrease within 48 h.

Efforts to install a heterologous Wood-Ljungdahl pathway in Clostridium acetobutylicum enable the identification of the native tetrahydrofolate (THF) cycle and result in early induction of solvents.

Here, we report the construction of a Clostridium acetobutylicum strain ATCC 824 (pCD07239) by heterologous expression of carbonyl branch genes (CD630_0723∼CD630_0729) from Clostridium difficile, aimed at installing a heterologous Wood-Ljungdahl pathway (WLP). As part of this effort, in order to validate the methyl branch of the WLP in the C. acetobutylicum, we performed 13C-tracing analysis on knockdown mutants of four genes responsible for the formation of 5-methyl-tetrahydrofolate (5-methyl-THF) from formate: CA_C3201, CA_C2310, CA_C2083, and CA_C0291. While C. acetobutylicum 824 (pCD07239) could not grow autotrophically, in heterotrophic fermentation, it began producing butanol at the early growth phase (OD600 of 0.80; 0.162 g/L butanol). In contrast, solvent production in the parent strain did not begin until the early stationary phase (OD600 of 7.40). This study offers valuable insights for future research on biobutanol production during the early growth phase.

Mediation of Bacterial Interactions via a Novel Membrane-Based Segregator to Enhance Biological Nitrogen Removal.

The regulation of microbial subpopulations in wastewater treatment plants (WWTPs) with desired functions can guarantee nutrient removal. In nature, "good fences make good neighbors," which can be applied to engineering microbial consortia. Herein, a membrane-based segregator (MBSR) was proposed, where porous membranes not only promote the diffusion of metabolic products but also isolate incompatible microbes. The MBSR was integrated with an anoxic/aerobic membrane bioreactor (i.e., an experimental MBR). The long-term operation showed that the experimental MBR exhibited higher nitrogen removal (10.45 ± 2.73 mg/L total nitrogen) than the control MBR (21.68 ± 4.23 mg/L) in the effluent. The MBSR resulted in much lower oxygen reduction potential in the anoxic tank of the experimental MBR (-82.00 mV) compared to that of the control MBR (83.25 mV). The lower oxygen reduction potential can inevitably aid in the occurrence of denitrification. The 16S rRNA sequencing showed that the MBSR significantly enriched acidogenic consortia, which yielded considerable volatile fatty acids by fermenting the added carbon sources and allowed efficient transfer of these small molecules to the denitrifying community. Moreover, the sludge communities of the experimental MBR harbored a higher abundance of denitrifying bacteria than those of the control MBR. Metagenomic analysis further corroborated these sequencing results. The spatially structured microbial communities in the experimental MBR system demonstrate the practicability of the MBSR, achieving nitrogen removal efficiency superior to that of mixed populations. Our study provides an engineering method for modulating the assembly and metabolic division of labor of subpopulations in WWTPs. IMPORTANCE This study provides an innovative and applicable method for regulating subpopulations (activated sludge and acidogenic consortia), which contributes to the precise control of the metabolic division of labor in biological wastewater treatment processes.

Strategies to control pH in the dark fermentation of sugarcane vinasse: Impacts on sulfate reduction, biohydrogen production and metabolite distribution.

pH is notably known as the main variable defining distinct metabolic pathways during sugarcane vinasse dark fermentation. However, different alkalinizing (e.g. sodium bicarbonate; NaHCO3) and/or neutralizing (e.g. sodium hydroxide; NaOH) approaches were never directly compared to understand the associated impacts on metabolite profiles. Three anaerobic structured-bed reactors (AnSTBR) were operated in parallel and subjected to equivalent operational parameters, except for the pH control: an acidogenic-sulfidogenic (R1; NaOH + NaHCO3) designed to remove sulfur compounds (sulfate and sulfide), a hydrogenogenic (R2; NaOH) aimed to optimize biohydrogen (bioH2) production, and a strictly fermentative system without pH adjustment (R3) to mainly evaluate lactic acid (HLa) production and other soluble metabolites. NaHCO3 dosing triggered advantages not only for sulfate reduction (up to 56%), but also to enhance the stripping of sulfide to the gas phase (75-96% of the theoretical sulfide produced) by the high and constant biogas flow resulting from the CO2 released during NaHCO3 dissociation. Meanwhile, molasses-based vinasse presented higher potential for bioH2 (up to 4545 mL-H2 L-1 d-1) and HLa (up to 4800 mg L-1) production by butyric-type and capnophilic lactic fermentation pathways. Finally, heterolactic fermentation was the main metabolic route established when no pH control was provided (R3), as indicated by the high production of both HLa (up to 4315 mg L-1) and ethanol (1987 mg L-1). Hence, one single substrate (from which one single source of inoculum was originated) offers a wide range of metabolic possibilities to be exploited, providing substantial versatility to the application of anaerobic digestion in sugarcane biorefineries.

Hydrogen and organic acid production from dark fermentation of cheese whey without buffers under mesophilic condition.

BioH2 production from cheese whey (CW) was evaluated in two acidogenic reactors, UASB and structured fixed-bed (FB), without pH adjustment, under mesophilic conditions, and OLR of 25-90 g COD/L.d. Stage 1 was conducted as a control experiment using sucrose. BioH2 production occurred under pH < 3.0 with maximum yields of 5.8 and 3.0 mol H2/mol sucroseconsumed for UASB and FB reactors, respectively. In Stage 2, CW was the only substrate and a negligible bioH2 production was observed. Nevertheless, a maximum lactic acid concentration of 9.6 g/L was obtained, indicating that pH adjustment can be non-essential for lactic acid production from CW. In Stage 3, a strategy to enrich hydrogenogenic biomass was conducted by initially feeding the reactors with sucrose and gradually replacing it by CW. This strategy brought better bioH2 results compared to Stage 2, but it could not bear over the long-term, as non-hydrogen producing bacteria became predominant.

Dark fermentative volatile fatty acids production from food waste: A review of the potential central role in waste biorefineries.

Volatile fatty acids (VFAs) are high-value chemicals that are increasingly demanded worldwide. Biological production via food waste (FW) dark fermentation (DF) is a promising option to achieve the sustainability and environmental benefits typical of biobased chemicals and concurrently manage large amounts of residues. DF has a great potential to play a central role in waste biorefineries due to its ability to hydrolyze and convert complex organic substrates into VFAs that can be used as building blocks for bioproducts, chemicals and fuels. Several challenges must be faced for full-scale implementation, including process optimization to achieve high and stable yields, the development of efficient techniques for selective recovery and the cost-effectiveness of the whole process. This review aims to critically discuss and statistically analyze the existing relationships between process performance and the main variables of concern. Moreover, opportunities, current challenges and perspectives of a FW-based and fermentation-centred biorefinery layout are discussed.

Potassium ferrate pretreatment promotes short chain fatty acids yield and antibiotics reduction in acidogenic fermentation of sewage sludge.

During the acidogenic fermentation converting waste activated sludge (WAS) into short-chain fatty acids (SCFA), hydrolysis of complex organic polymers is a limiting step and the transformation of harmful substances (such as antibiotics) during acidogenic fermentation is unknown. In this study, potassium ferrate (K2FeO4) oxidation was used as a pretreatment strategy for WAS acidogenic fermentation to increase the hydrolysis of sludge and destruct the harmful antibiotics. Pretreatment with K2FeO4 can effectively increase the SCFA production during acidogenic fermentation and change the distribution of SCFA components. With the dosage of 0.2 g/g TS, the maximum SCFA yield was 4823 mg COD/L, which is 28.3 times that of the control group; acetic acid accounts for more than 90% of the total SCFA. The higher dosage (0.5 g/g TS) can further increase the proportion of acetic acid, but inhibit the overall performance of SCFA production. Apart from the promotion of hydrolysis and acidogenesis, K2FeO4 pretreatment can also simultaneously oxidizes and degrades part of the antibiotics in the sludge. When the dosage is 0.5 g/g TS, the degradation efficacy of antibiotics is the most significant, and the contents of ofloxacin, azithromycin, and tetracycline in the sludge are reduced by 69%, 42%, and 50%, respectively. In addition, K2FeO4 pretreatment can also promote the release of antibiotics from sludge flocs, which is conducive to the simultaneous degradation of antibiotics in the subsequent biological treatment process.

Effects of prepartum vaccination timing relative to pen change with an acidogenic diet on serum and colostrum immunoglobulins in Holstein dairy cows.

The objective of the present study was to assess the effects of prepartum vaccination timing relative to pen change with an acidogenic diet at 28 or 21 d before expected parturition (dpp) on colostral and serum IgG concentrations at calving in pregnant Holstein dairy cows. Pregnant multiparous Holstein cows (n = 308) from one large dairy herd were randomly allocated into 1 of 3 treatment groups at 35 ± 3 dpp: (1) vaccination at 28 dpp and pen change at 21 dpp (V28PC21; n = 108), (2) vaccination and pen change at 28 dpp (V28PC28; n = 99), and (3) vaccination and pen change at 21 dpp (V21PC21; n = 101). An acidogenic diet was fed when cows changed pens at 28 or 21 dpp. Blood and colostral samples were collected within 1 h following parturition. The total number of clinical mastitis (CM) cases within the first 150 d in milk (DIM) were recorded. The V28PC21 cows had greater colostral IgG concentrations at calving (160.4 ± 7.0 g/L) compared with V21PC21 cows (134.4 ± 7.0 g/L), and V28PC28 cows were intermediate (148.3 ± 7.2 g/L). At calving, V28PC21 cows had lower serum IgG concentrations (29.1 ± 1.2 g/L) compared with V21PC21 cows (32.2 ± 1.2 g/L) or V28PC28 cows (32.6 ± 1.3 g/L). Overall, 41% of V21PC21 cows received the booster vaccinations with at least 21 d before actual calving compared with V28PC21 or V28PC28 cows (88 and 86% respectively). The shorter the interval from prepartum booster vaccination to calving, the lower the colostral IgG at calving, regardless of treatment groups. Vaccinating at 28 dpp and pen change with an acidogenic diet at 21 dpp tended to reduce the rate of CM within the first 150 DIM compared with V21PC21. These findings provide evidence that vaccinating cows at 28 dpp, followed by pen change with an acidogenic diet at 21 dpp, improved the concentrations of colostral IgG at calving and tended to reduce the rate of CM. The interaction of prepartum vaccination timing relative to feeding an acidogenic diet should be considered when implementing an effective vaccination program to enhance overall herd health.

Effect of timing of prepartum vaccination relative to pen change with an acidogenic diet on lying time and metabolic profile in Holstein dairy cows.

The objective was to assess the effect of prepartum vaccination timing relative to pen change with an acidogenic diet at 28 or 21 d before expected parturition (dpp) on lying time (LT), prepartum serum energy status (glucose, IGF-1, and nonesterified fatty acids), urine pH, and serum Ca at calving in pregnant Holstein dairy cows. Pregnant multiparous Holstein cows (n = 308) from 1 large dairy herd were randomly allocated into 1 of 3 treatment groups at 35 ± 3 dpp as follows: (1) vaccination at 28 dpp and pen change at 21 dpp (V28PC21; n = 108), (2) vaccination and pen change at 28 dpp (V28PC28; n = 99), and (3) vaccination and pen change at 21 dpp (V21PC21; n = 101). When cows changed pens, an acidogenic diet was introduced. Every other week, a group of 43 to 53 animals were enrolled and electronic data loggers (IceQube, IceRobotics) were fitted to the hind leg of individual cows to assess their LT. Blood samples were collected at 28, 26, 21, 19, 14 dpp and at calving. Parity, body condition score, days dry, and gestation length were not different among groups. Overall, V28PC28 cows had 7 additional days in prepartum pens consuming an acidogenic diet compared with V28PC21 or V21PC21 cows. Regardless of treatment group, cows in the far-off pen had 43 min/d less LT (709 vs. 753 min/d) and increased day-to-day coefficient of variation of LT (0.21 vs. 0.10) compared with cows within the prepartum pen. On average, for the 7 d following vaccination alone (28 to 22 dpp period), V28PC21 cows had ~22 min/d less LT compared with V21PC21 cows. Serum concentrations of glucose, nonesterified fatty acids, and IGF-1 were altered following vaccination alone, pen change alone, or vaccination plus pen change with an acidogenic diet before calving. At calving, V28PC21 cows had greater glucose concentrations (6.45 mmol/L) compared with V21PC21 cows (5.76 mmol/L), with V28PC28 cows intermediate (6.11 mmol/L). The assessment of Ca status at calving revealed that V28PC21 cows had greater Ca concentration (2.34 mmol/L) with lower subclinical hypocalcemia (<2.0 mmol/L; 17.3%) compared with V21PC21 cows (2.17 mmol/L and 31.9%), with V28PC28 cows intermediate (2.28 mmol/L and 25.2%). Serum concentrations of IGF-1 at calving were also greater for V28PC21 (3.43 nmol/L) cows compared with V21PC21 (2.69 nmol/L), with V28PC28 cows intermediate (3.07 nmol/L). Overall, V28PC21 cows had greater serum glucose, IGF-1, and ~46% reduction in subclinical hypocalcemia (from 31.9 to 17.3%) compared with V21PC21 cows but did not differ from V28PC28 cows (25.2%). These findings provided evidence that vaccinating cows at 28 dpp, followed 7 d later by pen change with an acidogenic diet at 21 dpp, would be beneficial.

Duration and degree of diet-induced metabolic acidosis prepartum alter tissue responses to insulin in dairy cows.

The objective of this experiment was to determine the effects of altering the dietary cation-anion difference (DCAD) fed for the last 21 or 42 d of gestation on glucose metabolism and tissue insulin responsiveness. Ninety parous Holstein cows at 232 d of gestation were assigned randomly to dietary treatments with 2 levels of DCAD (-70 or -180 mEq/kg) fed for 2 durations (short: the last 21 d of gestation; long: the last 42 d of gestation). For the short treatments, a diet with +110 mEq/kg was fed from 232 to 254 d of gestation. Intravenous glucose tolerance tests (IVGTT) were performed at either 250 or 270 d of gestation by infusing 0.25 g of dextrose/kg of body weight within 1 min. The following day, cows underwent an insulin challenge (IC) and received 0.1 IU of insulin/kg of body weight intravenously. Blood was sampled at min -15, -5, and 0 to establish a baseline and from 5 to 180 min relative to infusions; plasma concentrations of glucose, insulin, and fatty acids were determined, and the respective areas under the curves (AUC) were calculated. Liver was sampled after the IVGTT, and adipose tissue was sampled after the IVGTT and IC for quantification of mRNA expression and protein abundance. Reducing the DCAD altered acid-base balance compatible with a compensated metabolic acidosis. At 250 d, reducing the DCAD increased the AUC for glucose and reduced that of insulin following the IVGTT, whereas during the IC, clearance rate decreased and time to half-life of insulin increased with reducing DCAD, resulting in a tendency to a larger AUC for fatty acids. At 270 d, quantitative insulin sensitivity check index and the revised quantitative insulin sensitivity check index were smaller in cows fed the acidogenic diets for the last 42 d of gestation compared with the last 21 d of gestation, thereby suggesting reduced insulin sensitivity. In addition, cows fed for the long duration tended to have greater AUC for glucose but smaller AUC for insulin following an IVGTT than those fed for the short duration, thereby suggesting reduced insulin release and glucose disposal. Treatments did not affect hepatic mRNA expression of G6PC, PCK1, PCK2, and PC or adipose tissue mRNA expression of ATGL, ACC, B2AR, HSL, and PLIN1. On the other hand, for proteins, reducing the DCAD linearly reduced abundance of rabbit anti-mouse protein kinase B (AKT) and tended to reduce rabbit anti-human phosphorylated (Ser-9) glycogen synthase kinase-3 ß (pGSK) and the pGSK:rabbit anti-human glycogen synthase kinase-3 ß (GSK) ratio in hepatic tissue, whereas a linear increase in rabbit anti-human hormone-sensitive lipase (HSL) and rabbit anti-mouse phosphorylated (Ser-660) hormone-sensitive lipase (pHSL) in adipose tissue was observed after the IVGTT at 250 d. Moreover, reducing the DCAD resulted in a linear reduction of AKT and tended to reduce rabbit anti-human acetyl-CoA carboxylase (ACC) but increased pHSL linearly in adipose tissue after an IC at 250 d. Cows fed acidogenic diets for a short duration tended to have less pHSL in adipose tissue than those fed for a long duration after an IVGTT at 270 d. Associations were observed between blood pH and mRNA and protein abundance in hepatic and adipose tissues. Diet-induced metabolic acidosis altered insulin release and insulin signaling, resulting in a shift in adipose tissue metabolism that would favor lipolysis over lipogenesis.

Prepartum level of dietary cation-anion difference fed to nulliparous cows: Lactation and reproductive responses.

Objectives were to determine the effects of 3 levels of dietary cation-anion difference (DCAD) fed prepartum to nulliparous cows on productive and reproductive performance. We enrolled 132 pregnant nulliparous Holstein cows at 250 (248-253) d of gestation in a randomized block design. Cows were blocked by genomic merit of energy-corrected milk yield and assigned randomly to diets varying in DCAD, +200 (P200; n = 43), -50 (N50; n = 45), or -150 (N150; n = 44) mEq/kg of dry matter (DM). Dietary treatments were fed during the last 22 d of gestation and, after calving, postpartum cows received the same lactation diet. Productive performance was evaluated for the first 14 wk of lactation, and reproduction was assessed until 305 d postpartum. Intake of DM prepartum decreased linearly (results presented in sequence as least squares means ± standard error of the mean, P200 vs. N50 vs. N150) with a reduction in DCAD (9.0 vs. 8.9 vs. 8.4 ± 0.1 kg/d), which resulted in linear decreases in net energy balance (0.34 vs. 0.20 vs. -0.36 ± 0.20 Mcal/d), body weight change (1.1 vs. 0.8 vs. 0.3 ± 0.1 kg/d), and mean body weight (652 vs. 649 vs. 643 ± 2 kg) prepartum. Treatment did not affect yield of colostrum (6.3 vs. 5.8 vs. 5.1 ± 0.6 kg) or the contents or yields of fat, protein, lactose, IgG, Ca, or Mg in colostrum. Intake of DM (19.4 vs. 19.2 vs. 19.0 ± 0.2 kg/d), yields of milk (36.6 vs. 36.7 vs. 35.8 ± 0.6 kg/d) or energy-corrected milk (36.7 vs. 36.3 vs. 35.9 ± 0.5 kg/d), feed efficiency (1.93 vs. 1.92 vs. 1.93 ± 0.03 kg of energy-corrected milk per kilogram of DM intake), and content and yield of milk components did not differ among treatments during the first 14 wk of lactation. Prepartum DCAD did not affect the cumulative milk yield by 305 d of lactation (9,653 vs. 10,005 vs. 9,918 ± 196 kg). Of the 132 cows, 40 P200, 45 N50, and 43 N150 received at least 1 artificial insemination (AI), and treatment did not affect pregnancy per AI at first (32.5 vs. 35.6 vs. 37.2%) or all AI (30.6 vs. 33.9 vs. 40.2%), although reducing the DCAD increased the proportion of cows pregnant by 305 d postpartum (76.7 vs. 88.9 vs. 93.2%) without altering the rate of pregnancy. Collectively, manipulating the DCAD of prepartum diets, from +200 to -150 mEq/kg of DM, fed to late gestation nulliparous cows did not affect subsequent lactation productive performance, but may have provided some benefit to reproduction, which warrants further confirmation.

High dietary acid load score is not associated with the risk of metabolic syndrome in Iranian adults.

Background and objective: The association between dietary acid load and metabolic syndrome (MetS) risk is not well-known. Therefore, we aimed to investigate the relationship between dietary acid load and the risk of MetS among Iranian adults. Methods: This cross-sectional study was carried out on 1430 Iranian adults. Dietary intakes were assessed using a validated food frequency questionnaire. Dietary acid load was estimated using potential renal acid load (PRAL) and net endogenous acid production (NEAP). MetS was defined according to the ATP-III criteria. The risk of MetS and its components was explored using logistic regression test. Results: Totally, 205 individuals were identified to have MetS. No significant association for MetS was found across the quartiles of PRAL and NEAP either in the crude model [Q4 PRAL: OR (95% CI): 0.94 (0.67-1.32), and NEAP: OR (95% CI): 0.88 (0.63-1.25)] or fully-adjusted model [Q4 PRAL: OR (95% CI): 0.90 (0.61-1.33), and NEAP: OR (95% CI): 1.05 (0.70-1.57)]. Amongst the components of MetS, higher scores of NEAP was associated with an increased risk of impaired blood sugar after adjustment for potential confounders [OR (95% CI): 1.35 (0.93-1.96)]. No significant association was found for other components either with PRAL or with NEAP. Conclusion: Our findings suggest no association between dietary acid load and MetS risk in Iranian adults. However, higher dietary acid load, measured by NEAP, but not PRAL, was associated with increased risk of impaired fasting blood sugar. Longitudinal studies are warranted to explore whether a diet low in potential acid load could reduce MetS risk.

New insights into the evaluation of anaerobic properties of sludge: Biodegradability and stabilization.

Evaluating anaerobic biodegradability of sludge and then identifying the stabilization of digestate is necessary in sludge treatment and disposal. 48 sludge samples from 24 typical waste water treatment plants (WWTPs) in 11 provinces in China were selected to investigate the relationship between Biochemical Acidogenic Potential (BAP) test and Biochemical Methane Potential (BMP) test. The volatile fatty acid (VFA) production obtained from BAP tests was found linearly related to the ultimate methane production from corresponding BMP tests. Satisfying results were obtained with Pearson correlation coefficient as 0.929 and R2 value as 0.76. Furthermore, the physio-chemical characteristics (FCI, SUVA254, E4/E6) of supernatant, which were associated with humic-like substances (HS), were investigated before and after BMP tests. Through which a new criterion (FCI>1.50, SUVA254>1.10, E4/E6<4.0) was proposed to evaluate the stabilization level of anaerobic digested sludge.

Energy recovery and carbon/nitrogen removal from sewage and contaminated groundwater in a coupled hydrolytic-acidogenic sequencing batch reactor and denitrifying biocathode microbial fuel cell.

Developing cost-effective technology for treatment of sewage and nitrogen-containing groundwater is one of the crucial challenges of global water industries. Microbial fuel cells (MFCs) oxidize organics from sewage by exoelectrogens on anode to produce electricity while denitrifiers on cathode utilize the generated electricity to reduce nitrogen from contaminated groundwater. As the exoelectrogens are incapable of oxidizing insoluble, polymeric, and complex organics, a novel integration of an anaerobic sequencing batch reactor (ASBR) prior to the MFC simultaneously achieve hydrolytic-acidogenic conversion of complex organics, boost power recovery, and remove Carbon/Nitrogen (C/N) from the sewage and groundwater. The results obtained revealed increases in the fractions of soluble organics and volatile fatty acids in pretreated sewage by 52 ± 19% and 120 ± 40%, respectively. The optimum power and current generation with the pretreated sewage were 7.1 W m-3 and 45.88 A m-3, respectively, corresponding to 8% and 10% improvements compared to untreated sewage. Moreover, the integration of the ASBR with the biocathode MFC led to 217% higher carbon and 136% higher nitrogen removal efficiencies compared to the similar system without ASBR. The outcomes of the present study represent the promising prospects of using ASBR pretreatment and successive utilization of solubilized organics in denitrifying biocathode MFCs for simultaneous energy recovery and C/N removal from both sewage and nitrate nitrogen-contaminated groundwater.

Technical note: Evaluation of a colorimetric point-of-care test for measuring urine ammonium concentration in periparturient dairy cattle.

Urine ammonium concentration ([NH4+]) provides a clinically useful indicator of the magnitude of nutritionally induced systemic acidification in dairy cattle when urine pH < 6.1. The objective of this study was to evaluate the analytical performance of a low-cost point-of-care colorimetric test in measuring urine [NH4+] in dairy cattle consuming an acidogenic ration. A method comparison study was performed using 154 urine samples from 43 periparturient Holstein-Friesian cows. We compared urine [NH4+] measured by an indophenol blue colorimetric test (MColortest, Merck KGaA, Billerica, MA; test method) with levels measured by formaldehyde titration (reference method). Diagnostic performance was evaluated using Pearson correlation coefficient (r), Passing-Bablok regression, Bland-Altman plot, and binary logistic regression. Urine [NH4+] measured by the colorimetric test was strongly correlated (r = 0.98) with urine [NH4+] measured by formaldehyde titration. Method comparison studies indicated that the colorimetric test provided acceptable test performance when urine [NH4+] < 80 mmol/L. Logistic regression analysis indicated that the area under the receiver operating characteristic curve for the colorimetric ammonium test was high at 0.985 when used to identify formaldehyde titration [NH4+] > 10 mmol/L, equivalent to urine pH <6.1. At the optimal cut point ([NH4+] > 11 mmol/L) for the colorimetric test, sensitivity = 0.94, specificity = 0.97, positive likelihood ratio = 27.6, and κ = 0.89, indicating excellent test performance. We conclude that the indophenol blue colorimetric test provided an accurate, low-cost, and practical on-farm test for measuring urine [NH4+] in diluted urine samples from dairy cattle consuming an acidogenic ration.

Recent advances in n-butanol and butyrate production using engineered Clostridium tyrobutyricum.

Acidogenic clostridia naturally producing acetic and butyric acids has attracted high interest as a novel host for butyrate and n-butanol production. Among them, Clostridium tyrobutyricum is a hyper butyrate-producing bacterium, which re-assimilates acetate for butyrate biosynthesis by butyryl-CoA/acetate CoA transferase (CoAT), rather than the phosphotransbutyrylase-butyrate kinase (PTB-BK) pathway widely found in clostridia and other microbial species. To date, C. tyrobutyricum has been engineered to overexpress a heterologous alcohol/aldehyde dehydrogenase, which converts butyryl-CoA to n-butanol. Compared to conventional solventogenic clostridia, which produce acetone, ethanol, and butanol in a biphasic fermentation process, the engineered C. tyrobutyricum with a high metabolic flux toward butyryl-CoA produced n-butanol at a high yield of > 0.30 g/g and titer of > 20 g/L in glucose fermentation. With no acetone production and a high C4/C2 ratio, butanol was the only major fermentation product by the recombinant C. tyrobutyricum, allowing simplified downstream processing for product purification. In this review, novel metabolic engineering strategies to improve n-butanol and butyrate production by C. tyrobutyricum from various substrates, including glucose, xylose, galactose, sucrose, and cellulosic hydrolysates containing the mixture of glucose and xylose, are discussed. Compared to other recombinant hosts such as Clostridium acetobutylicum and Escherichia coli, the engineered C. tyrobutyricum strains with higher butyrate and butanol titers, yields and productivities are the most promising hosts for potential industrial applications.

Metagenomic analysis and characterization of acidogenic microbiome and effect of pH on organic acid production.

Organic acid production including lactate and acetate is an economically attractive technology that has gained momentum worldwide over the past years. These series of action need to be performed by an esoteric and complex microbial community, in which different members have distinct roles in the establishment of a collective organization. In this study, we analyzed the bioma from bioreactors with various pH conditions of 4.0, 5.0 and 6.0 (R1, R2 and R3), respectively, involved in acidogenic digestion for stable production of various organic acids by means of high-throughput Illumina sequencing, disclosing thousands of genes and extracting more than 53 microbial genomes. At pH 5.0, the hydrolysis reaction was enhanced and thus the lactic acid fermentation was stably improved to 45.96 mm/L and acetic acid to 73.77 mm/L. R2 was found with the most suitable pH condition for stable organic acids production as Lactobacilli and Bifidobacteria were the major members. Both the members have the key roles in heterofermentation and produce higher transcripts of key encoding enzymes involved in the dominant heterofermentation pathways.

Measurement of urine pH and net acid excretion and their association with urine calcium excretion in periparturient dairy cows.

Urine pH (UpH) and net acid excretion (NAE) are used to monitor the degree of systemic acidification and predict the magnitude of resultant hypercalciuria when feeding an acidogenic ration to control periparturient hypocalcemia in dairy cattle. The objectives of this study were to evaluate the diagnostic performance of urine dipstick and pH paper for measuring UpH, and to characterize the UpH-NAE relationship and the association of urine Ca concentration ([Ca]) with UpH and NAE. Urine samples (n = 1,116) were collected daily from 106 periparturient Holstein-Friesian cows fed an acidogenic ration during late gestation. Net acid excretion was measured by titration, and UpH was measured by a glass-electrode pH meter (reference method), Multistix-SG urine dipsticks (Siemens Medical Solutions Inc., Ann Arbor, MI), and Hydrion pH paper (Micro Essential Laboratory Inc., Brooklyn, NY). Diagnostic performance was evaluated using Spearman correlation coefficient (rs), Bland-Altman plots, and logistic regression. Urine pH measured by urine dipstick (rs = 0.94) and pH paper (rs = 0.96) were strongly associated with UpH. Method-comparison studies indicated that the urine dipstick measured an average of 0.28 pH units higher, and pH paper 0.10 pH units lower, than UpH. Urine [Ca] was more strongly associated with UpH (rs = -0.65) than NAE (rs = 0.52). Goals for controlling periparturient hypocalcemia under the study conditions were UpH <6.22 and <6.11, based on achieving urine [Ca] ≥5 mmol/L and estimated urinary Ca excretion ≥4 g/d, respectively. Urine pH was as accurate at predicting urine [Ca] as NAE when UpH >6.11. We conclude that pH paper is an accurate, practical, and low-cost cow-side test for measuring UpH and provides a clinically useful estimate of urine [Ca].

Enhancement of Volatile Fatty Acids Production from Food Waste by Mature Compost Addition.

Food waste (FW) collected from a university canteen was treated in acidogenic fermenters to produce volatile fatty acids (VFA) under biological pretreatment with mature compost. Batch assays working at pH 6 revealed an increment of 9.0%, 7.9%, and 4.1% (on COD basis) of VFA concentration when adding 2.5%, 3.5%, and 4.5% w/w of mature compost, respectively, even though the volatile solids (VS) concentration of food waste was lower in the tests with increasing doses of mature compost. For batch tests at pH 7, this VFA generation improvement was lower, even though enhanced COD solubilization was recorded. Operating in semi-continuous conditions at 35 °C, pH of 6, and hydraulic retention time (HRT) of 3.5 days, the addition of 2.5% w/w of mature compost led to a VFA concentration up to 51.2 ± 12.3% more (on VS basis) when compared to a reference reactor without compost addition. Moreover, the percentage of butyric acid on VS basis in the fermentation broth working at a pH of 6 increased from up to 12.2 ± 1.9% (0% compost addition) to up to 23.5 ± 2.7% (2.5% compost addition). The VFA production was not improved when a higher percentage of mature compost was used (3.5% instead of 2.5% w/w), and it slightly decreased when mature compost addition was lowered to 1.5% w/w. When working at a pH of 7 in the semi-continuous fermenters with the addition of 2.5% w/w mature compost at an HRT of 3.5 days, an improvement of 79% and 104% of the VFA concentration (on VS basis) were recorded as compared to fermenters working at a pH of 6 with 2.5% and 0% w/w of mature compost addition, respectively. At a pH of 7, higher production of propionic and valeric acids was found with respect to the reactor working at a pH of 6. The effect of pH on VFA generation was estimated to have greater contribution than that of only biological pretreatment using mature compost. At a pH of 7, the VFA yield was higher for the fermenter working with 2.5% w/w mature compost but at a pH of 7 and HRT of 5 days, the effect of mature compost on VFA production improvement was lower than that obtained at a pH of 6. Moreover, higher solubilization in terms of soluble chemical oxygen demand and total ammonium was detected when biological pretreatment using mature compost was applied at both a pH of 6 and a pH of 7, which indicates enhanced hydrolysis in both conditions.

Comparison of Two Process Schemes Combining Hydrothermal Treatment and Acidogenic Fermentation of Source-Separated Organics.

This study compares the effects of pre- and post-hydrothermal treatment of source- separated organics (SSO) on solubilization of particulate organics and acidogenic fermentation for volatile fatty acids (VFAs) production. The overall COD solubilization and solids removal efficiencies from both schemes were comparable. However, the pre-hydrolysis of SSO followed by acidogenic fermentation resulted in a relatively higher VFA yield of 433 mg/g VSS, which was 18% higher than that of a process scheme with a post-hydrolysis of dewatered solids from the fermentation process. Regarding the composition of VFA, the dominance of acetate and butyrate was comparable in both process schemes, while propionate concentration considerably increased in the process with pre-hydrolysis of SSO. The microbial community results showed that the relative abundance of Firmicutes increased substantially in the fermentation of pretreated SSO, indicating that there might be different metabolic pathways for production of VFAs in fermentation process operated with pre-treated SSO. The possible reason might be that the abundance of soluble organic matters due to pre-hydrolysis might stimulate the growth of more kinetically efficient fermentative bacteria as indicated by the increase in Firmicutes percentage.