Analysis Of Volatile Fatty Acids Research Articles

Maternal laminarin supplementation decreases Salmonella Typhimurium shedding and improves intestinal health in piglets following an experimental challenge with S. Typhimurium post-weaning

During the period immediate post-weaning (PW), piglets become vulnerable to pathogenic bacteria such as Salmonella Typhimurium, the most common Salmonella serovar. Laminarin (LAM), a β-glucan derived from seaweed, is known to have antimicrobial and anti-inflammatory activity when supplemented maternally or directly to the piglet. The aim of this study was to investigate the effects of supplementing sows and/or piglets post-weaning with LAM after an experimental challenge with S. Typhimurium. Gestating sows (n=24) were supplemented with LAM (0 vs. 1.0g/d) from day 107 of gestation until weaning (day 20). The lactation and PW diet were arranged in a 2×2 factorial to provide four treatment groups: (1) pigs weaned from basal fed sows and offered a basal diet post-weaning (n=12; BB); (2) pigs weaned from basal fed sows and offered a LAM supplemented diet post-weaning (n=12; BL); (3) pigs weaned from LAM supplemented sows and offered a basal diet post-weaning (n=12; LB); (4) pigs weaned from LAM supplemented sows and offered a LAM supplemented diet post-weaning (n=12; LL). The LAM supplemented post-weaning diets contained 300ppm LAM/t of feed. On day 10 PW, piglets were challenged with 108 CFU S. Typhimurium and euthanized on day 26 PW to facilitate sample collection for microbial populations, volatile fatty acid (VFA) and immune related gene expression analysis. Pigs were weighed at birth, at weaning, at challenge and at slaughter and feed intake post-weaning was recorded. Faeces were visually scored for diarrhoea. Piglets weaned from LAM supplemented sows had improved average daily gain (ADG) and feed efficiency post-challenge (P<0.05). The faecal score was improved when LAM was supplemented maternally or post-weaning (P<0.001). Maternal supplementation of LAM reduced faecal counts of S. Typhimurium by 0.35 log gene copy numbers post-challenge (measured on day 2, 5 and 7; P<0.05). Total VFA production in the colon was increased after maternal or post-weaning LAM supplementation (P<0.05). The expression of IL-22 in the colon was reduced after maternal LAM supplementation (P<0.05). It can be concluded that, after an experimental challenge with S. Typhimurium, maternal LAM supplementation resulted in improved feed efficiency, faecal scores and increased the VFA production in the colon, while reducing faecal counts of S. Typhimurium and gene expression of IL-22 in the colon.

Examination of the molecular control of ruminal epithelial function in response to dietary restriction and subsequent compensatory growth in cattle.

BackgroundThe objective of this study was to investigate the effect of dietary restriction and subsequent compensatory growth on the relative expression of genes involved in volatile fatty acid transport, metabolism and cell proliferation in ruminal epithelial tissue of beef cattle. Sixty Holstein Friesian bulls (mean liveweight 370 ± 35 kg; mean age 479 ± 15 d) were assigned to one of two groups: (i) restricted feed allowance (RES; n = 30) for 125 d (Period 1) followed by ad libitum access to feed for 55 d (Period 2) or (ii) ad libitum access to feed throughout (ADLIB; n = 30). Target growth rate for RES was 0.6 kg/d during Period 1. At the end of each dietary period, 15 animals from each treatment group were slaughtered and ruminal epithelial tissue and liquid digesta harvested from the ventral sac of the rumen. Real-time qPCR was used to quantify mRNA transcripts of 26 genes associated with ruminal epithelial function. Volatile fatty acid analysis of rumen fluid from individual animals was conducted using gas chromatography.ResultsDiet × period interactions were evident for genes involved in ketogenesis (BDH2, P = 0.017), pyruvate metabolism (LDHa, P = 0.048; PDHA1, P = 0.015) and cellular transport and structure (DSG1, P = 0.019; CACT, P = 0.027). Ruminal concentrations of propionic acid (P = 0.018) and n-valeric acid (P = 0.029) were lower in RES animals, compared with ADLIB, throughout the experiment. There was also a strong tendency (P = 0.064) toward a diet × period interaction for n-butyric with higher concentrations in RES animals, compared with ADLIB, during Period 1.ConclusionsThese data suggest that following nutrient restriction, the structural integrity of the rumen wall is compromised and there is upregulation of genes involved in the production of ketone bodies and breakdown of pyruvate for cellular energy. These results provide an insight into the potential molecular mechanisms regulating ruminal epithelial absorptive metabolism and growth following nutrient restriction and subsequent compensatory growth.

Feeding distillers dried grains in replacement of forage in limit-fed dairy heifer rations: Effects on growth performance, rumen fermentation, and total-tract digestibility of nutrients

The objective of this study was to determine the effects of increasing dietary concentration of distillers dried grains (DDGS) in dairy heifer rations. A 16-wk randomized complete block design study was conducted using 48 Holstein heifers [199±2 d of age; body weight (BW) 206±2kg] to evaluate effects of dietary treatment on dry matter (DM) intake, average daily gain, growth performance, rumen fermentation, and nutrient digestibility. Treatments were (1) 30% DDGS with the diet fed at 2.65% of BW, (2) 40% DDGS with the diet fed at 2.50% of BW, and (3) 50% DDGS with the diet fed at 2.35% of BW. The remainder of the diet consisted of grass hay and 1.5% mineral mix. Heifers were individually limit-fed using Calan gates. Heifers were weighed every 2 wk and the ration amount offered was adjusted accordingly. Frame measurements and body condition score were recorded every 2wk. Rumen fluid was collected via esophageal tubing during wk 12 and 16 for pH, ammonia N, and volatile fatty acid analysis. Total-tract digestibility of nutrients was evaluated during wk 16 using fecal grab sampling. No treatment by week interactions were found for any of the growth parameters measured, and growth parameters did not differ among treatments. Heifer DM intake linearly decreased with increasing concentrations of DDGS. Body weight and average daily gain were similar among treatments, whereas gain:feed linearly increased across treatments, with a tendency for a treatment by time interaction. As the dietary concentrations of DDGS increased, rumen ammonia N linearly increased. Acetate proportion and acetate:propionate linearly decreased as DDGS increased, whereas propionate linearly increased. There were treatment by time interactions for propionate proportion and acetate:propionate. Increasing dietary concentrations of DDGS linearly increased total-tract digestibility of DM, organic matter, and crude protein. Limit-feeding diets with greater concentrations of DDGS improved gain:feed and total-tract digestibility of DM and crude protein, and maintained frame growth without increasing body condition score. Results demonstrated that replacing forage with DDGS at up to a 50% inclusion rate in limit-fed rations can maintain heifer growth performance.

Authentication of Italian PDO lard using NIR spectroscopy, volatile profile and fatty acid composition combined with chemometrics

This study analysed the usefulness of near infrared spectroscopy (NIRS), combined with volatile compound (VOC) and fatty acid (FA) analyses, for the authentication of the unique Italian Valle d’Aosta Arnad Protected Designation of Origin (PDO) lard. Ensuring the authenticity of high value meat products remains an emerging topic within the food sector. This study validated a FA, VOC and NIRS model for use in the authentication of Arnad PDO lard. The model showed a high potential rate to recognize patterns in lard samples. In particular the sensitivity and specificity calibration values were both 100%, and cross-validation models were performed using FAs and VOCs separately. The NIRS model obtained sensitivity and specificity values of 98.2% in the calibration data set, and 94.4% in the cross-validation step. This analytical approach may represent an effective tool to prevent food fraud, which is crucial for meat derived products with a high commercial value.

Influence of steam explosion pretreatment on the anaerobic digestion of vinegar residue.

Vinegar residue is the by-product in the vinegar production process. The large amount of vinegar residue has caused a serious environmental problem owing to its acidity and corrosiveness. Anaerobic digestion is an effective way to convert agricultural waste into bioenergy, and a previous study showed that vinegar residue could be treated by anaerobic digestion but still had room to improve digestion efficiency. In this study, steam explosion at pressure of 0.8, 1.2, and 1.5 MPa and residence time of 5, 10, 15, and 20 min were used to pretreat vinegar residue to improve methane production, respectively. Scanning electron microscopy and X-ray diffraction analyses were applied to validate structural changes of vinegar residue after steam explosion. Results showed that steam explosion pretreatment could destroy the structure of lignocellulose by removing the hemicellulose and lignin, and improve the methane yield effectively. Steam explosion-treated vinegar residue at 0.8 MPa for 5 min produced the highest methane yield of 153.58 mL gVS (-1), which was 27.65% (significant, α < 0.05) more than untreated vinegar residue (120.31 mL gVS (-1)). The analyses of pH, total ammonia-nitrogen, total alkalinity, and volatile fatty acids showed that steam explosion did not influence the stability of anaerobic digestion. This study suggested that steam explosion pretreatment on vinegar residue might be a promising approach and it is worth further study to improve the efficiency of vinegar residue waste utilisation.

Effect of starchy or fibrous carbohydrate supplementation of orchardgrass on ruminal fermentation and methane output in continuous culture

A 4-unit dual-flow continuous culture fermentor system was used to assess the effects of supplementing orchardgrass (Dactylis glomerata L.) with 2 levels [5 and 10% of total dry matter (DM) fed] of starchy (barley grain, BAR) or fibrous (beet pulp, BP) carbohydrate sources on nutrient digestibility, volatile fatty acid (VFA) production, bacterial protein synthesis, and CH4 output. Treatments were randomly assigned to fermentors in a 4×4 Latin square design with a 2×2 factorial arrangement using 7 d for microbial adaptation and 3 d for sample collection. Treatments included (1) 57g of DM orchardgrass + 3g of DM BAR, (2) 54g of DM orchardgrass + 6g of DM BAR, (3) 57g of DM orchardgrass + 3g of DM BP, or (4) 54g of DM orchardgrass + 6g of DM BP. Feedings occurred at 0900, 1030, 1400, and 1900h throughout four 10-d periods. Gas samples for CH4 analysis were collected 6 times daily at 0725, 0900, 1000, 1355, 1530, and 1630h. Fermentor samples for pH, NH3-N, and VFA analysis were taken on d 8, 9, and 10. Samples were also analyzed for DM, organic matter, crude protein, purines, neutral detergent fiber, and acid detergent fiber to determine nutrient digestibilities and estimation of bacterial protein synthesis. Apparent and true DM and organic matter digestibilities were not affected by supplement source. Apparent neutral and acid detergent fiber digestibilities were greater for BAR than BP. Conversely, apparent crude protein digestibility was greater for BP than BAR. Mean and maximum pH tended to be greatest for BAR than BP. Minimum pH was greater at the lower level (5% of diet DM) of supplementation. Barley produced greater concentrations of total VFA and acetate, whereas BP had greater daily outputs of CH4. Significant supplement type × level interactions were found for bacterial N flow and efficiency. Overall, supplementing orchardgrass with BP improved crude protein digestibility, reduced fiber digestibility and total VFA concentration, but increased CH4 output compared with BAR.

Rumen papillae morphology of beef steers relative to gain and feed intake and the association of volatile fatty acids with kallikrein gene expression

Feed costs are the most expensive input in beef production. Improvement in the ability of beef cattle to convert feed into meat would lower feed inputs and reduce the cost of production. The rumen epithelium is responsible for absorption and metabolism of nutrients and microbial by-products, and may play a significant role in gain or feed intake. Our objective was to determine the relationships among rumen papillae morphology, gene expression, volatile fatty acid concentrations, and gain and feed intake. Average daily gain (ADG) and average daily feed intake (ADFI) were collected on a crossbred population of beef steers over three feeding trials. Based on feed intake and weight gain differences, 48 steers were selected for the project (16 from each feeding study). At harvest, rumen epithelial samples were taken from three locations in the rumen of each animal. The number of papillae on 1cm2 of epithelium was counted to determine density. Papillae (n=30) from each sample were measured for length and width. The density, length, and width were combined to determine surface enlargement factor (SEF). None of the morphological characteristics of the papillae (length, width, density or SEF) were associated with feed intake or gain (P≥0.10). Ruminal fluid was collected from steers (n=15) in the third trial for volatile fatty acid (VFA) analysis to determine if a relationship between VFA and ADG or ADFI existed. No differences in volatile fatty acid (VFA) concentrations were associated (P≥0.17) with ADFI or ADG. VFA variation was also evaluated for a relationship with kallikrein (KLK) genes since ruminal butyrate concentrations have previously been associated with the transcript abundance KLK genes. Additionally, qRT-PCR data showed that variation in the transcript abundance of KLK6,8-10, and 13 associated with feed intake in our first trial of steers. Thus, we evaluated the transcript abundance of eight KLK genes in rumen papillae from steers in the third trial (n=16). Expression levels of KLK5 correlated with valerate concentrations in the rumen (P<0.05), KLK10 correlated with acetate concentrations in the rumen (P<0.05), KLK12 correlated with acetate and butyrate concentrations in the rumen (P<0.05), and the expression levels of KLK9 and KLK10 were associated with gain. While rumen papillae morphology was not associated with beef steer gain or intake, our data suggests members of the kallikrein gene family have a relationship with the VFA environment in the rumen, and also appear to play a role in the highly correlated traits of ADG and ADFI.

Enhanced Anaerobic Digestion of Food Waste by Supplementing Trace Elements: Role of Selenium (VI) and Iron (II)

This paper discusses the potential to enhance the anaerobic digestion of food waste FW by supplementing trace elements (Fe, Co, Ni, Zn, Mn, Cu, Se, and Mo) individually as well as in cocktails. A series of batch experiments on the biomethane potential of synthetic food waste were performed with low (FW-A) and high (FW-B) trace element background concentrations prepared in, respectively, Delft (The Netherlands) and Tampa (Florida, USA). The most effective trace elements for FW-A were Fe with an increase of 39.2 (± 0.6) % of biomethane production, followed by Se (34.1 ± 5.6 % increase), Ni (26.4 ± 0.2 % increase) and Co (23.8 ± 0.2 % increase). For FW-B supplementing these trace elements did not result in enhancement of the biomethane production, except for Se. FW-B had a Se concentration of 1.3 (± 0. 5) µg/gTS, while it was below the detection limit for FW-A. Regardless of the FW source, Se resulted in 30 – 35% increase of biomethane production at a concentration range of 25-50 µg/L (0.32 – 0.63 µM). Volatile fatty acids analysis revealed that TE supplementation enhances their consumption, thus yielding a higher biomethane production. Moreover, additional experiments on sulfide inhibition showed the enhancing effects of trace elements on the anaerobic digestion of food waste were not related with sulfide toxicity, but with the enzymatic reactions and/or microbial biomass aggregation.

Evaluation of titration methods for volatile fatty acids measurement: effect of the bicarbonate interference and feasibility for the monitoring of anaerobic reactors

The analysis of volatile fatty acids (VFA) is of utmost importance for monitoring the stability of anaerobic reactors. For routine control, titration methods are advantageous because of their simplicity, speed and low cost. However, bicarbonate, which is usually present in relatively high concentrations in anaerobic reactors, influences the potentiometric titration. In this paper, the methods of Ripley, Kapp, DiLallo, DiLallo with modified calculations, and DiLallo with sonication were evaluated regarding the effects of bicarbonate interference and their suitability for monitoring an anaerobic reactor containing high concentrations of organic matter and bicarbonate. Standard solutions with various combinations of acetic acid and bicarbonate, ranging from 100 to 4,000 mg/l, and anaerobically treated stillage containing VFA in concentrations between 5,500 and 30 mg/l were analyzed. The methods of DiLallo and Kapp were little affected by sodium bicarbonate interference, unlike Ripley's and DiLallo's methods with sonication, which overestimated VFA concentrations as the relative concentrations of bicarbonate were high compared to the acids. When the anaerobic reactor was starting, the Ripley, Kapp, DiLallo and modified DiLallo methods were statistically equal. As the system achieved stability, with VFA levels up to 200 mg/l, Ripley's method gave significantly higher VFA values. Among the methods evaluated, Kapp and original DiLallo presented the closest results, indicating their feasibility for analysis of VFA over a wide concentration range. Kapp's method stands out due to its greater simplicity and standardization.

Formate, acetate, and propionate as substrates for sulfate reduction in sub-arctic sediments of Southwest Greenland.

Volatile fatty acids (VFAs) are key intermediates in the anaerobic mineralization of organic matter in marine sediments. We studied the role of VFAs in the carbon and energy turnover in the sulfate reduction zone of sediments from the sub-arctic Godthåbsfjord (SW Greenland) and the adjacent continental shelf in the NE Labrador Sea. VFA porewater concentrations were measured by a new two-dimensional ion chromatography-mass spectrometry method that enabled the direct analysis of VFAs without sample pretreatment. VFA concentrations were low and surprisingly constant (4–6 μmol L−1 for formate and acetate, and 0.5 μmol L−1 for propionate) throughout the sulfate reduction zone. Hence, VFAs are turned over while maintaining a stable concentration that is suggested to be under a strong microbial control. Estimated mean diffusion times of acetate between neighboring cells were <1 s, whereas VFA turnover times increased from several hours at the sediment surface to several years at the bottom of the sulfate reduction zone. Thus, diffusion was not limiting the VFA turnover. Despite constant VFA concentrations, the Gibbs energies (ΔGr) of VFA-dependent sulfate reduction decreased downcore, from −28 to −16 kJ (mol formate)−1, −68 to −31 kJ (mol acetate)−1, and −124 to −65 kJ (mol propionate)−1. Thus, ΔGr is apparently not determining the in-situ VFA concentrations directly. However, at the bottom of the sulfate zone of the shelf station, acetoclastic sulfate reduction might operate at its energetic limit at ~ −30 kJ (mol acetate)−1. It is not clear what controls VFA concentrations in the porewater but cell physiological constraints such as energetic costs of VFA activation or uptake could be important. We suggest that such constraints control the substrate turnover and result in a minimum ΔGr that depends on cell physiology and is different for individual substrates.

Fermentation of Pretreated High-Biomass Sorghum Hydrolysates to Biohydrogen by Mixed Consortia

In the present study, hydrolysate generated during pretreatment of high-biomass sorghum as a carbon source for biohydrogen production was investigated. The high-biomass sorghum bagasse (HBS) was pretreated using acid (H2SO4) and alkali (NaOH) at various concentrations (0.5–5 % w/v) for the residence time of 30 min at 121 °C, 15 lbs pressure at 10 % (w/v) solid loading. At the optimal acid load of 2 % (w/v) H2SO4 yielded 78.0 g COD/L of hydrolysate. The hydrolysate generated during this pretreatment was analyzed and noticed to contain glucose 10 g L−1, xylose 23 g L−1, arabinose 2.0 g L−1, HMF 1.9 g L−1, furfural 3.5 g−1, acetic acid 9.3 g L−1, formic acid 5.0 g L−1, and phenols 1.9 g L−1. The fermentation studies were conducted in dark conditions using all the hydrolysates by heat-treated mixed microbial consortia. Maximum H2 production rate (HPR), cumulative H2 production (CHP), and specific H2 yield (SHY) were measured. Maximum CHP (328 mL) and SHY (4.68 mol/kg CODr) were registered with acid treatment-resulted hydrolysate, and volatile fatty acid analysis indicated higher acetic acid concentration (1.6 g L−1) showing acidogenic microenvironment directing fermentation toward acetate pathway. The present study assumes importance in safe disposal and simultaneous production of value-added byproducts during lignocellulosic biorefinery.

Organic building block based microporous network SNW-1 coating fabricated by multilayer interbridging strategy for efficient enrichment of trace volatiles.

Microporous organic polymers (MOPs) are an emerging class of functional porous materials for diverse potential applications. Typically, tailored microporous structures of MOPs are generated by linkages of organic polymerizable monomer building blocks, providing high permanent porosity and excellent stability. Herein, we reported the first example of the application of organic building block based MOPs (OBB-MOPs) as efficient enrichment media for sample preparation. A novel multilayer interbridging strategy was proposed to fabricate OBB-MOP coatings, and hereby SNW-1 (a kind of OBB-MOPs) was coated on silica substrate with well-controlled thickness. Strong covalent bonds throughout the network and interlayer bridging improved the durability of the coating significantly. Outstanding chemical stability was observed in diverse solvents as well as solutions with a wide range of pH or high ionic strength and even under extremely harsh conditions like boiling water. The SNW-1 coating possessed a microporous network structure constructed of conjugated and nitrogen-rich building blocks. Thus, the coating exhibited a superior enrichment performance of polycyclic aromatic hydrocarbons and volatile fatty acids (VFAs) over commercial coatings based on interactions including π-π affinity and acid-base interaction. For further application, this coating was combined with gas chromatography/mass spectrometry for the noninvasive analysis of VFAs from tea leaf and tobacco shred samples. The low detection limits of 0.014-0.026 μg/L were achieved with the relative standard deviations (RSDs) between 4.3 and 9.0%. Consequently, trace original VFAs from the samples were detected. Good recoveries were obtained in the range of 90-129% and 77-118% with the corresponding RSDs (n = 3) of 2.6-9.3% and 1.9-10%, respectively.

Monitoring anaerobic digestion: a 2-year brewery case study

Operational data from an anaerobic wastewater treatment plant (expanded granular sludge bed (EGSB) reactor) were analysed before and after a defect with the solids separator. The results presented suggest that a newly available method for the analysis of total volatile fatty acids (VFAs) was ideal as a rapid, onsite, operational indicator of reactor stability. These total VFAs were shown to provide an earlier warning of the separator problem than the other rapid routine methods of monitoring digesters such as alkalinity and suspended solids. Chemical oxygen demand (COD) removal, pH and gas yield were not as useful for monitoring because of their slow response. The results are from a high rate reactor; the loads were 18 kg COD/m3/d in the first year and 26 in the second with 4·4 d hydraulic retention time. The results for both years of operation demonstrate a 95% conversion of COD into gas with an additional contribution from solids digestion (specific gas yield of 0·4 l methane (CH4)/g CODrem). This high performance was attributed to the solubility of the COD and the efficient EGSB mixing.

Effect of sprouted barley grain supplementation of an herbage-based or haylage-based diet on ruminal fermentation and methane output in continuous culture

A 4-unit dual-flow continuous-culture fermentor system was used to assess the effect of supplementing 7-d sprouted barley (SB) or barley grain (BG) with an herbage-based or haylage-based diet on nutrient digestibility, volatile fatty acid (VFA) profiles, bacterial protein synthesis, and methane (CH4) output. Treatments were randomly assigned to fermentors in a 4×4 Latin square design with a 2×2 factorial arrangement using 7 d for diet adaptation and 3 d for sample collection. Experimental diets were (1) 55.5g of herbage dry matter (DM) + 4.5g of SB DM, (2) 56.0g of herbage DM + 4.0g of BG DM, (3) 55.5g of haylage DM + 4.5g of SB DM, and (4) 56.0g of haylage DM + 4.0g of BG DM. Forages were fed at 0730, 1030, 1400, and 1900h, whereas SB and BG were fed at 0730 and 1400h. Gas samples for CH4 analysis were collected at 0725, 0900, 1000, 1355, 1530, and 1630h on d 8, 9, and 10. Fluid samples were taken once daily on d 8, 9, and 10 for pH measurements and for ammonia-N and VFA analysis and analyzed for DM, organic matter, crude protein, neutral detergent fiber, and acid detergent fiber for determination of nutrient digestibilities and estimation of bacterial protein synthesis. Orthogonal contrasts were used to compare the effect of forage source (haylage vs. herbage), supplement (BG vs. SB), and the forage × supplement interaction. Apparent and true DM and organic matter digestibilities as well as apparent crude protein digestibility were not affected by forage source. However, true DM digestibility was greatest for diets supplemented with SB. Apparent neutral and acid detergent fiber digestibilities of herbage-based diets were higher than haylage-based diets but fiber digestibility was not affected by supplement. Diets supplemented with SB had higher mean and minimum pH than BG; however, maximum pH was not affected by diet. Supplementation with BG produced a greater concentration of total VFA compared with diets supplemented with SB. Haylage-based diets produced greater CH4 output compared with herbage-based diets but supplementation did not affect CH4 output. Efficiency of bacterial protein synthesis was greater for herbage-based diets compared with haylage-based diets, with no effect of supplementation. Overall, supplementation with SB marginally increased true DM digestibility of herbage- and haylage-based diets but did not affect fiber and crude protein digestibilities, CH4 output, and bacterial efficiency, compared with BG.

Effects on enteric methane production and bacterial and archaeal communities by the addition of cashew nut shell extract or glycerol—An in vitro evaluation

The objective of the study was to evaluate the effect of cashew nut shell extract (CNSE) and glycerol (purity >99%) on enteric methane (CH4) production and microbial communities in an automated gas in vitro system. Microbial communities from the in vitro system were compared with samples from the donor cows, in vivo. Inoculated rumen fluid was mixed with a diet with a 60:40 forage:concentrate ratio and, in total, 5 different treatments were set up: 5mg of CNSE (CNSE-L), 10mg of CNSE (CNSE-H), 15mmol of glycerol/L (glycerol-L), and 30mmol of glycerol/L (glycerol-H), and a control without feed additive. Gas samples were taken at 2, 4, 8, 24, 32, and 48h of incubation, and the CH4 concentration was measured. Samples of rumen fluid were taken for volatile fatty acid analysis and for microbial sequence analyses after 8, 24, and 48h of incubation. In vivo rumen samples from the cows were taken 2h after the morning feeding at 3 consecutive days to compare the in vitro system with in vivo conditions. The gas data and data from microbial sequence analysis (454 sequencing) were analyzed using a mixed model and principal components analysis. These analyses illustrated that CH4 production was reduced with the CNSE treatment, by 8 and 18%, respectively, for the L and H concentration. Glycerol instead increased CH4 production by 8 and 12%, respectively, for the L and H concentration. The inhibition with CNSE could be due to the observed shift in bacterial population, possibly resulting in decreased production of hydrogen or formate, the methanogenic substrates. Alternatively the response could be explained by a shift in the methanogenic community. In the glycerol treatments, no main differences in bacterial or archaeal population were detected compared with the in vivo control. Thus, the increase in CH4 production may be explained by the increase in substrate in the in vitro system. The reduced CH4 production in vitro with CNSE suggests that CNSE can be a promising inhibitor of CH4 formation in the rumen of dairy cows.

Direct analysis of volatile fatty acids in marine sediment porewater by two‐dimensional ion chromatography‐mass spectrometry

Volatile fatty acids (VFAs) are key intermediates in the microbial food web. However, the analysis of low concentrations of VFAs in marine porewater is hampered by interference from high concentrations of inorganic ions. Published methods often use sample pretreatment, including distillation or derivatization, to overcome this problem. This is not only labor intensive but also increases the risk of contamination. We have developed an analytical procedure that enables the direct quantification of several VFAs (formate, acetate, propionate, butyrate, valerate, pyruvate, and lactate) in marine porewater by two‐dimensional ion chromatography‐mass spectrometry (2D IC‐MS). The first chromatographic dimension is used to separate the VFAs from the inorganic ions whereas the second dimension separates the individual VFAs. Quantification and identification are achieved by online mass spectrometry in selected ion monitoring mode. No sample pretreatment is required and statistically determined detection limits are below 25 ppb (µg L−1). The method can also be used without the online coupling to a mass spectrometer. In the latter case, quantification of the VFAs can be done by the conductivity detector signal of the second dimension with the limitation that lactate cannot be quantified due to signal interference with an unidentified peak and pyruvate and valerate have slightly higher detection limits (&lt;100 ppb).

An improved titration model reducing over estimation of total volatile fatty acids in anaerobic digestion of energy crop, animal slurry and food waste

Titration methodologies have been used for the many years for low cost routine monitoring of full scale anaerobic digestion plants. These methodologies have been correlated to indicate the carbonate alkalinity and the volatile fatty acids (VFA) content within digesters. Two commonly used two end-point titration methods were compared using a dataset of 154 samples from energy crop and animal slurry digestates and were shown to be inaccurate in the estimation of tVFA. Using this dataset correlated with HPLC VFA analysis, two empirical bivariate linear regression equations were derived, where the validation dataset showed an absolute tVFA mean error improvement from ±3386 and ±3324 mg kg−1 tVFA to ±410 and ±286 mg kg−1 tVFA, respectively. The same equation was then applied to a food waste dataset where an absolute tVFA mean error was improved from ±3828 to ±576 mg kg−1 tVFA. The newly derived titration equations can provide greater confidence in digester performance monitoring and are tools that can improve digester management.

Effects of a gradually increased load of fish waste silage in co-digestion with cow manure on methane production

This study examined the effects of an increased load of nitrogen-rich organic material on anaerobic digestion and methane production. Co-digestion of fish waste silage (FWS) and cow manure (CM) was studied in two parallel laboratory-scale (8L effective volume) semi-continuous stirred tank reactors (designated R1 and R2). A reactor fed with CM only (R0) was used as control. The reactors were operated in the mesophilic range (37°C) with a hydraulic retention time of 30days, and the entire experiment lasted for 450days. The rate of organic loading was raised by increasing the content of FWS in the feed stock. During the experiment, the amount (volume%) of FWS was increased stepwise in the following order: 3% – 6% – 13% – 16%, and 19%. Measurements of methane production, and analysis of volatile fatty acids, ammonium and pH in the effluents were carried out. The highest methane production from co-digestion of FWS and CM was 0.400L CH4 gVS−1, obtained during the period with loading of 16% FWS in R2. Compared to anaerobic digestion of CM only, the methane production was increased by 100% at most, when FWS was added to the feed stock. The biogas processes failed in R1 and R2 during the periods, with loadings of 16% and 19% FWS, respectively. In both reactors, the biogas processes failed due to overloading and accumulation of ammonia and volatile fatty acids.

Effects of ethyl-3-nitrooxy propionate and 3-nitrooxypropanol on ruminal fermentation, microbial abundance, and methane emissions in sheep

The aim of this work was to investigate the effect of feeding ethyl-3-nitrooxy propionate (E3NP) and 3-nitrooxypropanol (3NP), 2 recently developed compounds with potential antimethanogenic activity, in vitro and in vivo in nonlactating sheep on ruminal methane production, fermentation pattern, the abundance of major microbial groups, and feed degradability. Three experiments were conducted, 1 in vitro and 2 in vivo. The in vitro batch culture trial (experiment 1) tested 2 doses of E3NP and 3NP (40 and 80μL/L), which showed a substantial reduction of methane production (up to 95%) without affecting concentration of volatile fatty acids (VFA). The 2 in vivo trials were conducted over 16 d (experiment 2) and 30 d (experiment 3) to study their effects in sheep. In experiment 2, 6 adult nonpregnant sheep, with permanent rumen cannula and fed alfalfa hay and oats (60:40), were treated with E3NP at 2 doses (50 and 500mg/animal per day). After 7, 14, and 15 d of treatment, methane emissions were recorded in respiration chambers and rumen fluid samples were collected for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. Methane production decreased by 29% compared with the control with the higher dose of E3NP on d 14 to 15. A decrease in the acetate:propionate ratio was observed without detrimental effects on dry matter intake. In experiment 3, 9 adult nonpregnant sheep, with permanent rumen cannula and fed with alfalfa hay and oats (60:40), were treated with E3NP or 3NP at one dose (100mg/animal per day) over 30 d. On d 14 and d 29 to 30, methane emissions were recorded in respiration chambers. Rumen fluid samples were collected on d 29 and 30 for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. In addition, on d 22 and 23, samples of oats and alfalfa hay were incubated in the rumen of sheep to determine dry matter ruminal degradation over 24 and 48h, respectively; no effect was observed (78.6, 78.3, and 78.8% of alfalfa and 74.2, 74.0, and 70.6% of oats in control, E3NP, and 3NP groups, respectively). A reduction in methane production was observed for both additives at d 14 and d 29 to 30. In both treatments, the acetate:propionate ratio was significantly decreased. Likewise, total concentrations of the analyzed microbial groups in the rumen showed no difference among treatments and doses for both experiments. Both tested compounds showed promise as methane inhibitors in the rumen, with no detrimental effects on fermentation or intake, which would need to be confirmed in lactating animals.

Effect of a high strength chemical industry wastewater on microbial community dynamics and mesophilic methane generation

A high strength chemical industry wastewater was assessed for its impact on anaerobic microbial community dynamics and consequently mesophilic methane generation. Cumulative methane production was 251 mL/g total chemical oxygen demand removed at standard temperature and pressure at the end of 30 days experimental period with a highest recorded methane percentage of 80.6% of total biogas volume. Volatile fatty acids (VFAs) analysis revealed that acetic acid was the major intermediate VFAs produced with propionic acid accumulating over the experimental period. Quantitative analysis of microbial communities in the test and control groups with quantitative real time polymerase chain reaction highlighted that in the test group, Eubacteria (96.3%) was dominant in comparison with methanogens (3.7%). The latter were dominated by Methanomicrobiales and Methanobacteriales while Methanosarcinaceae in test groups increased over the experimental period, reaching a maximum on day 30. Denaturing gradient gel electrophoresis profile was performed, targeting the 16S rRNA gene of Eubacteria and Archaea, with the DNA samples extracted at 3 different time points from the test groups. A phylogenetic tree was constructed for the sequences using the neighborhood joining method. The analysis revealed that the presence of organisms resembling Syntrophomonadaceae could have contributed to increased production of acetic and propionic acid intermediates while decrease of organisms resembling Pelotomaculum sp. could have most likely contributed to accumulation of propionic acid. This study suggested that the degradation of organic components within the high strength industrial wastewater is closely linked with the activity of certain niche microbial communities within eubacteria and methanogens.