Effects of nitrate addition to a diet on fermentation and microbial populations in the rumen of goats,with special reference to Selenomonas ruminantium having the ability to reduce nitrate and nitrite

This study investigated the effects of dietary nitrate addition on ruminal fermentation characteristics and microbial populations in goats. The involvement of Selenomonas ruminantium in nitrate and nitrite reduction in the rumen was also examined. As the result of nitrate feeding, the total concentration of ruminal volatile fatty acids decreased, whereas the acetate : propionate ratio and the concentrations of ammonia and lactate increased. Populations of methanogens, protozoa and fungi, as estimated by real‐time PCR, were greatly decreased as a result of nitrate inclusion in the diet. There was modest or little impact of nitrate on the populations of prevailing species or genus of bacteria in the rumen, whereas Streptococcus bovis and S. ruminantium significantly increased. Both the activities of nitrate reductase (NaR) and nitrite reductase (NiR) per total mass of ruminal bacteria were increased by nitrate feeding. Quantification of the genes encoding NaR and NiR by real‐time PCR with primers specific for S. ruminantium showed that these genes were increased by feeding nitrate, suggesting that the growth of nitrate‐ and nitrite‐reducing S. ruminantium is stimulated by nitrate addition. Thus, S. ruminantium is likely to play a major role in nitrate and nitrite reduction in the rumen.     

Effect of ethanol on nitrate and nitrite reduction and methanogenesis in the ruminal microbiota

The effect of ethanol on nitrate and nitrite reduction was examined by conducting in vitro experiments with mixed ruminal microbes. The addition of ethanol to cultures of mixed ruminal microbes stimulated nitrate reduction, and, to a greater extent, nitrite reduction, which resulted in a decrease in nitrite accumulation. However, known nitrate‐reducing ruminal bacteria, such as Selenomonas ruminantium, Veillonella parvula and Wolinella succinogenes, were unable to utilize ethanol directly as an electron donor for nitrate reduction. No nitrate‐reducing bacterium capable of utilizing ethanol was found in the rumen of goats. However, when mixed ethanol‐utilizing, hydrogen gas (H₂)‐producing bacteria (Ruminococcus albus and Ruminococcus flavefaciens) were added to the culture of the mixed nitrate‐reducing bacteria described above, nitrate and nitrite reduction was observed. These results suggest that the nitrate‐reducing bacteria utilized the H₂ that was produced from ethanol oxidation by the ethanol‐utilizing bacteria as an electron donor. It is conceivable that the stimulation of nitrate and nitrite reduction by ethanol, observed in the culture of mixed ruminal microbes, was a result of electron transfer from ethanol to nitrate, and nitrite through H₂, that is, ‘interspecies hydrogen transfer’ from ethanol‐metabolizing bacteria to nitrate‐reducing bacteria. Thus, the addition of ethanol to high‐nitrate diets may be effective for preventing nitrate poisoning. Furthermore, methane production was reduced to less than one‐third by the addition of mixed nitrate‐reducing bacteria to the co‐culture of mixed methanogens with mixed ethanol‐utilizing bacteria incubated in a medium containing ethanol and nitrate. Therefore, the addition of ethanol and nitrate may decrease methanogenesis without suppressing overall fermentation in the rumen.     

Ability to utilize lactate and related enzymes of a ruminal bacterium, Selenomonas ruminantium

The ability of a nitrate‐reducing and nitrite‐reducing strain of Selenomonas ruminantium ssp. lactilytica (TH1) to utilize lactate was examined at the cell and enzyme levels. The TH1 strain was found to possess NAD‐independent D‐lactate dehydrogenase (iD‐LDH), with little or no lactate racemase or L‐lactate dehydrogenase, implying that TH1 virtually utilizes only the D‐form of lactate. Therefore, the introduction of lactate racemase to TH1 may enhance its ability to utilize lactate in the rumen where both D‐lactate and L‐lactate are produced. Because lactate utilization by Megasphaera elsdenii in the rumen may increase methanogenesis, it is desirable to increase lactate utilization by S. ruminantium, which may decrease methanogenesis. However, the specific activity of iD‐LDH, which represents the amount of enzyme per cell, in TH1 was approximately threefold lower than M. elsdenii. Properties of iD‐LDH, such as optimal pH and temperature, affinity for D‐lactate, and effect of metal ions, did not differ greatly between TH1 and M. elsdenii. The specific activity of iD‐LDH in TH1 increased as the D‐lactate concentration in the medium increased, suggesting that iD‐LDH synthesis is regulated in response to D‐lactate. On the contrary, no iD‐LDH activity was detected when TH1 was grown in the presence of glucose, even when D‐lactate was present. This result suggests that iD‐LDH synthesis is strongly suppressed by glucose. In order to improve the ability of S. ruminantium to utilize lactate and reduce nitrate and nitrite, it is important to enhance iD‐LDH synthesis in addition to the introduction of lactate racemase.     

Effect of high oral doses of nitrate on salivary recirculation of nitrates and nitrites and on bacterial diversity in the saliva of young pigs

Ingested nitrate is absorbed in the small intestine, recirculated into the saliva and reduced to nitrite by oral bacteria. In pigs receiving a moderate dietary addition of nitrate, the recirculation into the saliva is modest, so we aimed to assess the effect of higher nitrate doses to find out how the animal reacts to this new situation and to evaluate if a higher nitrate level could enhance the nitrate reduction process, improving the nitrite production Trial 1. Six piglets received 100 g of a commercial diet with 2.45% KNO₃. In relation to baseline values, nitrate in blood serum and saliva increased 15 times, and declined after 6 h vs. 2 h. Salivary nitrite increased seven times after the addition and declined after 6 h vs. 2 h. Trial 2. Six piglets were fed a diet with or without 1.22% KNO₃ for 2 weeks. Salivary nitrate and nitrite increased with the addition of KNO3: nitrate increased from d0 to the end of the trial, nitrite increased 15 times after 1 week, but decreased after 2 weeks to 4.5‐fold the control. After 2 weeks, nitrate reduced Shan diversity index of salivary microbiota. The present results indicate that the long exposure to high quantities of nitrates impairs the oral reduction of nitrate to nitrite and engenders a reduction of the mouth’s microbiota diversity.     

Numbers of nitrate-reducing bacteria in the rumen as estimated by competitive polymerase chain reaction

Cell numbers of known species of nitrate- and nitrite-reducing bacteria, Selenomonas ruminantium, Veillonella parvula and Wollinella succinogenes , in the rumen of goats (25–30 kg) were estimated by competitive polymerase chain reaction (PCR). The number of S. ruminantium was the largest of the three species examined, and tended to be greater in goats fed a high-concentrate diet (5.6 × 10⁷ cells/mL rumen fluid) than in goats fed a high-roughage diet (1.3 × 10⁷ cells/mL). The number of V. parvula tended to be greater when goats were fed a high-roughage diet (6.7 × 10³/mL) than when fed a high-concentrate diet (3.2 × 10³/mL). The number of W. succinogenes was below the detectable level (< 1.0 × 10²/mL) when a high-concentrate diet was fed, but was significantly increased by feeding a high-roughage diet (1.6 × 10³/mL). Addition of potassium nitrate (6 g/day) to the high-concentrate diet tended to increase V. parvula , and significantly increased W. succinogenes , indicating that these two bacteria can be increased by feeding a diet containing nitrate.     

Synergistic fibrolysis in the rumen by cellulolytic Ruminococcus flavefaciens and non-cellulolytic Selenomonas ruminantium: Evidence in defined cultures

To investigate the rumen bacterial interaction between cellulolytic Ruminococcus flavefaciens and non‐cellulolytic Selenomonas ruminantium, fiber digestibility and fermentation products were determined in defined cultures consisting of these two species. Avicel, orchardgrass hay, rice straw and alfalfa hay were used as substrates for 72 h incubation to monitor digestibility, volatile fatty acids, succinate, lactate and bacterial number. In monoculture, R. flavefaciens digested the fiber sources at 21–32%, while S. ruminantium strains did not. When R. flavefaciens was cocultured with one of three different strains (GA192, S137 and S150) of S. ruminantium, fiber digestion exceeded the value recorded by R. flavefaciens alone. In particular, cocultures with S. ruminantium S137 showed significantly higher digestibility for all the fiber sources than R. flavefaciens alone (P < 0.05). Propionate production and growth of S. ruminantium was notable in all cocultures but not in monocultures. Succinate was accumulated in monoculture of R. flavefaciens, while the accumulation was not observed in cocultures. These results indicate that R. flavefaciens provides fiber hydrolysis products to S. ruminantium as growth substrates. In addition, S. ruminantium could activate R. flavefaciens by rapidly consuming the products. Such cross‐feeding between cellulolytic and non‐cellulolytic bacteria could enhance fiber digestion, although the extent of the enhancement may depend on strain combinations.     

The bio‐surfactant mannosylerythritol lipid acts as a selective antibacterial agent to modulate rumen fermentation

Methyl‐mannosylerythritol lipid (MEL), a new sugar esterified lipid synthesized by Pseudozyma aphidis, was assessed for its functionality in modulating rumen fermentation and microbiota toward more propionate and less methane production. A pure culture study using rumen representatives showed that MEL selectively inhibited the growth of most Gram‐positive bacteria including Streptococcus bovis, ruminococci, and Fibrobacter succinogenes, but not Gram‐negative bacteria such as Megasphaera elsdenii, Succinivibrio dextrinosolvens, and Selenomonas ruminantium. A batch culture study revealed that MEL significantly decreased methane production in a dose‐dependent manner with accumulation of hydrogen, while propionate production was enhanced. A continuous culture (Rusitec) study confirmed all of these changes. A feeding study revealed that sheep fed a MEL diet showed an increased proportion of propionate, while proportions of acetate and butyrate were decreased without affecting total VFA level. These changes disappeared after cessation of MEL feeding. Based on these results, dietary application of MEL can favorably modify rumen fermentation in terms of the efficiency of dietary energy utilization.     

The effect of dietary Chlorella vulgaris supplementation on micro‐organism community,enzyme activities and fatty acid profile in the rumen liquid of goats

Microalgae might be considered as an alternative source of fat and/or protein for ruminant's diets. However, changes in populations of ruminal micro‐organisms associated with biohydrogenation process, methane and ammonia production in response to microalgae dietary supplementation have not been well characterized. Thus, 16 cross‐bred goats were divided into two groups. Each goat of both groups was fed individually with alfalfa hay and concentrates separately. The concentrates of the control group had no microalgae while those of the treated group were supplemented with 10 g lyophilized Chlorella vulgaris/kg concentrate (chlor). On the 30th experimental day, samples of rumen fluid were collected for microbial DNA extraction, fatty acid profile and enzyme activity analyses. The results showed that the chlor diet compared with the control increased significantly the populations of Methanosphaera stadtmanae, Methanobrevibacter ruminantium and Methanogens bacteria and protozoa in the rumen of goats. A significant reduction in the cellulase activity and in the abundance of Ruminococcus albus, and a significant increase in the protease activity and in the abundance of Clostridium sticklandii in the rumen liquid of goats fed with the chlor diet, compared with the control, were found. Chlorella vulgaris supplementation promoted the formation of trans C_18:1, trans‐11 C_18:1 and monounsaturated fatty acids (MUFA), while the proportions of C_18:0 and long‐chain fatty acids (LCFA) reduced significantly in the rumen liquid of goats. This shift in ruminal biohydrogenation pathway was accompanied by a significant increase in Butyrivibrio fibrisolvens trans C_18:1‐producing bacteria. In conclusion, the supplementation of diets with microalgae needs further investigation because it enhances the populations of methane‐producing bacteria and protozoa.     

Partial characterization of phylogeny,ecology and function of the fibrolytic bacterium Ruminococcus flavefaciens OS14, newly isolated from the rumen of swamp buffalo

The fibrolytic rumen bacterium Ruminococcus flavefaciensOS14 was isolated from swamp buffalo and its phylogenetic, ecological and digestive properties were partially characterized. Isolates from rumen contents of four swamp buffalo were screened for fibrolytic bacteria; one of the 40 isolates showed a distinctive feature of solubilizing cellulose powder in liquid culture and was identified as R. flavefaciens based on its 16S ribosomal DNA sequence. This isolate, OS14, was employed for detection and digestion studies, for which a quantitative PCR assay was developed and defined cultures were tested with representative forages in Thailand. OS14 was phylogenetically distant from other isolated and uncultured R. flavefaciens and showed limited distribution among Thai ruminants but was absent in Japanese cattle. OS14 digested rice straw and other tropical forage to a greater extent than the type strain C94 of R. flavefaciens. OS14 produced more lactate than C94, and digested para grass to produce propionate more extensively in co‐culture with lactate‐utilizing Selenomonas ruminantium S137 than a co‐culture of C94 with S137. These results indicate that phylogenetically distinct OS14 could digest Thai local forage more efficiently than the type strain, possibly forming a symbiotic cross‐feeding relationship with lactate‐utilizing bacteria. This strain might be useful for future animal and other industrial applications.     

Validation and application of real-time polymerase chain reaction assays for representative rumen bacteria

Real‐time polymerase chain reaction (PCR) assays for 11 representative rumen bacterial species were validated. The sensitivity was tested by using the serially diluted target 16S rDNA from respective bacterial species. The recovery of the target DNA and the assay reproducibility were determined using DNA from rumen fluid spiked with different quantities of the target. Minimum detection levels for the target were 10–100 copies in pure culture. The recovery of the added target ranged from 82.4 to 116.6%. The intra‐ and inter‐assay variations of each assay were <9.4 and <12.6%, respectively. Therefore, the real‐time PCR assays evaluated in the present study are considered to be sufficiently reliable for monitoring all 11 bacterial species in the rumen. The assays were then applied to the monitoring of the bacterial species attached to ruminally incubated rice straw. Among the monitored fibrolytic species, Fibrobacter succinogenes was found to be the most dominant, accounting for 2.61% of total bacteria after 24 h incubation. Selenomonas ruminantium and Streptococcus bovis, non‐fibrolytics, were detected on the rice straw at 8.96% and 1.16% of total bacteria, respectively. Such high levels of non‐fibrolytics on the plant fiber suggest a synergistic relationship between fibrolytics and non‐fibrolytics.     

Effect of non‐starch‐polysaccharide‐degrading enzymes as feed additive on the rumen bacterial population in non‐lactating cows quantified by real‐time PCR

The effects of non‐starch‐polysaccharide‐degrading enzymes, added to a maize silage‐ and grass silage‐based total mixed ration (TMR) at least 14 h before feeding, on the rumen bacterial population were investigated. Six non‐lactating Holstein Friesian cows were allocated to three treatment groups using a duplicate 3 × 3 Latin square design with three 31‐day periods (29 days of adaptation and 2 days of sampling). Treatments were control TMR [69% forage and 31% concentrates on a dry matter (DM) basis] or TMR with 13.8 or 27.7 ml/kg of feed DM of Roxazyme G2 liquid with activities (U/ml enzyme preparation) of xylanase 260 000, β‐glucanase 180 000 and cellulase 8000 (DSM Nutritional Products, Basel, Switzerland). The concentrations of 16S rDNA of Anaerovibrio lipolytica, Fibrobacter succinogenes, Prevotella ruminicola, Ruminococcus flavefaciens, Selenomonas ruminantium and Treponema bryantii, and their relative percentage of total bacteria in rumen samples obtained before feeding and 3 and 7 h after feeding and from two rumen fractions were determined using real‐time PCR. Sampling time had only little influence, but bacterial numbers and the composition of the population differed between the transition layer between rumen fluid and the fibre mat (fraction A) and the rumen fluid (fraction B) highlighting the importance to standardize sampling. The 16S rDNA copies of total bacteria and the six bacterial species as well as the population composition were mainly unaffected by the high levels of exogenous enzymes supplemented at all sampling times and in both rumen fractions. Occasionally, the percentages of the non‐fibrolytic species P. ruminicola and A. lipolytica changed in response to enzyme supplementation. Some increases in the potential degradability of the diet and decreases in lag time which occurred collaterally indicate that other factors than changes in numbers of non‐particle‐associated bacteria are mainly responsible for the effects of exogenous enzymes.     

Oral administration of Lactobacillus plantarum and Bacillus subtilis on rumen fermentation and the bacterial community in calves

The objective of this study was to assess the effect of dietary probiotics on rumen fermentation and the bacterial community in dairy calves. Twelve Holstein calves were randomly allocated to three treatments: a basal diet, the basal diet supplemented with Lactobacillus plantarum GF103 (LB) or basal diet supplemented with a mixture of Lactobacillus plantarum GF103 and Bacillus subtilis B27 (LBS). A milk replacer was fed to calves from 8 days of age. A starter and alfalfa hay was offered ad libitum from 21 and 28 days of age, respectively, and the orts were weighted daily. The ruminal fluid was sampled at 56 and 83 days of age to determine the rumen fermentation characteristics. The bacterial community was analyzed by denaturing gradient gel electrophoresis (DGGE) and the number of certain bacteria was quantified by real‐time polymerase chain reaction. The ratio of total dry matter intake to average body wieght was higher in the control (P < 0.05). The DGGE fingerprint of the 16S ribosomal RNA gene was affected by the blended probiotics at 83 days of age. The number of Ruminococcus albus was lower in the LB and LBS treatment (P < 0.05). Oral administration of the probiotics affected the rumen bacterial community and the numbers of cellulolytic bacteria decreased.     

The effects of dietary calcium iodate on productive performance,egg quality and iodine accumulation in eggs of laying hens

The aim of this study was to examine the effects of various levels of supplemental calcium iodate (CI) on productive performance, egg quality, blood indices and iodine (I) accumulation in the eggs in commercial laying hens. A total of 240 White Leghorn layers (Hy‐line W36) were divided through a completely randomized design into six treatments with five replicates and eight hens per each at 32 weeks of age. This experiment lasted for 12 weeks. Concentrations of I in the mash diets were 0.74, 3.13, 5.57, 8.11, 10.65 and 12.94 mg I/kg of feed in treatments 1–6 respectively. The added doses of CI were included 0.0 (control), 2.5, 5.0, 7.5, 10.0 and 12.5 mg/kg of diet for treatments 1–6 respectively. There were no significant differences in productive performance among the treatments. The highest eggshell strength was observed in group fed diet containing 3.13 mg I/kg (p = .014). The highest percentage of calcium and lowest percentage of phosphorus in eggshell were observed in group fed diet containing 12.94 mg I/kg (p = .0001). Feeding hens with diet containing 12.94 mg I/kg increased serum triiodothyronine‐to‐thyroxine ratio (p = .0001). Serum alanine aminotransferase activity in hens fed diet containing 12.94 mg I/kg was significantly more than control (p = .041). Blood Serum triglycerides in hens fed diet containing 8.11 mg I/kg were significantly higher than control (p = .0001). Edible fraction of the eggs of birds fed diet containing 12.94 mg I/kg was enriched by I almost 3 times more than those fed diet containing 0.74 mg I/kg. The results suggested that egg production, egg mass, feed intake and feed conversion ratio were not significantly affected by dietary I levels. Iodine accumulation in the eggs were increased by increasing dietary I levels and the level of 10 mg/kg CI could supply I enrichment of the eggs.     

3种饲草组合对湖羊瘤胃还原硝酸盐过程的影响

采用3×3拉丁方试验设计,研究以燕麦(Avena sativa)或苜蓿(Medicago sativa)干草等比例替代羊草(Leymus chinensis)后,对饲草中硝酸盐动态消失率及湖羊瘤胃发酵参数的影响。晨饲后0、1、2、3、4、5和6h通过瘤胃瘘管采集湖羊瘤胃液,测定瘤胃pH值以及硝酸盐(NO_3~-)、亚硝酸盐(NO_2~-)、氨态氮(NH_3-N)的浓度;同时,晨饲后2h经颈静脉采血测定血清高铁血红蛋白含量。结果表明,1)3组瘤胃液NO_3~-和NO_2~-浓度分别在喂后1和2h达到最大值,之后迅速降低;2)pH值、NO_3~-、NO_2~-浓度各组间均无显著差异(P0.05);NH_3-N仅在饲喂3h后全羊草组(A组)显著低于其它两组(P=0.018);3)高铁血红蛋白含量均较低,且无显著差异(P0.05)。本研究选用的牧草组合对湖羊瘤胃还原硝酸盐过程没有显著影响。     

Effect of maize starch concentration in the diet on starch and cell wall digestion in the dairy cow

An in vivo experiment was performed to determine the effect of level of maize starch in the diet on digestion and site of digestion of organic matter, starch and neutral detergent fibre (NDF). In a repeated change‐over design experiment, three cows fitted with a rumen cannula and T‐piece cannulae in duodenum and ileum received a low‐starch (12% of ration dry matter) and a high‐starch (33% of ration dry matter) diet. Starch level was increased by exchanging dried sugar beet pulp by ground maize. After a 2‐week adaptation period, feed intake, rumen fermentation parameters (in vivo and in situ), intestinal flows, faecal excretion of organic matter, starch and NDF were estimated. When the high‐starch diet was fed, dry matter intake was higher (19.0 kg/day vs. 17.8 kg/day), and total tract digestibility of organic matter, starch and NDF was lower when the low‐starch diet was fed. Maize starch concentration had no significant effect on rumen pH and volatile fatty acid concentration nor on the site of digestion of organic matter and starch and rate of passage of ytterbium‐labelled forage. On the high‐starch diet, an extra 1.3 kg of maize starch was supplied at the duodenum in relation to the low‐starch diet, but only an extra 0.3 kg of starch was digested in the small intestine. Digestion of NDF was only apparent in the rumen and was lower on the high‐starch diet than on the low‐starch diet, mainly attributed to the reduction in sugar beet pulp in the high‐starch diet. It was concluded that without the correction for the reduction in NDF digestion in the rumen, the extra supply of glucogenic (glucose and propionic acid) and ketogenic nutrients (acetic and butyric acid) by supplemented starch will be overestimated. The mechanisms responsible for these effects need to be addressed in feed evaluation.     

日粮硝酸盐水平对湖羊瘤胃硝态氮动态消失率、发酵参数及血液高铁血红蛋白含量的影响

本试验研究日粮硝酸盐的添加量对湖羊瘤胃还原硝态氮程度和对发酵参数及血液高铁血红蛋白的影响。试验选取6头湖羊,采用随机区组试验设计,每期分别饲喂含0%,1%,2%,3%,4%,5%硝酸钾的日粮。测定喂后0,0.5,1.0,1.5,2.0,4.0,6.0,8.0 h瘤胃液硝酸盐和亚硝酸盐含量及瘤胃发酵参数。晨饲后2.0 h通过颈静脉采血,测定高铁血红蛋白含量。结果表明,湖羊在采食1.0 h后硝酸盐含量达到最高(0.952.14 g/L),之后快速消失,亚硝酸盐含量在喂后1.5 h达到最高(0.936.22 μmol/L),之后降低。高铁血红蛋白的含量随硝酸盐添加量的增加而显著升高(P<0.01),最高达0.32%。添加硝酸盐显著提高瘤胃液pH值(P<0.01)和2.0 h的氨态氮含量(P<0.01)以及乙酸/丙酸,添加量高于3%会降低微生物蛋白和总挥发性脂肪酸的浓度,2%的硝酸盐添加量微生物蛋白和总挥发性脂肪酸的合成量最高,最有利于微生物发酵。     

Ground transport stress affects bacteria in the rumen of beef cattle: A real‐time PCR analysis

Transport stress syndrome often appears in beef cattle during ground transportation, leading to changes in their capacity to digest food due to changes in rumen microbiota. The present study aimed to analyze bacteria before and after cattle transport. Eight Xianan beef cattle were transported over 1000 km. Rumen fluid and blood were sampled before and after transport. Real‐time PCR was used to quantify rumen bacteria. Cortisol and adrenocorticotrophic hormone (ACTH) were measured. Cortisol and ACTH were increased on day 1 after transportation and decreased by day 3. Cellulolytic bacteria (Fibrobacter succinogenes and Ruminococcus flavefaciens), Ruminococcus amylophilus and Prevotella albensis were increased at 6 h and declined by 15 days after transport. There was a significant reduction in Succinivibrio dextrinosolvens, Prevotella bryantii, Prevotella ruminicola and Anaerovibrio lipolytica after transport. Rumen concentration of acetic acid increased after transport, while rumen pH and concentrations of propionic and butyric acids were decreased. Body weight decreased by 3 days and increased by 15 days after transportation. Using real‐time PCR analysis, we detected changes in bacteria in the rumen of beef cattle after transport, which might affect the growth of cattle after transport.     

Effects of thiosulfate addition on ammonia and nitrogen removal in biofilters packed with Oyaishi (pumice tuff)

Ammonia removal is achieved partly by absorption and nitrification in biofilters, resulting in the accumulation of nitrogen and the necessity of treating the effluent water. We investigated the effects of thiosulfate addition to a biofilter containing pumice tuff for ammonia and nitrogen removal in a laboratory‐scale experiment. The addition of thiosulfate to the circulating water led to a decreased nitrate and nitrite along with an increase of sulfate. The inorganic nitrogen in the circulating water decreased by up to 44% with thiosulfate addition compared to without thiosulfate. Batch experiments revealed that denitrification activity decreased exponentially along with increases in dissolved oxygen; however, approximately 30% of denitrification activity was maintained at dissolved oxygen concentration of 3.3 mg/L. Metabarcoding of 16S rRNA genes indicated that the genus Thiobacillus had a relative abundance of 0.002%–0.016% of total bacteria in the biofilter packing material. The circulating water pH was decreased below 5 with sulfur oxidation, and ammonium was accumulated without pH control resulting in a decrease in the relative abundance of the family Nitrosomonadaceae. Its relative abundance increased with control of pH to near neutral, indicating that ammonia‐oxidizing activity could be maintained by adjusting pH. Thiosulfate addition could stimulate nitrogen removal by sulfur‐dependent denitrification in biofiltration systems.     

Effects of incremental amounts of fish oil on trans fatty acids and Butyrivibrio bacteria in continuous culture fermenters

Previous studies have shown that adding fish oil (FO) to ruminant animal diets increased vaccenic acid (VA; t11 C18:1) accumulation in the rumen. Therefore, the objective of this study was to evaluate the effect of dietary FO amounts on selected strains of rumen bacteria involved in biohydrogenation. A single‐flow continuous culture system consisting of four fermenters was used in a 4 × 4 Latin square design with four 9 days consecutive periods. Treatment diets were as follows: (i) control diet (53:47 forage to concentrate; CON), (ii) control plus FO at 0.5% (DM basis; FOL), (iii) control plus FO at 2% (DM basis; FOM) and (iv) control plus FO at 3.5% (DM basis; FOH). Fermenters were fed treatment diets three times daily at 120 g/day. Samples were collected from each fermenter on day 9 of each period at 1.5, 3 and 6 h post‐morning feeding and then composited into one sample per fermenter. Increasing dietary FO amounts resulted in a linear decrease in acetate and isobutyrate concentrations and a linear decrease in acetate‐to‐propionate ratio. Propionate, butyrate, valerate and isovalerate concentrations were not affected by FO supplementation. Concentrations of C18:0 in fermenters linearly decreased, while concentrations of t10 C18:1 and VA linearly increased as dietary FO amounts increased. The concentrations of c9t11 and t10c12 conjugated linoleic acid were not affected by FO supplementation. The DNA abundance for Butyrivibrio fibrisolvens, Butyrivibrio vaccenic acid subgroup, Butyrivibrio stearic acid subgroup and Butyrivibrio proteoclasticus linearly decreased as dietary FO amounts increased. In conclusion, FO effects on trans fatty acid accumulation in the rumen may be explained in part by FO influence on Butyrivibrio group.