Effect of cellulase‐producing bacteria on the metabolic activities and functional diversity of bacterial communities in Chinese liquor fermenting grains

To study the roles of cellulase‐producing bacteria in the fermentation of Chinese liquor fermenting grains, a strain of cellulase‐producing bacteria was added into the fermenting grains at different levels, and the substrate utilization was profiled to evaluate the bacterial communities’ functional diversity in fermenting grains during fermentation. The results showed that, when the addition level of the cellulase‐producing bacteria was at or below 10² cfu/g, the average well colour development (AWCD_590nm) of the fermenting grains was not affected significantly. When the addition level was more than 10⁴ cfu/g, the average well colour development (AWCD_590nm) of the fermenting grains increased significantly, and the maximum AWCD_590nm was obtained when the addition was 10⁶ cfu/g. The diversity index analysis showed that the addition of the cellulase‐producing bacteria helped to increase the uniformity of bacterial communities in fermenting grains significantly, while the richness and dominance of the microbial communities were not affected. Copyright © 2015 The Institute of Brewing & Distilling     

Investigation of physicochemical composition and microbial communities in traditionally fermented vrum from Inner Mongolia

Mongolian traditionally fermented vrum is known for its functional characteristics, and indigenous microbial flora plays a critical role in its natural fermentation. However, studies of traditionally fermented vrum are still rare. In this study, we investigated the artisanal production of traditionally fermented vrum from Inner Mongolia. In general, its physicochemical composition was characterized by 34.5 ± 8% moisture, 44.9 ± 12.1% fat, 10.6 ± 3.2% protein, and 210 ± 102°T. The total lactic acid bacteria and yeast counts ranged from 50 to 2.8 × 10⁸ cfu/g and from 0 to 1.1 × 10⁶ cfu/g, respectively. We studied bacterial and fungal community structures in 9 fermented vrum; we identified 5 bacterial phyla represented by 11 genera (an average relative abundance >1%) and 8 species (>1%), and 3 fungal phyla represented by 8 genera (>1%) and 8 species (>1%). Relative abundance values showed that Lactococcus and Lactobacillus were the most common bacterial genera, and Dipodascus was the predominant fungal genus. This scientific investigation of the nutritional components, microbial counts, and community profiles in Mongolian traditionally fermented vrum could help to develop future functional biomaterials and probiotics.     

酱香型白酒第四轮次酒酿造过程中细菌多样性分析

该研究应用Illumina MiSeq高通量测序技术解析酱香型白酒第四轮次酒酿造过程中细菌多样性,并阐明其优势真菌微生物群落结构及其随酿造工艺动态变化。结果表明,在酒曲中主要优势菌有芽孢杆菌科（Bacillaceae）、海洋芽胞杆菌属（Oceanobacillus）;堆积发酵中主要优势菌有芽孢杆菌科（Bacillaceae）、变形菌纲（Proteobacteria）;在窖内发酵中乳杆菌属（Lactobacillus）占绝对优势;窖泥中主要优势菌有放线菌（Actinomyces）和乳杆菌属（Lactobacillus）和醋杆菌属（Acetobacter）。同一酒厂和不同酒厂新老车间酒曲、堆积、窖内发酵、窖泥之间细菌组成相似度较高,但其优势菌群丰度差异显著。发酵车间使用年限及窖龄影响着酿酒微生物多样性;使用时间长的车间和窖池其环境微生物种群结构更稳定,优势菌群更突出。     

Inoculant effects on alfalfa silage: fermentation products and nutritive value

The effect of 14 microbial inoculants on the fermentation and nutritive value of alfalfa silages was studied under laboratory conditions. The first cut (477 g of dry matter/kg) and second cut (393 g of dry matter/kg) of a second-year alfalfa stand were ensiled in 2 trials. In both trials alfalfa was harvested with standard field equipment. All inoculants were applied at 1.0 × 10⁶ cfu/g of crop. Uninoculated silages served as controls. After inoculants were added, the chopped forages were ensiled in 1.0- and 0.5-L anaerobic glass jars, respectively, at a density of 500 g/L. Each trial had 15 treatments (uninoculated control and 14 inoculants), with 4 silos per treatment. Silos were stored for a minimum of 30 d at room temperature (∼22°C). In first-cut silage, all inoculants but one reduced pH relative to the uninoculated control, and all but 2 of the homofermentative strains shifted fermentation toward lactic acid. In second-cut silage, the epiphytic lactic acid bacterial population was 2.7 × 10⁷ cfu/g, and only commercial inoculants produced significant shifts in fermentation. Overall, microbial inoculants generally had a positive effect on alfalfa silage characteristics in terms of lower pH and shifting fermentation toward lactic acid with homofermentative lactic acid bacteria or toward acetic acid with heterofermentative lactic acid bacteria, Lactobacillus buchneri. These effects were stronger in the commercial products tested. In spite of the positive effects on silage fermentation, 48-h in vitro true DM digestibility was not improved by inoculation with lactic acid bacteria.     

牛栏山二锅头大茬酒醅发酵过程中细菌群落结构分析

为解析牛栏山二锅头大茬酒醅发酵过程中细菌群落结构及多样性,确定发酵过程中细菌种属变化及优势菌群。 该研究采用 传统可培养分离方法和高通量测序技术对牛栏山二锅头大茬酒醅发酵过程中的细菌群落进行多样性分析。 研究结果表明,入池阶段, 大茬酒醅内含有大量乳酸菌、芽孢杆菌和其余细菌,随着发酵的进行,乳酸菌数量快速增长,成为发酵过程中的主体细菌,仅在出池 阶段数量出现降低。而芽孢杆菌和其余细菌数量基本稳定,放线菌数量较少且无规律性变化;高通量测序共获得559种细菌类操作分 类单元（OUT）,分别为乳杆菌科、高温放线菌科、明串珠菌科和醋杆菌科。发酵温度表现出前缓、中挺、后缓落3个阶段,不同种乳酸菌 分别成为大茬酒醅中的优势菌群,同时在牛栏山二锅头发酵过程中首次发现了高温放线菌（Thermoactinomyces）。     

“FD工艺”对酱香型白酒堆积过程的影响

为探究酱香型白酒酿造堆积过程中酒醅堆各位点发酵状态及细菌群落结构的变化规律,将“FD工艺”应用于酱香型白酒第二轮次酒生产堆积发酵过程,对堆积过程中的酒醅理化因子和细菌群落变化进行探索和研究。结果表明:相较于传统酒醅堆积发酵而言,“FD工艺”处理后的酒醅酸度能够有效降低,酸度下降了1.72 mmol/10 g,同时能有效控制酒醅中水分含量,使水分含量保持在±0.8%;“FD工艺”能有效促进淀粉转化（增加转化了0.76%）,同时酒醅中还原糖含量也增加（增长了0.46%）;酒醅细菌群落Shannon指数、Sobs指数和Ace指数显著（P<0.05）增加;“FD工艺”处理后的酒醅微生物属分类水平的丰富度和多样性明显增加,有利于好氧菌属如芽孢杆菌属Bacillus、高温放线菌属Thermoactinomyces等微生物生长。综上,“FD工艺”能改善堆积发酵过程中酒醅发酵不均的情况,酒醅细菌群落丰富度和多样性均明显增加,有利于优势好氧及耐高温细菌群落的生长。     

The effect of the feed-to-buffer ratio on bacterial diversity and ruminal fermentation in single-flow continuous-culture fermenters

Eight single-flow continuous-culture fermenters were used in a completely randomized block design with a 2 × 4 factorial arrangement of treatments to investigate the effects of the feed-to-buffer ratio (F/B) on ruminal fermentation, the diversity and community structure of bacteria, nutrient digestibility, and N metabolism. Four diets with forage-to-concentrate ratios of 70:30 or 30:70 with alfalfa or grass hay as forage were supplied to fermenters twice per day at 2 different F/B (23.5 and 35 g of DM/L). The dilution rate was kept constant (5.3%) among all fermenters by infusing the same volume of buffer. An increase in the total volatile fatty acid (VFA) concentration and a decrease in the average pH were observed with an increased F/B. In addition, the molar proportions of all individual VFA found in fermenters differed, depending on the F/B. A terminal restriction fragment length polymorphism analysis showed that the community structure and diversity of bacteria were highly influenced by the F/B. Both diversity and the number of peaks in the electropherograms were lower in most fermenters receiving diets at a high F/B, whereas the similarity percentage of the bacterial communities across diets was higher as the F/B increased. Moreover, the high reduction of neutral detergent fiber digestibility (15.3% ± 3.65) in fermenters with high F/B suggested a pH-related decrease in the cellulolytic bacterial community as the F/B increased. The crude protein degradation found in fermenters receiving diets with a high F/B was lower compared with that from fermenters with a low F/B. The VFA concentration and purine bases flow response patterns to diets were similar to in vivo conditions only in the case of fermenters with a low F/B. The results suggested that the community structure and diversity of bacteria, as well as the in vitro fermentation parameters, may be affected by the F/B that is used, most likely through a pH effect. In addition, several fermentation parameters showed different response patterns to diets according to the F/B used. Therefore, the amount of feed supplied to single-flow continuous-culture fermenters in which pH is not under control should be carefully chosen according to the volume of buffer infused for the purpose of simulating ruminal fermentation.     

Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives

The first objective of this study was to examine effects of adding Escherichia coli O157:H7 with or without chemical or microbial additives on the bacterial diversity and composition of alfalfa silage. The second objective was to examine associations between the relative abundance of known and unknown bacterial species and indices of silage fermentation quality. Alfalfa forage was harvested at 54% dry matter, chopped to a theoretical length of cut of 19 mm, and ensiled in quadruplicate in laboratory silos for 100 d after the following treatments were applied: (1) distilled water (control); (2) 1 × 10⁵ cfu/g of E. coli O157:H7 (EC); (3) EC and 1 × 10⁶ cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1 × 10⁶ cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 0.22% propionic acid (EC+PA). After 100 d of ensiling, the silage samples were analyzed for bacterial diversity and composition via the Illumina MiSeq platform (Illumina Inc., San Diego, CA) and chemically characterized. Overall, Firmicutes (74.1 ± 4.86%) was the most predominant phylum followed by Proteobacteria (20.4 ± 3.80%). Relative to the control, adding E. coli O157:H7 alone at ensiling did not affect bacterial diversity or composition but adding EC+LP or EC+LB reduced the Shannon index, a measure of diversity (3.21 vs. 2.63 or 2.80, respectively). The relative abundance of Firmicutes (69.2 and 68.8%) was reduced, whereas that of Proteobacteria (24.0 and 24.9%) was increased by EC+LP and EC+PA treatments, relative to those of the control (79.5 and 16.5%) and EC+LB (77.4 and 18.5%) silages, respectively. Compared with the control, treatment with EC+LP increased the relative abundance of Lactobacillus, Sphingomonas, Pantoea, Pseudomonas, and Erwinia by 426, 157, 200, 194, and 163%, respectively, but reduced those of Pediococcus, Weissella, and Methylobacterium by 5,436, 763, and 250%, respectively. Relative abundance of Weissella (9.19%) and Methylobacterium (0.94%) were also reduced in the EC+LB silage compared with the control (29.7 and 1.50%, respectively). Application of propionic acid did not affect the relative abundance of Lactobacillus, Weissella, or Pediococcus. Lactate concentration correlated positively (r = 0.56) with relative abundance of Lactobacillus and negatively (r = ?0.41) with relative abundance of Pediococcus. Negative correlations were detected between ammonia-N concentration and relative abundance of Sphingomonas (r = ?0.51), Pantoea (r = ?0.46), Pseudomonas (r = ?0.45), and Stenotrophomonas (r = ?0.38). Silage pH was negatively correlated with relative abundance of Lactobacillus (r = ?0.59), Sphingomonas (r = ?0.66), Pantoea (r = ?0.69), Pseudomonas (r = ?0.69), and Stenotrophomonas (r = ?0.50). Future studies should aim to speciate, culture, and determine the functions of the unknown bacteria detected in this study to elucidate their roles in silage fermentation.     

Fate of Escherichia coli O157:H7 and bacterial diversity in corn silage contaminated with the pathogen and treated with chemical or microbial additives

Inhibiting the growth of Escherichia coli O157:H7 (EC) in feeds may prevent the transmission or cycling of the pathogen on farms. The first objective of this study was to examine if addition of propionic acid or microbial inoculants would inhibit the growth of EC during ensiling, at silo opening, or after aerobic exposure. The second objective was to examine how additives affected the bacterial community composition in corn silage. Corn forage was harvested at approximately 35% dry matter, chopped to a theoretical length of cut of 10 mm, and ensiled after treatment with one of the following: (1) distilled water (control); (2) 1 × 10⁵ cfu/g of EC (ECCH); (3) EC and 1 × 10⁶ cfu/g of Lactobacillus plantarum (ECLP); (4) EC and 1 × 10⁶ cfu/g of Lactobacillus buchneri (ECLB); and (5) EC and 2.2 g/kg (fresh weight basis) of propionic acid, containing 99.5% of the acid (ECA). Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, and 120 d and analyzed for EC, pH, and organic acids. Samples from d 0 and 120 were also analyzed for chemical composition. Furthermore, samples from d 120 were analyzed for ammonia N, yeasts and molds, lactic acid bacteria, bacterial community composition, and aerobic stability. The pH of silages from all treatments decreased below 4 within 3 d of ensiling. Escherichia coli O157:H7 counts were below the detection limit in all silages after 7 d of ensiling. Treatment with L. buchneri and propionic acid resulted in fewer yeasts and greater aerobic stability compared with control, ECCH, and ECLP silages. Compared with the control, the diversity analysis revealed a less diverse bacterial community in the ECLP silage and greater abundance of Lactobacillus in the ECLP and ECA silages. The ECLB silage also contained greater abundance of Acinetobacter and Weissella than other silages. Subsamples of silages were reinoculated with 5 × 10⁵ cfu/g of EC either immediately after silo opening or after 168 h of aerobic exposure, and EC were enumerated after 6 or 24 h, respectively. All silages reinoculated with EC immediately after silo opening (120 h) had similar low pH values (<4.0) and EC counts were below the detection limit. The ECCH and ECLP silages reinoculated with EC after 168 h of aerobic exposure had relatively high pH values (>5.0) and EC counts (5.39 and 5.30 log cfu/g, respectively) 24 h later. However, those treated with L. buchneri or propionic acid had lower pH values (4.24 or 3.96, respectively) and lower EC counts (1.32 log cfu/g or none, respectively). During ensiling, EC was eliminated from all silages at pH below 4.0. During aerobic exposure, the growth of EC was reduced or prevented in silages that had been treated with L. buchneri or propionic acid at ensiling, respectively.     

清酱香型白酒冬季发酵细菌群落演替及堆积过程细菌来源解析

利用高通量测序对清酱香型白酒窖池发酵过程中细菌群落结构进行分析,从清酱香型白酒发酵酒醅中共检出14 个菌门,113 个菌属,发酵开始（0 d）时,Firmicutes、Proteobacteria及Cyanobacteria在酒醅占主导地位,平均相对丰度均大于10%。随着发酵的进行,逐渐演替为单一的Firmicutes为主导。发酵过程相对丰度前10的细菌属为Lactobacillus、Bacillus、Weissella、Acetobacter、Gluconobacter、Pediococcus、Kroppenstedtia、Staphylococcus、Enterobacter、Scopulibacillus,随着发酵的进行,逐渐演替为单一的Lactobacillus为主导。为了说明薄层堆积发酵过程酒醅中细菌来源,对发酵用曲中细菌群落结构进行解析,并设置不添加酒曲的堆积样品为空白,通过分析酒醅样品（发酵0 d）、酒曲、环境样品（不添加大曲堆积酒醅）的细菌群落结构,结果表明酒醅中的细菌属31.67%来自于环境,50%来自于酒曲,23.33%由酒曲及环境共同提供,5%的细菌属只由环境提供。此外,优势细菌属中Gluconobacter仅来自于环境。本研究系统解析了清酱香型白酒窖池发酵过程细菌群落结构,初步分析了堆积发酵过程酒醅中细菌来源,有助于完善白酒发酵微生态的研究,并对提升白酒产品质量、安全及生产可控性具有重要的意义。     

Bacterial communities during the process of high‐temperature Daqu production of roasted sesame‐like flavour liquor

Bacterial communities in fermentative high‐temperature Daqu play an important role in the Chinese roasted sesame‐like flavour liquor brewing process. A culture‐independent method of 16S rDNA clone library was applied to investigate the changes in bacterial diversity during the process of a Daqu fermentation. The results indicated that the types of bacteria species gradually increased with prolonged fermentation time. The first dominant bacterium, Pantoea sp. (73.68%), in the raw material was only detected at the beginning of the fermentation stage (8 days) in small quantities. The second dominant bacterium, Weissella sp. (16.99%) and the third, Lactobacillus sp. (4.57%), were both detected in the beginning fermentation sample (3.22, 11.29%), in the ripening fermentation sample (24 days; 6.89, 25.11%) and in the drying stage (49 days; 3.74, 7.47%). In the beginning fermentation, Thermoactinomyces sp. (47.57%) was detected as the first predominant bacterium, and it was also detected in the ripening fermentation stage (4.93%) and in the drying stage (15.68%). The bacterial communities in the Daqu fermentation process affected the physicochemical index of Daqu. A molecular analysis of the bacterial community from the Daqu fermentation process will benefit studies on the aroma production mechanisms from these bacteria. Copyright © 2015 The Institute of Brewing & Distilling     

不同形态外源硒元素对白酒酒醅菌群的影响

探究不同形态硒元素的添加对白酒酒醅微生物菌群的影响。在白酒酿造酒醅中添加外源硒进行发酵,采用高通量测序对发酵完成的酒醅样品解析其真菌和细菌微生物群落特征。结果表明,未添加外源硒、添加有机硒和添加无机硒的三组白酒酒醅样品之间的细菌菌群的α-多样性指数存在显著性差异（P＜0.05）,添加外源硒样品的操作分类单元（OTUs）数目和Shannon指数数值较高,Chao1指数较低,在细菌的β-多样性中,硒的添加增加了变形菌门（Proteobacteria）在群落中的占比,添加无机硒后放线菌门（Actinobacteria）成为酒醅中优势菌门,外源硒的添加提高了白酒酒醅细菌菌群的多样性;真菌菌群的α、β多样性不具有显著性差异（P＞0.05）。研究结果表明,外源硒的添加（5 mg/kg）对白酒酒醅细菌菌群的群落结构有一定影响。     

Microbial succession and metabolite changes during the fermentation of Chinese light aroma‐style liquor

A combination of culture‐dependent and culture‐independent methods and SPME–GC–MS were used to monitor changes of bacterial and yeast communities, and flavour compounds during the fermentation process of Chinese light aroma‐style liquor. Bacillus and Lactobacillus were the main bacterial genera. Pichia anomala, Saccharomyces cerevisiae and Issatchenkia orientalis were the dominant yeast species. There was a close relationship between fermentation time and the shift of microbial community. Compared with the microbiota in the fermentation of other style liquors, higher bacterial diversity and different non‐Saccharomyces composition led to a variety of metabolites. Metabolite analysis showed that esters, acids, alcohols, aromatic compounds and phenols were the main flavour components and most of them were synthesised in the latter phase of fermentation. Principal component analysis further demonstrated that Bacillus and yeast were the most influential microorganisms in the first 10 days of fermentation, and lactic acid bacteria predominated in the later phase. Lactic acid bacteria regulated the composition of other bacteria and yeast, and synthesised flavour compounds to affect the organoleptic properties of liquor. S. cerevisiae and P. anomala were two important yeast species responsible for the characteristic aroma of liquor. These results present a comprehensive understanding of microbial interaction and potential starter cultures to produce desirable liquor quality. © 2018 The Institute of Brewing & Distilling     

Spatial Distribution of Bacterial Communities and Related Biochemical Properties in Luzhou‐Flavor Liquor‐Fermented Grains

Grain fermenting with separate layers in a fermentation pit is the typical and experiential brewing technology for Chinese Luzhou‐flavor liquor. However, it is still unclear to what extent the bacterial communities in the different layers of fermented grains (FG) effects the liquor's quality. In this study, the spatial distributions of bacterial communities in Luzhou‐flavor liquor FG (top, middle, and bottom layers) from 2 distinctive factories (Jiannanchun and Fenggu) were investigated using culture‐independent approaches (phospholipid fatty acid [PLFA] and polymerase chain reaction–denaturing gel electrophoresis [DGGE]). The relationship between bacterial community and biochemical properties was also assessed by Canonical correspondence analysis (CCA). No significant variation in moisture was observed in spatial samples, and the highest content of acidity and total ester was detected in the bottom layer (P < 0.05). A high level of ethanol was observed in the top and middle layers of Fenggu and Jiannanchun, respectively. Significant spatial distribution of the total PLFA was only shown in the 50‐y‐old pits (P < 0.05), and Gram negative bacteria was the prominent community. Bacterial 16S rDNA DGGE analysis revealed that the most abundant bacterial community was in the top layers of the FG both from Fenggu and Jiannanchun, with Lactobacillaceae accounting for 30% of the total DGGE bands and Lactobacillus acetotolerans was the dominant species. FG samples from the same pit had a highly similar bacterial community structure according to the hierarchal cluster tree. CCA suggested that the moisture, acidity, ethanol, and reducing sugar were the main factors affecting the distribution of L. acetotolerans. Our results will facilitate the knowledge about the spatial distribution of bacterial communities and the relationship with their living environment.     

Isomaltooligosaccharide increases the Lactobacillus rhamnosus viable count in Cheddar cheese

Five batches of Cheddar cheese were manufactured containing different levels of isomaltooligosaccharide (IMO) and a probiotic strain of Lactobacillus rhamnosus to study the effect of IMO on the survival of starter lactococci and probiotic micro‐organisms, on proteolytic patterns, cheese composition and sensory properties. The cheese was exposed to conditions simulating those found in the gastrointestinal tract to evaluate the survival of Lb. rhamnosus. Results demonstrated that the addition of Lb. rhamnosus and IMO did not affect the main compositional variables of Cheddar cheese. The counts of starter culture and probiotic organisms increased in cheese which contained Isomaltooligosaccharide (Batches 3, 4 and 5) more than in the control (Batches 1 and 2) during the fermentation. The probiotic counts in fresh cheese (B‐4) was 9.23 log₁₀ cfu/g which was more than one log cycle greater than in the control (B‐2). The probiotic counts remained above 8 log₁₀ cfu/g at the end of the manufacturing process. Primary proteolysis was not affected by the addition of probiotic bacteria and IMO, but the level of secondary proteolysis was slightly higher compared with the control group. The addition of IMO improved the texture and sensory quality of the cheese and the probiotic bacterium had the same effect. Under conditions that simulated the gastrointestinal tract, the probiotic bacteria in cheese (B‐4) exhibited good survival and remained above the recommended 6–7 log₁₀ cfu/g.     

Bacillus and Paenibacillus species associated with extended shelf life milk during processing and storage

Characterisation of spore formers associated with extended shelf life milk was performed by analysing the bacteriological quality of milk samples collected at various processing stages and during storage. Isolates were identified with MALDI‐TOF‐MS. Milk had spore counts <2 log₁₀ cfu/mL and 4 log₁₀ cfu/mL during processing and storage, respectively. Bacillus pumilus dominated the bacterial population. Bacterial species were inoculated into sterile milk for a shelf life study, and the population change was observed over 42 days at 7 °C. Although the extended shelf life milk process was effective in reducing bacterial counts and species diversity, the presence of Bacillus cereus shows a potential safety problem in extended shelf life milk.     

Effects of bacterial direct-fed microbials on ruminal characteristics,methane emission,and milk fatty acid composition in cows fed high- or low-starch diets

This study investigated the effects of bacterial direct-fed microbials (DFM) on ruminal fermentation and microbial characteristics, methane (CH₄) emission, diet digestibility, and milk fatty acid (FA) composition in dairy cows fed diets formulated to induce different ruminal volatile fatty acid (VFA) profiles. Eight ruminally cannulated dairy cows were divided into 2 groups based on parity, days in milk, milk production, and body weight. Cows in each group were fed either a high-starch (38%, HS) or a low-starch (2%, LS) diet in a 55:45 forage-to-concentrate ratio on a dry matter (DM) basis. For each diet, cows were randomly assigned to 1 of 4 treatments in a Latin square design of (1) control (CON); (2) Propionibacterium P63 (P63); (3) P63 plus Lactobacillus plantarum 115 (P63+Lp); (4) P63 plus Lactobacillus rhamnosus 32 (P63+Lr). Strains of DFM were administered at 10¹⁰ cfu/d. Methane emission (using the sulfur hexafluoride tracer technique), total-tract digestibility, dry matter intake, and milk production and composition were quantified in wk 3. Ruminal fermentation and microbial characteristics were measured in wk 4. Data were analyzed using the mixed procedure of SAS (SAS Institute Inc., Cary, NC). The 2 diets induced different ruminal VFA profiles, with a greater proportion of propionate at the expense of acetate and butyrate for the HS diet. Greater concentrations of total bacteria and selected bacterial species of methanogenic Archaea were reported for the HS diet, whereas the protozoa concentration in HS decreased. For both diets, bacterial DFM supplementation raised ruminal pH (+0.18 pH units, on average) compared with CON. Irrespective of diet, P63+Lp and P63+Lr increased ruminal cellulase activity (3.8-fold, on average) compared with CON, but this effect was not associated with variations in ruminal microbial numbers. Irrespective of diet, no effect of bacterial DFM on ruminal VFA was observed. For the LS diet, supplementing cows with P63+Lr tended to decrease CH₄ emission (26.5%, on average, when expressed per kilogram of milk or 4% fat-corrected milk). Only P63 supplementation to cows fed the HS diet affected the concentration of some milk FA, such as cis isomers of 18:1 and intermediates of ruminal biohydrogenation of polyunsaturated FA. Overall, bacterial DFM could be useful to stabilize ruminal pH. Their effects on CH₄ production mitigation and milk FA profile depended on DFM strain and diet and should be confirmed under a greater variation of dietary conditions.     

Influence of farmyard manure on the quality of grass silage

The influence of adding small aggregates (5 g) of farmyard manure (FYM) and/or a bacteria suspension (BS), containing Escherichia coli, and spores of Bacillus cereus and Clostridium tyrobutyricum, to silage was studied in two successive years. Direct cut (approximately 200 g DM kg⁻¹), precision-chopped grass herbage was ensiled in 1·6 l experimental silos. At ensiling the BS was either well distributed in the crop or added directly to 5 g of grass or FYM, which was placed in the centre of the green material when the silos were filled. Silage was further treated with formic acid (4 kg t⁻¹ FM), or an inoculant of lactic acid bacteria (10⁶ cfu g⁻¹ FM), and compared with no additive. Only minor influences on silage quality resulted from the addition of BS, although the number of clostridial spores increased slightly. Silages given additions of small aggregates of FYM were of poor quality. The inferior silage with a high number of clostridial spores (⩾3·9 cfu g⁻¹) and a high concentration of butyric acid (⩾2·8 g kg⁻¹) was found in the immediate vicinity of the addition. Application of silage additives did not improve silage quality. The poor quality of ‘FYM-silages’ could not solely be explained by the high number of organisms, but other factors in the manure and/or crops may contribute to the poor silage fermentation. © 1997 SCI.     

Changes in rumen fermentation and bacterial community in lactating dairy cows with subacute rumen acidosis following rumen content transplantation

Rumen microbiota intervention has long been used to cure ruminal indigestion in production and has recently become a research hotspot. However, how it controls the remodeling of rumen bacterial homeostasis and the restoration of rumen fermentation in cows of subacute ruminal acidosis (SARA) remains poorly understood. This study explored changes in rumen fermentation and bacterial communities in SARA cows following rumen content transplantation (RCT). The entire experiment comprised 2 periods: the SARA induction period and the RCT period. During the SARA induction period, 12 ruminally cannulated lactating Holstein cows were selected and allocated into 2 groups at random, fed either a conventional diet [CON; n = 4; 40% concentrate, dry matter (DM) basis] or a high-grain diet (HG; n = 8; 60% concentrate, DM basis). After the SARA induction period, the RCT period began. The HG cows were randomly divided into 2 groups: the donor-recipient (DR) group and the self-recipient (SR) group, and their rumen contents were removed completely before RCT. For the DR group, cows received 70% rumen content from the CON cows, paired based on comparable body weight; for the SR group, each cow received 70% rumen content, self-derived. The results showed that nearly all rumen fermentation parameters returned to the normal levels that the cows had before SARA induction after 6 d of transplantation, regardless of RCT. The concentrations of acetate, valerate, and total volatile fatty acids (VFA) were not recovered in the SR cows, whereas all of them were recovered in the DR cows. The amplicon sequencing results indicated that both the SR and DR cows rebuild their rumen bacterial homeostasis quickly within 4 d after RCT, and the DR group showed a higher level of bacterial community diversity. At the genus level, the DR cows displayed an improved proportion of unclassified Ruminococcaceae and Saccharofermentans compared with the SR cows. Correlation analysis between the rumen bacteria and rumen fermentation suggested some potential relationships between the predominant transplantation-sensitive operational taxonomic units and VFA. Co-occurrence network analysis revealed that RCT affected only those rumen bacterial taxa that showed weak interactions with other taxa and did not affect the pivotal rumen bacteria with high levels of co-occurrence. Our findings indicate that RCT contributes to the restoration of rumen bacterial homeostasis and rumen fermentation in cows suffering from SARA without affecting the core microbiome.