Effect of a blend of essential oil compounds on the colonization of starch-rich substrates by bacteria in the rumen

AIMS: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. METHODS AND RESULTS: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 x 4 latin square design and receiving a 60:40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. CONCLUSIONS: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.     

28种禾本科植物种子形态学特征及其萌发对绵羊瘤胃消化的反应

采用瘘管羊瘤胃瘘管尼龙袋法,对北疆地区常见的28种禾本科植物种子进行不同时间段的瘤胃消化处理,对处理前后种子的长、宽、厚、形状指数、百粒重和萌发率进行了测量,研究种子对绵羊瘤胃消化的反应,以期丰富北疆地区植物种子消化道传播的内容.结果表明: 除臭草、剪股颖、鳞茎早熟禾、猫尾草和小麦以外,其余23种植物的种子均为椭圆或扁平状种子;小麦和燕麦种子百粒重分别为3.52和1.69 g,其余种子的百粒重均为0.01～1 g,属中等或偏小类型.种子经瘤胃消化后,颜色变深,种子结构被破坏,种子的芒、稃和颖等附属结构的长度随消化时间的延长逐渐减小;种子长、宽、厚、百粒重随消化时间的延长呈逐渐减小趋势,但变化不显著;种子经绵羊瘤胃消化后,萌发率显著降低,经瘤胃消化6 h后,除鹅观草和燕麦种子萌发率降为0外,其余26种植物仍有部分种子保持活力.     

Quantitative analysis of cellulose degradation and growth of cellulolytic bacteria in the rumen

Ruminant animals digest cellulose via a symbiotic relationship with ruminal microorganisms. Because feedstuffs only remain in the rumen for a short time, the rate of cellulose digestion must be very rapid. This speed is facilitated by rumination, a process that returns food to the mouth to be rechewed. By decreasing particle size, the cellulose surface area can be increased by up to 10⁶-fold. The amount of cellulose digested is then a function of two competing rates, namely the digestion rate ( K _d) and the rate of passage of solids from the rumen ( K _p). Estimation of bacterial growth on cellulose is complicated by several factors: (1) energy must be expended for maintenance and growth of the cells, (2) only adherent cells are capable of degrading cellulose and (3) adherent cells can provide nonadherent cells with cellodextrins. Additionally, when ruminants are fed large amounts of cereal grain along with fiber, ruminal pH can decrease to a point where cellulolytic bacteria no longer grow. A dynamic model based on stella ^® software is presented. This model evaluates all of the major aspects of ruminal cellulose degradation: (1) ingestion, digestion and passage of feed particles, (2) maintenance and growth of cellulolytic bacteria and (3) pH effects.     

Structure of a cellulose degrading bacterial community during anaerobic digestion

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells.     

Three-reaction model for the anaerobic digestion of microalgae

Coupling an anaerobic digester to a microalgal culture has received increasing attention as an alternative process for combined bioenergy production and depollution. In this article, a dynamic model for anaerobic digestion of microalgae is developed with the aim of improving the management of such a coupled system. This model describes the dynamics of inorganic nitrogen and volatile fatty acids since both can lead to inhibition and therefore process instability. Three reactions are considered: Two hydrolysis–acidogenesis steps in parallel for sugars/lipids and for proteins, followed by a methanogenesis step. The proposed model accurately reproduces experimental data for anaerobic digestion of the freshwater microalgae Chlorella vulgaris with an organic loading rate of 1 gCOD L^?1 d^?1. In particular, the three‐reaction pathway allows to adequately represent the observed decoupling between biogas production and nitrogen release. The reduced complexity of this model makes it suitable for developing advanced, model‐based control and monitoring strategies. Biotechnol. Bioeng. 2012; 109:415–425. © 2011 Wiley Periodicals, Inc.     

Fate of bacterial pathogens in cattle dung slurry subjected to anaerobic digestion

The survival of certain pathogenic bacteria was studied in anaerobic batch digesters at room temperature (18–25 °C) as well as at 35 °C under laboratory conditions. The survival of Escherichia coli and Salmonella typhi at room temperature was upto 20 days whereas at 35 °C it was only upto 10 days. Shigella dysenteriae was found to be the most sensitive organism which could survive upto 10 days at room temperature and upto 5 days at 35 °C. The longest survival was observed in case of Streptococcus faecalis which could survive upto 35 days at room temperature and 15 days at 35 °C. The survival time of Salmonella typhi increased when the solid contents of the digester were elevated from 9% to 15%.     

Effects of dietary crude protein and tannic acid on rumen fermentation,rumen microbiota and nutrient digestion in beef cattle

The objectives of the trial were to study the effects of dietary crude protein (CP) and tannic acid (TA) on rumen fermentation, microbiota and nutrient digestion in beef cattle. Eight growing beef cattle (live weight 350 ± 25 kg) were allocated in a 2 × 2 crossover design using two levels of dietary CP [111 g/kg dry matter (DM) and 136 g/kg DM] and two levels of TA (0 and 16.9 g/kg DM) as experimental treatments. Each experimental period lasted 19 d, consisting of 14-d adaptation and 5-d sampling. The impacts of dietary CP and TA on ruminal microbiota were analysed using high-throughput sequencing of 16S rRNA gene. Results indicated that no interactions between dietary CP and TA were found on rumen fermentation and nutrient digestibility. Increasing dietary CP level from 111 to 136 g/kg DM increased the ruminal concentrations of ammonia nitrogen (NH₃-N) (p < 0.01) and improved the CP digestibility (p < 0.001). Adding TA at 16.9 g/kg DM inhibited rumen fermentation and decreased the digestibility of dietary CP (p < 0.001), DM (p < 0.05) and organic matter (p < 0.01). Increasing the dietary CP level or adding TA did not affect the relative abundances of the major bacteria Firmicutes and Proteobacteria at the phylum level and Prevotella_1 and Christensenellaceae_R-7_group at the genus level, even though adding TA increased the Shannon index of the ruminal bacterial community. TA was partly hydrolysed to pyrogallol, gallic acid and resorcinol in rumen fluid and the inhibitory effects of TA on rumen fermentation and nutrient digestibility could have been resulted from the TA metabolites including pyrogallol, gallic acid and resorcinol as well as the protein-binding effect.     

Biosynthesis of plant hormones during anaerobic digestion of instant coffee waste

A large amount of solid waste remains after the production of instant coffee. This waste has to be moved to dumps, where it poses a threat of environmental pollution. Treatment of this waste by anaerobic methanogenic thermophilic digestion produced, besides biogas, a digested slurry which was used as a growth medium for horticulture, and proved to be a suitable and economical substitute for peat moss. Biological tests with mung bean cuttings and Grevillea plantlets showed promotional effects on rooting of the slurry and its sieved fraction extract, washed with water (Capul). Green coffee beans, instant coffee waste, its anaerobically-digested slurry and Capul were extracted by various methods and the extracts were analyzed by TLC, HPLC and GC/MS. Examinations showed clearly the presence of IAA and IBA in free and bound forms in all the substrates. The values of free and bound IAA were calculated by use of an internal standard and GC/MS. The amount of conjugated IAA was found to be much higher than that of free IAA, in both the coffee beans and instant coffee waste (11.1 vs 2.7 nmol g^–1, respectively). In the digested slurry and Capul, however, most of the IAA was present as the free form and was approximately 23.5–33.0 nmol g^–1, which is almost ten times more than in the waste, and almost twice the total amount of IAA in coffee beans. It is postulated that the high levels of free IAA in the digested instant coffee waste are a result of catabolism of tryptophan by anaerobic bacteria.     

Comparison of the effects of lanthanum,cerium and praseodymium on rumen fermentation,nutrient digestion and plasma biochemical parameters in beef cattle

The objectives of the trial were to compare the effects of supplementing rare earth elements (REE) lanthanum (La), cerium (Ce) and praseodymium (Pr) on rumen fermentation, nutrient digestion, methane (CH₄) production, nitrogen (N) balance and plasma biochemical parameters in beef cattle. Four Simmental male cattle, aged 12 months, with initial average liveweight of 333 ± 9 kg and fitted with rumen cannulas, were fed with a basal ration composed of concentrate mixture and maize silage. Animals received a basal ration without adding REE (Control) or three treatments, i.e. supplementing LaCl₃, CeCl₃ or PrCl₃ at 204 mg/kg DM to the basal ration, respectively, which were allocated in a 4 × 4 Latin square design. Each experimental period lasted 15 d, consisting of 12 d for pre-treatment and three subsequent days for sampling. Results showed that all tested levels of REE tended to increase neutral detergent fibre digestibility (p = 0.064) and tended to decrease rumen CH₄ production (p = 0.056). Supplementing LaCl₃ and CeCl₃ decreased total N excretion and urinary N excretion, increased N retention (p < 0.05), tended to increase total urinary purine derivatives (PD) (p = 0.053) and microbial N flow (p = 0.095), whereas supplementing PrCl₃ did not affect N retention, urinary PD and microbial N flow. No differences were found in the effects of nutrient digestibility, CH₄ production and plasma biochemical parameters among LaCl₃, CeCl₃ and PrCl₃. Further trials using graded levels of LaCl₃, CeCl₃ and PrCl₃ in a wide range are needed to obtain more pronounced results for comparing effects of La, Ce and Pr on rumen fermentation and nutrient digestion in beef cattle.     

Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen

Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.     

Electrolysis-enhanced anaerobic digestion of wastewater

This study demonstrates enhanced methane production from wastewater in laboratory-scale anaerobic reactors equipped with electrodes for water electrolysis. The electrodes were installed in the reactor sludge bed and a voltage of 2.8-3.5 V was applied resulting in a continuous supply of oxygen and hydrogen. The oxygen created micro-aerobic conditions, which facilitated hydrolysis of synthetic wastewater and reduced the release of hydrogen sulfide to the biogas. A portion of the hydrogen produced electrolytically escaped to the biogas improving its combustion properties, while another part was converted to methane by hydrogenotrophic methanogens, increasing the net methane production. The presence of oxygen in the biogas was minimized by limiting the applied voltage. At a volumetric energy consumption of 0.2-0.3 Wh/L_R, successful treatment of both low and high strength synthetic wastewaters was demonstrated. Methane production was increased by 10-25% and reactor stability was improved in comparison to a conventional anaerobic reactor.     

Evaluation of group-specific, 16S rRNA-targeted scissor probes for quantitative detection of predominant bacterial populations in dairy cattle rumen

AIMS: To develop a suite of group-specific, rRNA-targeted oligonucleotide scissor probes for the quantitative detection of the predominant bacterial groups within the ruminal microbial community with the rRNA cleavage reaction-mediated microbial quantification method. METHODS AND RESULTS: Oligonucleotides that complement the conserved sites of the 16S rRNA of phylogenetically defined groups of bacteria that significantly contribute to the anaerobic fermentation of carbohydrates in ruminal ecosystems were selected from among published probes or were newly designed. For each probe, target-specific rRNA cleavage was achieved by optimizing the formamide concentration in the reaction mixture. The set of scissor probes was then used to analyse the bacterial community in the rumen fluids of four healthy dairy cows. In the rumen fluid samples, the genera Bacteroides/Prevotella and Fibrobacter and the Clostridium coccoides-Eubacterium rectale group were detected in abundance, accounting for 44-48%, 2.9-10%, and 9.1-10% of the total 16S rRNA, respectively. The coverage with the probe set was 71-78% of the total bacterial 16S rRNA. CONCLUSIONS: The probe set coupled with the sequence-specific small-subunit rRNA cleavage method can be used to analyse the structure of a ruminal bacterial community. SIGNIFICANCE AND IMPACT OF THE STUDY: The probe set developed in this study provides a tool for comprehensive rRNA-based monitoring of the community members that dominate ruminal ecosystems. As the ruminal microbial community can be perturbed, it is important to track its dynamics by analysing microbiological profiles under specific conditions. The method described here will provide a convenient approach for such tracking.     

荷斯坦奶牛瘤胃微生物元基因组BAC文库的构建与分析

采用未培养技术和脉冲场电泳技术直接从瘤胃微生物提取到大小在2Mb左右混合微生物DNA,经HindⅢ不完全酶切获得50～100kbDNA片段,将其连接在pCC1BAC载体上,转化E.coliEPI300,得到瘤胃微生物BAC文库,经对文库的鉴定分析,该文库的平均插入片段54.5kb,空载体率小于2%,库容837Mb,共保存15360个克隆。通过对该文库进行部分酶活性筛选,获得具有淀粉酶活性的克隆16个;纤维素酶活性的克隆26个,而且能降解纤维素的克隆中25个呈现多酶活性。这些结果表明该文库具有重要研究价值。     

Detection and monitoring of anaerobic rumen fungi using an ARISA method

Aim: To develop an automated ribosomal intergenic spacer region analysis (ARISA) method for the detection of anaerobic rumen fungi and also to demonstrate utility of the technique to monitor colonization and persistence of fungi, and diet‐induced changes in community structure. Methods and Results: The method could discriminate between three genera of anaerobic rumen fungal isolates, representing Orpinomyces, Piromyces and Neocallimastix species. Changes in anaerobic fungal composition were observed between animals fed a high‐fibre diet compared with a grain‐based diet. ARISA analysis of rumen samples from animals on grain showed a decrease in fungal diversity with a dominance of Orpinomyces and Piromyces spp. Clustering analysis of ARISA profile patterns grouped animals based on diet. A single strain of Orpinomyces was dosed into a cow and was detectable within the rumen fungal population for several weeks afterwards. Conclusions: The ARISA technique was capable of discriminating between pure cultures at the genus level. Diet composition has a significant influence on the diversity of anaerobic fungi in the rumen and the method can be used to monitor introduced strains. Significance and Impact of the Study: Through the use of ARISA analysis, a better understanding of the effect of diets on rumen anaerobic fungi populations is provided.     

Effects of guanidinoacetic acid supplementation on growth performance,nutrient digestion,rumen fermentation and blood metabolites in Angus bulls

Guanidinoacetic acid (GAA) can improve the growth performance of bulls. This study investigated the influences of GAA addition on growth, nutrient digestion, ruminal fermentation and serum metabolites in bulls. Forty-eight Angus bulls were randomly allocated to experimental treatments, that is, control, low-GAA (LGAA), medium-GAA (MGAA) and high-GAA (HGAA), with GAA supplementation at 0, 0.3, 0.6 and 0.9 g/kg DM, respectively. Bulls were fed a basal diet containing 500 g/kg DM concentrate and 500 g/kg DM roughage. The experimental period was 104 days, with 14 days for adaptation and 90 days for data collection. Bulls in the MGAA and HGAA groups had higher DM intake and average daily gain than bulls in the LGAA and control groups. The feed conversion ratio was lowest in MGAA and highest in the control. Bulls receiving 0.9 g/kg DM GAA addition had higher digestibility of DM, organic matter, NDF and ADF than bulls in other groups. The digestibility of CP was higher for HGAA than for LGAA and control. The ruminal pH was lower for MGAA, and the total volatile fatty acid concentration was greater for MGAA and HGAA than for the control. The acetate proportion and acetate-to-propionate ratio were lower for MGAA than for LGAA and control. The propionate proportion was higher for MGAA than for control. Bulls receiving GAA addition showed decreased ruminal ammonia N. Bulls in MGAA and HGAA had higher cellobiase, pectinase and protease activities and Butyrivibrio fibrisolvens, Prevotella ruminicola and Ruminobacter amylophilus populations than bulls in LGAA and control. However, the total protozoan population was lower for MGAA and HGAA than for LGAA and control. The total bacterial and Ruminococcus flavefaciens populations increased with GAA addition. The blood level of creatine was higher for HGAA, and the activity of l-arginine glycine amidine transferase was lower for MGAA and HGAA, than for control. The blood activity of guanidine acetate N-methyltransferase and the level of folate decreased in the GAA addition groups. The results indicated that dietary addition of 0.6 or 0.9 g/kg DM GAA improved growth performance, nutrient digestion and ruminal fermentation in bulls.     

藏羊瘤胃内容物浸泡对11种高寒草甸植物种子萌发的影响

为明确藏系绵羊对青藏高原高寒草甸植物种子萌发特性的影响,利用藏羊瘤胃内容物对11种青藏高原东北缘常见植物种子浸泡处理12、24、36、48、60和72 h后进行萌发试验.结果表明:供试11种高寒草甸植物种子的萌发因藏羊瘤胃内容物处理时间、种皮(果皮)完整性以及植物不同而异.划破种皮的黄花棘豆(Oxytropis ochrocephala)、去除果皮的无脉苔草(Carex enervis)、完整或去除果皮的草玉梅(Anemone rivularis)种子经藏羊瘤胃内容物分别处理12、12、12 ～ 36 h的发芽率显著高于对照(P＜0.05).藏羊瘤胃内容物处理均显著抑制了破皮和完整的垂穗披碱草(Elymus nutans)、甘肃马先蒿(Pedicularis kansuensis)、醉马草(Achnatherum inebrians)、冷地早熟禾(Poa crymophila)、阴山扁蓿豆(Medicago ruthenia var.inschanica)种子的发芽率.短时间处理对矮生嵩草(Kobresia humilis)、酸模(Rumex acetosa)、西伯利亚蓼(Polygonum sibiricum)种子发芽率无影响而长时间则表现出抑制作用.破皮(划破种皮或去除果皮)种子的萌发响应比完整种子敏感,而完整种子变化趋势则相对平缓.短时间处理提高了去除果皮的草玉梅(12 ～24 h)、无脉苔草(12 h)和划破种皮黄花棘豆(12 h)种子的发芽指数(P＜0.05);随着处理时间的延长,发芽指数呈逐渐减小趋势.藏羊瘤胃内容物浸泡处理对高寒草甸种子萌发有促进、抑制和无影响3种作用,继而潜在影响高寒草甸幼苗的形态建成、种间竞争和群落结构.     

Regulation of methanol and methylamine dehydrogenases in Methylophilus methylotrophus

Abstract Methylophilus methylotrophus can use methylamine as sole source of carbon and nitrogen. Measurements of the specific activity of methylamine dehydrogenase (MNDH) in bacteria grown in batch or chemostat culture showed that MNDH was induced by methylamine and repressed when methanol or NH₄⁺ were provided as alternative carbon or nitrogen sources. The degree of repression varied with the growth conditions. Methanol dehydrogenase (MDH) was present in bacteria growtn on methylamine as sole carbon source, but the specific activity was low compared with that in bacteria grown on medium containing methanol, indicating that this enzyme is induced by methanol.     

The effect of rumen ciliates on chitinolytic activity,chitin content and the number of fungal zoospores in the rumen fluid of sheep

The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 10⁴ (D. affine) to 42.2 · 10⁴ cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 10⁵ cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.