瘤胃纤维素降解菌的分离鉴定及其纤维素降解特性

纤维素是地球上最丰富的可再生有机资源,但是其不溶于水和有机溶剂的难降解特性限制了它的利用.多年来,研究者们在利用纤维素资源方面做着努力,其中,利用微生物产生的纤维素酶降解纤维素,具有条件温和、产物产率高和无二次污染等特点,成为目前较有效且更接近自然的一种纤维素处理方法.同时,由微生物产生的纤维素酶在食品、酿酒、造纸、饲料和纺织等行业也有着广泛的应用.     

一株瘤胃纤维素降解菌的分离鉴定及其纤维素降解特性

从蒙古绵羊瘤胃内容物中分离到一株纤维素降解细菌WH-1, 通过形态、生理生化特征、G+C mol%含量和16S rRNA序列分析对分离菌株进行鉴定, 鉴定为溶纤维丁酸弧菌属(Butyrivibrio fibrisolvens)的溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)。同时, 用Mega 4.1软件构建的系统发育树显示分离菌株WH-1与多株溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)的亲缘关系最近。对该菌株纤维素降解特性的初步研究表明：当温度为37°C、     

不同碳源诱导下牦牛瘤胃厌氧真菌Orpinomyces sp. YF3的产酶机制

为探究体外发酵牦牛瘤胃源厌氧真菌Orpinomyces sp. YF3在不同碳源诱导下的产酶机制,本研究利用厌氧培养管在10 mL基础培养基中分别添加不同碳源复杂度的葡萄糖(glucose, Glu)、滤纸(filter paper, Flp)、微晶纤维素(avicel, Avi)各8 g/L作为唯一碳源进行体外发酵,检测发酵液中的纤维降解酶活性和挥发性脂肪酸,并利用转录组学探究Orpinomyces sp. YF3的产酶机制。结果表明葡萄糖诱导下的发酵液中羧甲基纤维素酶、微晶纤维素酶、滤纸酶和木聚糖酶的活性,及乙酸的比例显著升高(P＜0.05),丙酸、丁酸、异丁酸的比例显著降低(P＜0.05)。进一步分析发现与纤维降解酶相关的差异表达基因(differentially expressed genes, DEGs)在Glu组中显著上调。基因本体论(gene ontology, GO)功能富集显示DEGs主要集中在木聚糖酶、纤维素酶、葡萄糖和碳水化合物等的分解代谢过程及相关酶活性,京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)通路分析富集到的纤维降解酶相关的差异通路主要是淀粉和蔗糖代谢途径、其他聚糖降解途径。以上结果表明,以葡萄糖为碳源底物的Orpinomyces sp. YF3可增加纤维素降解酶活性,提高乙酸比例,通过调控纤维降解酶基因的表达及相关代谢通路来提高对底物的降解能力,提高能量利用效率。这为Orpinomyces sp. YF3在实际生产中的应用提供了理论基础。     

The effects of concentrate energy source on feed intake and rumen fermentation parameters of dairy cows offered a range of grass silages

The effects of concentrate energy source on feed intake and rumen fermentation parameters of lactating dairy cattle, offered one of three grass silages differing in fermentation and intake characteristics, were evaluated in a partially balanced changeover design experiment involving four rumen fistulated dairy cows. Three silages were harvested using different management practices prior to and at ensiling. Silages A and C and silage B were harvested from primary or secondary regrowths either untreated or treated with a bacterial inoculant. For silages A, B and C, dry matter (DM) concentrations were 334, 197 and 183 g/kg (S.E. 3.1), pH values 4.00, 4.79 and 4.80 (S.E. 0.042) and ammonia nitrogen (N) concentrations were 123, 319 and 295 g/kg total N (S.E. 20.0), respectively. Two concentrates were formulated to contain similar crude protein, effective rumen degradable protein, digestible undegradable protein and metabolisable energy concentrations but using different carbohydrate sources to achieve a wide range of starch concentrations. For the low and high starch concentrates starch concentrations were 17 and 304 g/kg DM and acid detergent fibre concentrations were 170 and 80 g/kg DM, respectively. The silages were offered ad libitum, supplemented with 10 kg fresh concentrate daily. For silages A, B and C, DM intakes were 10.9, 7.2 and 8.6 kg/day and concentrate energy sources did not alter (P>0.05) intake. Increasing the level of starch in the concentrate decreased the molar concentration of acetate (P<0.05) and tended to increase the molar concentration of propionate (P<0.1). Silage type altered the molar concentration of acetate (P<0.01) and the acetate:propionate ratio (P<0.05). There were no silagetype×concentrate interactions (P>0.05) on silage intake or rumen fermentation parameters. It is concluded that when concentrate and silage form equal proportions of the diet, the composition of the silage has an over-riding influence on rumen fermentation parameters. Furthermore, the changes in milk fat concentration, observed in a concurrent production study, due to changes in silage and concentrate types can be accounted for by changes in the ratio of lipogenic to glucogenic precursors in the rumen fluid.     

Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens

Competitive PCR assays were developed for the enumeration of the rumen cellulolytic bacterial species: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. The assays, targeting species-specific regions of 16S rDNA, were evaluated using DNA from pure culture and rumen digesta spiked with the relevant cellulolytic species. Minimum detection levels for F. succinogenes, R. albus and R. flavefaciens were 1-10 cells in pure culture and 10(3-4) cells per ml in mixed culture. The assays were reproducible and 11-13% inter- and intra-assay variations were observed. Enumeration of the cellulolytic species in the rumen and alimentary tract of sheep found F. succinogenes dominant (10(7) per ml of rumen digesta) compared to the Ruminococcus spp. (10(4-6) per ml). The population size of the three species did not change after the proportion of dietary alfalfa hay was increased. All three species were detected in the rumen, omasum, caecum, colon and rectum. Numbers of the cellulolytic species at these sites varied within and between animals.     

Effects of nitrate on methane production, fermentation, and microbial populations in in vitro ruminal cultures

The objective of this study was to examine the effects of nitrate on methane production, important fermentation characteristics, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, total bacteria, and methanogens using in vitro ruminal cultures. Potential adaptation of the above microbes and persistency of nitrate to mitigate CH₄ production were also evaluated. Methane production was reduced by 70% at 12 μmol ml⁻¹ and nearly completely at ?24 μmol ml⁻¹ nitrate. Production of volatile fatty acids (VFAs) was affected to different extents at different nitrate concentrations. Over a series of six consecutive cultures receiving 12 μmol ml⁻¹nitrate, production of CH₄ and VFA did not change significantly. R. albus and R. flavefaciens seemed to adapt to nitrate, while F. succinogenes and methanogens did not. Nitrate may be used in achieving persistent mitigation of CH4 production by ruminants.     

The ratio of fungi and bacteria in the biomass of different types of soil determined by selective inhibition

Tundra, chernozem (virgin and arable), soddy-podzolic (coniferous forest, meadow, and arable), and grey forest (larch forest) soils were used to separate the contributions of fungi and bacteria to substrate-induced respiration (SIR) with the help of antibiotics. For soils with a high content of organic matter (tundra and chernozem: 12 and 8%, respectively), the procedure of selective inhibition of SIR has been optimized. This procedure consists in application of high concentrations of streptomycin (50–120 mg/g of soil) and cycloheximide (50–80 mg/g of soil) and decreasing the weight of the analyzed soil sample. Soils under study have shown the predominant contribution of fungi (63–82%) to the total SIR. The fungal-bacterial ratio in the soils of natural ecosystems (0–5 cm, without litter) was 4.3, 2.2, 1.5, and 1.5 for tundra soil, virgin chernozem, coniferous (soddy-podzolic soil), and larch (grey forest soil) forests, respectively. The lower layers of soddy-podzolic (5–10 cm) and grey forest (48–58 cm) soils showed a decrease in the fungal and increase in the bacterial component in the total SIR.     

Lactic acid bacteria strains selected from fermented total mixed rations improve ensiling and in vitro rumen fermentation characteristics of corn stover silage

ObjectiveThis study identified the major lactic acid bacteria (LAB) strains from different fermented total mixed rations (FTMRs) via metataxonomic analysis and evaluated the ability of their standard strain as ensiling inoculants for corn stover silage.MethodsThe bacterial composition of eight FTMRs were analyzed by 16S rDNA sequencing. Corn stover was ensiled without LAB inoculation (control) or with 1×10⁶ cfu/g LAB standard strain (Lactobacillus vaginalis, Lactobacillus reuteri, Lactobacillus helveticus, or Lactobacillus paralimentarius) selected from the FTMRs or 10 g/t commercial silage inoculant (CSI) around 25°C for 56 days. For each inoculation, a portion of the silage was sampled to analyze ensiling characteristics at time intervals of 0, 1, 3, 7, 14, 28, and 56 days, gas production (GP), microbial crude protein and volatile fatty acids as the measurements of rumen fermentation characteristics were evaluated in vitro with the silages of 56 days after 72 h incubation.ResultsLactobacillus covered >85% relative abundance of all FTMRs, in which L. pontis, L. vaginalis, L. reuteri, L. helveticus, and L. paralimentarius showed >4% in specific FTMRs. CSI, L. helveticus, and L. paralimentarius accelerated the decline of silage pH. Silage inoculated with L. paralimentarius and CSI produced more lactic acid the early 14 days. Silage inoculated with L. paralimentarius produced less acetic acid and butyric acid. For the in vitro rumen fermentation, silage inoculated with CSI produced more potential GP, isobutyric acid, and isovaleric acid; silage inoculated with L. helveticus produced more potential GP and isovaleric acid, silage inoculated with L. paralimentarius or L. reuteri produced more potential GP only.ConclusionThe standard strain L. paralimentarius (DSM 13238) is a promising ensiling inoculant for corn stover silage. The findings provide clues on strategies to select LAB to improve the quality of silage.     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Effects of rumen fluid collection site on microbial population structure during in vitro fermentation of the different substrates quantified by 16S rRNA hybridisation

Rumen fluid samples from a cow were withdrawn manually from the feed mat (solid phase) or the liquid phase below this mat and incubated in vitro with wheat straw, sorghum hay and a concentrate mixture. From the inoculum and several samples collected during in vitro incubation RNA was extracted to assess microbial population size and structure. RNA content recovered from the solid phase rumen fluid was significantly higher than from the liquid phase. The composition of the microbial population in the solid phase material was characterised by a high proportion of Ruminococci. Neither the proportion of other cell wall degrading organisms (Fibrobacter and Chytridiomycetes) nor the Eukarya and Archaea populations differed between the two sampling sites. Gas production was higher when substrates were incubated with solid phase than with liquid phase rumen fluid regardless of sampling time. However, the higher level of gas production was not accompanied by a corresponding increase in true digestibility. The RNA probes showed that during in vitro incubation with liquid phase rumen fluid, the eukaryotic population was inactive no matter which substrate was used and the activity of methanogens (Archaea) was lower than with solid phase rumen fluid. The population pattern of the cell wall degrading organisms was influenced mainly by the substrate fermented, and to a smaller extent by the inoculum used for in vitro fermentation.     

Associative effect of cellulolytic fungi andAzospirillum lipoferum on yield and nitrogen uptake by wheat

The associative effect of cellulolytic fungi, such asAspergillus awamori andA. niger, with the nitrogen fixer,Azospirillum lipoferum was studied in a soil amended with rice straw. All the inoculants gave significantly higher grain and straw yield and nitrogen uptake by wheat crop than did the uninoculated treatment. The doubling of chemical nitrogen dose significantly increased the yield and nitrogen uptake. It was observed thatA awamori performed significantly better followed byA. niger andA. lipoferum. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum. Another experiment was conducted in the subsequent year in soil amended with and without rice straw using cellulolytic culture eitherA. awamori orSclerotium rolfsii, andA. lipoferum. Application of straw in soil significantly reduced the yield and N-uptake by wheat crop as compared to the controls. All the inoculants exceptS. rolfsii gave significantly higher grain yield. However, N-uptake by grain was significantly increased only by combined inoculation ofA. lipoferum and either one of the cellulolytic fungi. Similar trends on yield and N-uptake of straw due to inoculants were observed. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum followed byA. awamori alone on grain yield and only combined inoculants on N-uptake by the crop.