微生物介导反刍动物瘤胃氨生成及其对瘤胃功能的影响

反刍动物瘤胃中栖息着丰富多样的微生物,其在瘤胃内氨生成过程中发挥了重要的作用。微生物介导的氨基酸脱氨基作用和非蛋白氮水解作用是瘤胃内氨生成的主要途径。微生物介导了瘤胃内氨的生成,同时瘤胃内产生的氨也会反馈影响微生物菌群结构及瘤胃上皮功能,进而影响瘤胃发酵及宿主健康。本文主要综述了瘤胃微生物在介导氨生成中的作用和氨对瘤胃消化及瘤胃上皮功能的影响,以期对后续研究有所启发。     

瘤胃微生物甲烷生成的机理与调控

反刍动物瘤胃微生物产生的甲烷不但造成自身能量的大量损失,而且在地球温室效应中起着不可忽视的作用。在阐述了瘤胃中甲烷产生机理的基础上,详细论述了甲烷生成调控的各种途径、特点及其应用现状。     

奶牛瘤胃微生物研究进展和趋势

近年来在奶牛试验中,对瘤胃微生物的研究引起了人们越来越多的兴趣。这些研究的目的多是将微生物组成变化与日粮组成、宿主生产性能(如饲料效率,产奶量,乳脂等)、健康(如瘤胃酸中毒和亚急性酸中毒)以及环境(如甲烷排放)联系起来,另外还有一些研究则强调了微生物在多种反刍动物瘤胃发育中的作用。关于奶牛瘤胃微生物的大部分发现都是基于扩增子测序,可以揭示瘤胃微生物的分类组成,以及在不同处理条件下瘤胃菌群的变化。尽管新兴的宏基因组学和宏转录组学能够深入探索瘤胃微生物的功能,但在数据分析和解释方面也带来了更多的挑战,如目前大多数论文都严重依赖于相关性和推测分析。综述了奶牛瘤胃微生物研究的进展和局限,包括瘤胃微生物与产奶效率、甲烷排放以及瘤胃发育的关系,以及奶牛瘤胃微生物未来的研究趋势。     

瘤胃微生物对纤维素降解机理

城市有机垃圾中木质纤维素难以被降解的根本原因 ,在于其木质素的物理屏障作用及纤维素本身的结晶结构 ,瘤胃微生物能够高效降解木质纤维素 ,是因为瘤胃菌群中存在各种可以分别降解木素和结晶纤维素微生物 ,它们分泌的各种酶类是降解的关键所在。     

瘤胃微生物对纤维素的降解及其应用

瘤胃微生物主要包括细菌、真菌和原生动物。其中,瘤胃细菌和瘤胃真菌能分泌纤维素酶,对纤维素有较强的降解能力,主要介绍了瘤胃微生物对纤维素的降解作用及其广阔的应用前景。     

瘤胃微生物在有机废物处理中的应用研究

综述了瘤胃微生物在处理农业残余废物、城市有机垃圾和一些有毒物质方面的研究情况,并对影响其降解的环境条件、工艺条件和反应促进因素做了介绍,认为结合现代厌氧消化技术和瘤胃发酵技术,瘤胃微生物可以在有机废物处理中发挥较大作用.     

瘤胃微生物多样性及功能性研究方法概述

庞大的瘤胃微生物群系之间存在着共生关系,影响着宿主的代谢,是反刍动物营养学的研究热点之一.通过基于16S rRNA的分子生物学方法,如探针法、实时定量PCR法、DGGE/TGGE,RAPD和RFLP技术等研究瘤胃微生物多样性及组成结构,应用宏基因组学如建立YAC文库、BAC文库等研究方法对瘤胃微生物功能特征进行更深入的研究,实现改善反刍动物乳、肉产品品质的目的.     

近10年瘤胃微生物分离培养研究进展

瘤胃微生物的研究一直是反刍动物营养研究的重点领域,自20世纪中期以来,大量参与营养代谢的微生物被从瘤胃中分离和认识.当代,尽管分子生物学方法越来越广泛的应用于瘤胃微生物的研究,但传统的分离纯培养的研究方法,因能够获得新的微生物菌种,便于微生物生理功能与代谢的研究,仍具有不可或缺的作用.本文综述了2001年至2011年间中国知网和PubMed数据库中通过纯培养方法从瘤胃中分离微生物菌株(包括新属、种或首次从瘤胃中分离)的相关文献,并展示了这些微生物的形态和发酵特征,同时总结了瘤胃微生物的全基因组信息,旨在为今后瘤胃微生物的研究提供参考菌种和信息.     

Short communication: Saliva and salivary components affect goat rumen fermentation in short-term batch incubations

The research about the role of saliva in ruminants has been mainly focused on its buffering capacity together with facilitation of the rumination process. However, the role of salivary bioactive components on modulating the activity of the rumen microbiota has been neglected until recently. This study developed an in vitro approach to assess the impact of different components in saliva on rumen microbial fermentation. Four different salivary fractions were prepared from four goats: (i) non-filtrated saliva (NFS), (ii) filtrated through 0.25 µm to remove microorganisms and large particles (FS1), (iii) centrifuged through a 30 kDa filter to remove large proteins, (FS2), and (iv) autoclaved saliva (AS) to keep only the minerals. Two experiments were conducted in 24 h batch culture incubations with 6 ml of total volume consisting of 2 ml of rumen fluid and 4 ml of saliva/buffer mix. In Experiment 1, the effect of increasing the proportion of saliva (either NFS or FS1) in the solution (0%, 16%, 33% and 50% of the total volume) was evaluated. Treatment FS1 promoted greater total volatile fatty acids (VFA) (+8.4%) and butyrate molar proportion (+2.8%) but lower NH₃-N concentrations than NFS fraction. Replacing the bicarbonate buffer solution by increasing proportions of saliva resulted in higher NH₃-N, total VFA (+8.0%) and propionate molar proportion (+11%). Experiment 2 addressed the effect of the different fractions of saliva (NFS, FS1, FS2 and AS). Saliva fractions led to higher total VFA and NH₃-N concentrations than non-saliva incubations, which suggests that the presence of some salivary elements enhanced rumen microbial activity. Fraction FS1 promoted a higher concentration of total VFA (+7.8%) than the other three fractions, and higher propionate (+26%) than NFS and AS. This agrees with findings from Experiment 1 and supports that ‘microbe-free saliva’, in which large salivary proteins are maintained, boosts rumen fermentation. Our results show the usefulness of this in vitro approach and suggest that different salivary components can modulate rumen microbial fermentation, although the specific metabolites and effects they cause need further research.     

牦牛瘤胃中产脂肪酶微生物的分离与鉴定

【背景】脂肪酶是一类特殊的酯键水解酶,广泛应用于工业化生产中,微生物是工业脂肪酶的主要来源。瘤胃中微生物种类繁多、数量庞大,已有关于瘤胃微生物产纤维素酶的报道,尚无产脂肪酶瘤胃微生物的分离筛选报道。【目的】从牦牛瘤胃中分离筛选出能够产脂肪酶的微生物,并进行菌株鉴定及其酶学性质的研究。【方法】以橄榄油为唯一碳源,通过中性红油脂平板进行初步筛选后,用改进铜皂-分光光度法测定酶活力进行复筛;再经形态学观察、生理生化实验和16S rRNA基因序列分析进行菌种鉴定;研究3种脂肪酶的最适作用温度、pH值及金属离子、有机溶剂和表面活性剂对酶活力的影响。【结果】筛选出6株酶活力较高的菌株,其中3株为液化沙雷氏菌,2株为白地霉,1株为卷枝毛霉。脂肪酶的酶学性质研究表明:液化沙雷氏菌、白地霉和卷枝毛霉所产脂肪酶的最适作用温度为45、35和40°C;最适pH为8.0、7.0和7.0;Ca2+和Mg2+对3种脂肪酶均有激活作用;Zn2+对3种脂肪酶有不同程度的抑制作用,EDTA、SDS可使3种脂肪酶失活;3种脂肪酶对丙三醇的耐受力较高,卷枝毛霉脂肪酶对甲醇、乙醇、丙酮的耐受力较高。【结论】从牦牛瘤胃中分离出3种产脂肪酶的微生物,且证实瘤胃微生物在脂肪酶研究方面具有较高的价值。     

现代分子生物学技术在瘤胃微生态系统研究中的应用

瘤胃中栖息着大量的微生物,由于这些微生物组成复杂且有些细菌在体外无法培养,目前对这些微生物的了解仍然很少。现代分子生物学技术的发展为研究瘤胃微生物提供了有效的方法,利用核酸探针、基因序列分析、遗传指纹技术、全细胞杂交和实时定量PCR等技术可以对瘤胃微生物的分类及进化关系、区系结构图、重要酶的表达以及目的微生物的准确定量进行更为深入和透彻的研究。发展和利用这些技术不仅可以研究微生物之间的关系以及微生物与饲料颗粒之间时间与空间的关系,还能直接在细菌自然生长的环境中对其各种特征进行研究。     

香石竹设施栽培根际土壤微生物区系的16S rRNA系统遗传多样性

【目的】针对香石竹设施栽培土传病害的生物防治技术研究,探讨其根际土壤微生物与枯萎病害的关联性。【方法】采集香石竹健康植株与枯萎病植株根际土壤,采用不同培养基进行分离、纯化,并对分离菌株提取基因组DNA,用其16S rRNA序列的通用引物进行PCR扩增,进行blast同源分析。【结果】从采集样品中分离出的菌株分布于细菌域(Bacteria)中的4个门(Phyla)共15个属(Genera),其中从健康植株组土壤中培养出65株菌,分布于9个属,并以芽孢杆菌(Bacillus)、链霉菌(Streptomyces)及孢霉菌(Mortierella)为优势菌群;而枯萎病株组土样共培养出33株菌,分布于12个属,并且寡养单胞菌(Stenotrophomonas)、鞘氨醇杆菌(Sphingobacterium)、假单胞菌(Pseudomonas)、金黄杆菌(Chryseobacterium)、拟无枝菌酸菌(Amycolatopsis)及尖镰孢病原菌(Fusarium oxysporum)属的分离菌株仅从病株组土壤中分离到;分离菌株同源性在90%-98%的潜在新种(potential novel species)有13株。【结论】研究结果表明,根际土壤中真菌数与总菌数的百分比或Bacillus类群多样性的丰度,可作为评价区域香石竹种植土壤健康状况、栽培土壤演变及病害防治预测预报的参考指标。     

Effect of insulin on in vitro fermentation activity of microrganism community of rumen ciliate Entodinium caudatum culture

The influence of insulin (17.4 nmol l-1) on total gas and methane production, the concentration of total and individual fatty acids and dry matter degradability was investigated in the rumen ciliate culture of Entodinium caudatum. The experimental groups consisted of control group (without insulin) and two groups with insulin application--single shot and long-term application (over 30 days). Fermentation activity of each experimental group was observed on two subgroups: whole protozoan culture (protozoa plus bacteria) and bacterial fraction (bacteria without protozoa). Long-term application of insulin significantly increased methane production and DM degradability in the whole protozoan culture. Total VFA concentration was significantly increased by long-term as well as single-dose application of insulin (by 255% and 158%, respectively). The growth of the protozoa was not influenced by insulin treatments. It can be concluded that the fermentation activity of the community of the rumen ciliate Entodinium caudatum culture was marked stimulated by application of insulin.     

Metasecretome-selective phage display approach for mining the functional potential of a rumen microbial community

Background

In silico, secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences. For metasecretome (collection of surface, secreted and transmembrane proteins from environmental microbial communities) this approach is impractical, considering that the metasecretome open reading frames (ORFs) comprise only 10% to 30% of total metagenome, and are poorly represented in the dataset due to overall low coverage of metagenomic gene pool, even in large-scale projects.

Results

By combining secretome-selective phage display and next-generation sequencing, we focused the sequence analysis of complex rumen microbial community on the metasecretome component of the metagenome. This approach achieved high enrichment (29 fold) of secreted fibrolytic enzymes from the plant-adherent microbial community of the bovine rumen. In particular, we identified hundreds of heretofore rare modules belonging to cellulosomes, cell-surface complexes specialised for recognition and degradation of the plant fibre.

Conclusions

As a method, metasecretome phage display combined with next-generation sequencing has a power to sample the diversity of low-abundance surface and secreted proteins that would otherwise require exceptionally large metagenomic sequencing projects. As a resource, metasecretome display library backed by the dataset obtained by next-generation sequencing is ready for i) affinity selection by standard phage display methodology and ii) easy purification of displayed proteins as part of the virion for individual functional analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-356) contains supplementary material, which is available to authorized users.     

本科微生物学实验教学改革初探

微生物学是一门实验性很强的学科,加强实验教学改革对于培养学生的综合素质和创新精神具有重要意义。从实验内容选取,微生物实验教学方法和教学手段改革等三个方面,提出了实验教学改革的一些设想,目的在于提高实验教学质量,培养创新型人才。     

Using microbial fatty acids to improve understanding of the contribution of solid associated bacteria to microbial mass in the rumen

This study sought to distinguish liquid-(LAB) and detached (SAB₁) and undetached (SAB₂) solid-associated bacteria through their fatty acid (FA) and purine base (PB) profiles. Fatty acids and PB were also evaluated as internal microbial markers for estimating microbial biomass associated with rumen particles. Four merino rams fitted with rumen cannulae and fed dehydrated alfalfa pellets provided rumen contents. In 3 consecutive weeks, rumen contents were collected and samples of LAB and SAB₁, total rumen content (TRC), washed rumen particles (WRP) and rumen particles after SAB₁ extraction (ERP) were obtained and analysed for PB and FA. The SAB₂ biomass composition was estimated from the non-NDF organic matter (OM) remaining in ERP. The concentration of total SAB biomass in particles was estimated using both PB and odd and branched-chain fatty acids (OBCFA). Concentrations of PB and OBCFA were highly correlated among the different rumen fractions. Marked differences between LAB and SAB populations occurred with LAB having higher PB content, lower FA content and a higher proportion (g/100 g fatty acids) of OBCFA than did SAB. The chemical composition of SAB₁ and SAB₂ was similar, except for the 15% higher crude protein content of the latter. The concentration of OBCFA (mg/g microbial OM) did not differ between bacterial fractions. The PB/OBCFA ratio (mg/mg) was higher in LAB (2.08) than in SAB (0.94). The ratio between branched-chain and odd-linear-chain FA was higher in LAB (2.26) than in SAB (1.46). Extraction of PB and OBCFA from WRP with our SAB detachment procedure was 61% and 31%, respectively. Estimated SAB₁ and total SAB biomass (mg OM/g WRP) were 158 and 266, and 47 and 164, respectively, using PB and OBCFA as microbial markers. This study suggests that the OBCFA have potential as internal microbial markers in rumen ecosystem studies.     

本科生英文文献阅读能力培养的探索——“微生物生态学”课程教学中的改革实践

本文介绍了华中农业大学在"微生物生态学"教学过程中进行的双语教学和前沿英文文献开卷阅读测试的实践案例。连续三年的教改实践有效帮助本科生克服了英文文献阅读的恐惧心理,提高了本科生阅读专业前沿文献的理解能力,调动了学习的积极性,创新思维能力有明显提高。