Isolation and identification of adherent epimural bacteria during succession in young lambs.

Successive changes in aerobic and anaerobic bacterial counts and changes in the generic composition of the epimural community in lambs from 1 to 10 weeks were determined. Bacterial culture counts revealed a predominantly anaerobic community, with the mean anaerobic count being 1.4 X 10(7) CFU/cm2 of tissue surface. The aerobic count was highest at 1 week of age and declined significantly thereafter to a mean of 1.8 X 10(4) CFU/cm2, thus representing only 0.13% of the mean anaerobic count after week 1. Of the 345 strains isolated anaerobically at 1, 2, 4, 6, 8, and 10 weeks of age, 47, 32, 12, 32, 2, and 5% were capable of growth in a partially reduced medium, indicating a reduction in the number of facultative anaerobes with time. The majority of isolated strains were identified as belonging to genera commonly isolated from rumen contents. In some instances, however, strains did not correspond to previously described species, and some genera were present in proportions different from those expected in rumen fluid. At three of the sampling times, one genus was dominant, constituting 45 to 55% of the isolates. These dominant isolates were Streptococcus bovis, Bacteroides sp., and an anaerobic Streptococcus sp. for weeks 1, 2, and 10, respectively. During the transition period (weeks 4 to 8), two or more groups were codominant.     

Electron microscopy study of the bacteria adherent to the rumen wall in young conventional lambs

The lamb rumen walls were rapidly colonized by an abundant bacterial population after birth. This colonization was examined by electron microscopy in neonatal conventional lambs. The sequence of establishment of the epimural species during the 3 weeks following birth, and the distribution of bacteria on the different sacs of the rumen, were examined by scanning electron microscopy. The population was very dense and consisted of a limited number of morphological types by 2 days after birth. Three types of rods were dominant at that time. The microflora was more complex 2 weeks later. Observations by transmission electron microscopy of desquamated epithelial cells revealed the presence of adherent bacteria that are surrounded by fibrous carbohydrate coats and sometimes partially enclosed by invaginations of the epithelial cell.     

Colony counts and characterization of bacteria adherent to the rumen wall and desquamated epithelial cells in conventional young lambs

Characterization and enumeration of the adherent epimural community of the rumen wall of young, conventionally reared lambs were carried out from 2 to 21 days after birth. Three hundred strains were isolated by anaerobic procedures from three sites: dorsal, ventral, and caudal sacs, and from the sloughed epithelial cells. The population of epimural bacteria was very dense from the first days of the lamb's life. This population increased slightly with age. During the first week the counts were similar in the dorsal and ventral sacs, but they were 10 to 100 times lower in the caudal sac. Total counts for anaerobic bacteria were higher than the counts for aerobic bacteria. The isolated strains were distributed into 19 groups: 11 groups included aerotolerant strains, and 8 others, strictly anaerobic strains. During the first week the facultative microflora was mainly composed of Escherichia coli and Streptococci. Later, the epimural community was more complex and included Staphyloccus, Micrococcus, and Gaffkya. The strictly anaerobic microflora was mainly composed of Clostridium, Peptostreptococcus, Veillonella, Propionibacterium, and Acidaminococcus. Some of these strains appeared to be similar to those previously isolated from the rumen fluid of young lambs; however, the genera Micrococcus, Veillonella, Gaffkya, and Acidaminococcus, and E. coli seemed to be specific of the rumen wall tissues.     

Successive changes in the epimural bacterial community of young lambs as revealed by scanning electron microscopy.

Scanning electron microscopy was used to determine the time of initial colonization of the rumen epithelium of young lambs and successive changes with time in the morphological composition of the epimural community. Tissue samples were obtained from two groups of lambs at 1, 2, 4, 6, 8, and 10 weeks of age. Comparisons were made with the epimural communities observed at 12 well-distributed sites in the rumen of a mature wether. Epimural bacteria were already present on the epithelium at 1 week of age. The morphological composition of the epimural community changed with age, with the pattern of succession being similar in both groups of lambs. A total of 24 morphotypes were distinguished by scanning electron microscopy; 17 were rod shaped, 4 were cocci, 2 were spiral, and 1 was filamentous. These morphotypes were further subdivided into: (i) those persisting after their initial colonization in young lambs and present in the adult (7 morphotypes), (ii) those seen only in the adult (2 morphotypes), and (iii) those present only in young lambs (15 morphotypes). The seven morphotypes present in both the lamb and the adult could be considered indigenous members of the epimural community. Several morphotypes appeared restricted in their colonization to certain regions of the papillae, suggesting the presence of microhabitats within the epithelial habitat. Two rod-shaped bacteria were repeatedly seen specifically attached to one another, suggesting an interspecific association.     

Early feed restriction of lambs modifies ileal epimural microbiota and affects immunity parameters during the fattening period

Bacteria firmly attached to the gastrointestinal epithelium during the pre-weaning phase may show a significant impact on nutrient processing, immunity parameters, health and feed efficiency of lambs during post-weaning phases. Thus, the aim of this study was to describe the differences in the ileal epimural microbiota (e.g. total bacteria, Prevotella spp., Bifidobacterium spp. and Lactobacillus spp.) of fattening lambs promoted by early feed restriction during the suckling phase trying to elucidate some of the underlying mechanisms behind changes in feed efficiency during the fattening period. A total of 24 Merino lambs (average BW 4.81±0.256 kg) were used, 12 of them (ad libitum, ADL) kept permanently in individual pens with their mothers, whereas the other 12 lambs were separated from their dams for 9 h each day to be exposed to milk restriction (RES). After weaning (BW=15 kg) all the animals were penned individually, offered the same complete pelleted diet (35 g/kg BW per day) and slaughtered at a BW of 27 kg. During the fattening period, reduced gain : feed ratio (0.320 v. 0.261, P<0.001) was observed for the RES group. Moreover, increments of Prevotella spp. were detected in the ileal epimural microbiota of RES lambs (P<0.05). There were also higher numbers of infiltrated lymphocytes (T and B cells) in the ileal lamina propria (P<0.05), a higher M-cell labelling intensity in ileal Peyer’s patches domes (P<0.05) and a trend towards a thickening of the submucosa layer when compared with the ADL group (P=0.057). Some other immunological parameters, such as an increased immunoglobulin A (IgA) production (pg IgA/µg total protein) and increments in CD45+ cells were also observed in the ileum of RES group (P<0.05), whereas transforming growth factor β and toll-like receptor gene expression was reduced (P<0.05). In conclusion, early feed restriction during the suckling phase promoted changes in ileal epimural microbiota and several immunity parameters that could be related to differences in feed efficiency traits during the fattening period of Merino lambs.     

两种枝顶孢霉胞内细菌的分离鉴定及种群演替

分离、鉴定了枝顶孢霉(Acremonium strictum Gams.)2种胞内细菌,探讨了细菌在其宿主真菌菌丝细胞内的种群演替规律。结果表明,2种胞内细菌分别为不动杆菌Epbas6菌株(Acinetobacter sp.Epbas6)和地衣芽孢杆菌(Bacillus licheniformis)。镜检分析和原始分离物菌落统计表明,不动杆菌和地衣芽孢杆菌的比例为77.5∶1;然而,4℃保藏6个月的真菌分离物中不动杆菌的数量大大减少,地衣芽孢杆菌占据优势,二者的比例变为1∶50.45;而保藏12个月的真菌分离物中只有地衣芽孢杆菌。根据2种细菌的16SrDNA保守序列设计引物,分别扩增了保藏6个月和12个月的枝顶孢霉分离物,发现保藏6个月的分离物能够扩增出2种细菌的保守序列,而保藏12个月的分离物只能扩增出地衣芽孢杆菌保守序列。研究结果说明在枝顶孢霉人工培养和菌种保藏过程中,2种细菌在其宿主真菌菌丝细胞内发生着复杂的生态互作,存在着动态种群演替过程。     

Isolation and identification of synthetic pyrethroid-degrading bacteria

AIMS: To isolate, select, identify and assess the potential for the biodegradation of synthetic pyrethroids (SPs) in sheep dips. METHODS AND RESULTS: SP-degrading bacteria were isolated from a mixed soil sample consisting of garden soil and soils from farms where SPs had been used. The two largest in size were then identified using microscopy, biochemical and genetic techniques to be members of the genera Pseudomonas and Serratia. By comparing the 16S rRNA gene sequences, the Pseudomonas sp. discovered was shown to group within the Pseudomonas fluorescens intrageneric cluster. The Serratia isolated was closely related to Serratia plymuthica. Cell growth and degradation was greatest in the Pseudomonas sp. culture where there was breakdown of 60 mg l(-1) to 6 mg l(-1) technical cypermethrin in 20 days. Tolerance to the SPs was greater in the Pseudomonas sp. but was found to depend on the availability of other carbon sources and nutrients. CONCLUSIONS: The bacteria characterized show the potential to be used in a bioremediation application for the treatment of SP residues. SIGNIFICANCE AND IMPACT OF THE STUDY: The SP-degrading bacteria may have use in the disposal of used SP residues and with further research could lead to an alternative route of disposal for use in agriculture or industry.     

窗胸萤肠道细菌分离及鉴定研究

从窗胸萤的5龄幼虫中分离纯化获得10个细菌菌株,并对其菌体形态、染色反应、培养性状、生理生化性状进行了系统研究。鉴定结果表明,上述10个细菌菌株分别属于埃希氏菌属(Escherichia)、金色单胞菌属(Chryseomonas)、放线杆菌属(Actinobacillus)、摩根氏菌属(Morganella)、李斯特菌属(Listeria)、金杆菌属(Aureobacterium)、微杆菌属(Microbacterium)、地杆菌属(Terrabacter)、芽孢杆菌属(Bacillus)和黄杆菌属(Flavibacterium)。     

高效铁氧化鞘细菌的筛选与鉴定

【目的】铁氧化酶是氧化铁锰鞘细菌类在污水处理中最重要的酶,筛选铁氧化酶高产菌株,研究酶学特性及研究该菌株对水中铁、锰的吸附性能。【方法】利用尿素培养基富集、CGY平板从3份不同水样中分离产铁氧化酶菌株:富集培养铁氧化酶产生菌;摇瓶复筛铁氧化酶产量较高的菌株,通过菌落形态、镜检、生理生化试验和16S rRNA基因序列遗传分析对目的菌株进行鉴定。【结果】分离筛选到一株高产铁氧化酶的菌株S9,被鉴定为鞘细菌球衣菌属浮游球衣菌(Sphaerotilus natans);该菌株对铁锰具有较强的吸附性,当吸附时间为4h时,菌株对水中铁的吸附量为29.02mg/g,吸附率为66.77%;当吸附时间为6h时,菌株对锰的吸附量为34.49mg/g,吸附率为70.68%;该菌株产铁氧化酶的最适温度和p H分别为30℃和7.5,Mg2+、Na+、K+离子对铁氧化酶活性有一定的激活作用,Cu2+对铁氧化酶影响不大,而Mn2+、Zn2+对铁氧化酶具有很强的抑制作用,Pb2+、Ag+对铁氧化酶活性有一定的抑制作用。【结论】浮游球衣菌S9是一株具有较高铁氧化性的菌株,在治理污水和给排水工程中的应用具有潜在的应用价值。     

蟋蟀后肠纤维素降解细菌的分离与鉴定

近年来,具有农业、能源和环保价值的昆虫微生物种类和基因得到了开发,昆虫肠道微生物展示了其巨大的应用潜力,本研究旨在从蟋蟀后肠分离和鉴定纤维素降解细菌。首先采用羧甲基纤维素钠液体培养基对蟋蟀后肠中的微生物进行富集培养,然后使用羧甲基纤维素钠固体培养基分离和筛选单菌落,再通过16S rRNA测序对纤维素降解细菌进行分子鉴定,最后通过刚果红染色来进一步分析细菌降解纤维素的能力。从蟋蟀后肠中共分离出20株纤维素降解细菌,16S rRNA基因测序结果显示来自肠杆菌属(Enterobacter)9株,不动杆菌属(Acinetobacter)7株,克雷伯氏菌属(Klebsiella)2株,鞘氨醇杆菌属(Sphingobacterium)1株和葡萄球菌属(Staphylococcus)1株。刚果红染色试验结果显示,克雷伯氏菌属两株PDSCDXS_2B和8B,鞘氨醇杆菌属PDSCDXS_7C和不动杆菌属PDSCDXS_12C具有较高的纤维素降解能力。这是首次从蟋蟀后肠分离和筛选出来具有纤维素降解能力的细菌,为昆虫源纤维素降解细菌的研究提供了微生物资源。     

小碎斑鱼蛉幼虫肠道细菌分离及鉴定研究

目的从微生态学角度研究小碎斑鱼蛉幼虫的营养生理活动。方法从小碎斑鱼蛉幼虫肠道环境中进行分离、纯化和培养,获得3个细菌菌株,对其菌体形态、染色反应、培养性状和生理生化反应进行系统研究。结果上述3个菌株均属于芽胞杆菌属(Bacillus),1号菌株为巨大芽胞杆菌(Bacillus megaterium),2号菌株为枯草芽胞杆菌(Bacillus subtilis),3号菌株为地衣芽胞杆菌(Bacillus licheniformis)。结论小碎斑鱼蛉幼虫肠道环境中的不同细菌,其数量存在明显差异。     

内切葡聚糖酶产生菌的分离、筛选和识别

采用6种培养基对淡水水库、反刍动物粪便和植物组织共3种环境来源的110份样品进行微生物纯培养物的分离、培养,获得414株细菌纯培养物,其中171株来源于淡水水库环境,70株来源于反刍动物粪便,173株来源于植物内生环境.以羟甲基纤维素钠(CMC-Na)作为唯一碳源对新分离菌株进行内切葡聚糖酶活性筛选,在活性初筛中获得阳性菌197株,复筛验证确认149株为内切葡聚糖酶产生菌.通过16S rRNA基因序列分析初步确定了具内切葡聚糖酶活性菌株的分类学信息,其中64株产酶活性菌株来源于淡水水库环境,归属于10个科的11个属,19株活性菌株来源于反刍动物粪便,归属于10个科的10个属,66株活性菌株来源于植物内生环境,归属于16个科的19个属.实验表明在纤维素储备丰富的自然环境中,具内切葡聚糖酶活性的细菌资源丰富多样,值得进一步深入研究.     

多环芳烃降解菌的筛选、鉴定及降解特性

【目的】多环芳烃(PAHs)是一类普遍存在于环境中且具有高毒性的持久性有机污染物,高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。研究拟从供试菌株中筛选多环芳烃高效降解菌,并分析其降解特性,为多环芳烃污染环境的微生物修复提供资源保障和科学依据。【方法】采用平板法从25株供试菌株中筛选出以菲和芘为唯一碳源和能源的高效降解菌,经16S rRNA基因序列进行初步鉴定,通过单因素实验法分析其在液体培养基中的降解特性。【结果】筛选出的3株多环芳烃高效降解菌SL-1、02173和02830经16S rRNA基因序列分析,02173和02830分别与假单胞菌属中的Pseudomonas alcaliphila和Pseudomonas corrugate同源性最近,SL-1为本课题组发表新类群Rhizobium petrolearium的模式菌株;降解实验表明,菌株SL-1 3 d内对单一多环芳烃菲(100 mg/L)和芘(50 mg/L)的降解率分别达到100%和48%,5 d后能够降解74%的芘;而其3 d内对混合PAHs中菲和芘的降解率分别为75.89%和81.98%。菌株02173和02830 3 d内对混合多环芳烃中萘(200 mg/L)、芴(50 mg/L)、菲(100 mg/L)和芘(50 mg/L)的降解率均分别超过97%。【结论】筛选出的3株PAHs降解菌SL-1、02173和02830不仅可以高效降解低分子量PAHs,还对高分子量PAHs具有很好的降解潜力。研究表明,由于共代谢作用低分子量多环芳烃可促进高分子量多环芳烃的降解,而此时低分子量多环芳烃的降解将受到抑制。     

芝麻香型白酒发酵过程中乳酸菌多样性及其演替规律

【背景】乳酸菌是白酒发酵过程中一类非常重要的微生物,其种类及动态变化对于白酒品质具有重要影响。然而,目前对于芝麻香型白酒发酵过程中乳酸菌群落结构及其演替规律的认识并不全面。【目的】揭示芝麻香型白酒发酵过程中乳酸菌的多样性及菌群的演替规律,为更好地探索白酒酿造机理和控制白酒品质提供生物学依据。【方法】利用高通量测序技术对芝麻香型白酒发酵过程中乳酸菌菌群演替进行跟踪分析,同时采用实时荧光定量PCR对发酵过程中乳酸菌的生物量进行定量分析。【结果】高通量测序结果显示,芝麻香型白酒发酵过程涉及5个属的乳酸菌:魏斯氏菌属(Weissella)、片球菌属(Pediococcus)、乳杆菌属(Lactobacillus)、明串珠菌属(Leuconostoc)和乳球菌属(Lactococcus),共计43种乳酸菌。其中,在发酵过程中平均相对丰度大于0.5%的乳酸菌有10种,分别是类肠膜魏斯氏菌(Weissella paramesenteroides)、食窦魏斯氏菌(Weissella cibaria)、融合魏斯氏菌(Weissella confusa)、戊糖片球菌(Pediococcus pentosaceus)、假肠膜明串珠菌(Leuconostoc pseudomesenteroides)、发酵乳杆菌(Lactobacillus fermentum)、植物乳杆菌(Lactobacillus plantarum)、副干酪乳杆菌(Lactobacillus paracasei)、耐酸乳杆菌(Lactobacillus acetotolerans)和Lactobacillus sp.。在堆积发酵过程中,Weissella属占细菌总量的50%以上,其次是Pediococcus属和Lactobacillus属,而Leuconostoc属和Lactococcus属相对较少。在窖池发酵过程中Lactobacillus属的乳酸菌逐渐成为优势细菌,尤其是Lactobacillus sp.在窖池发酵中后期相对丰度达到80%以上。实时荧光定量PCR结果显示,在堆积发酵和窖池发酵前期乳酸菌总量变化不大;从窖池发酵5 d开始,乳酸菌总量迅速上升,30 d时达到最大值。【结论】对白酒发酵过程中乳酸菌种类及动态变化的研究有助于探究白酒酿造过程中乳酸菌功能,进而解析白酒酿造机理,最终达到控制白酒品质的目的。     

Urease activity of adherent bacteria and rumen fluid bacteria

In experiments on six sheep fed on a low nitrogen diet (3.7 g N/day), urease (EC 3.5.1.5) activity (nkat X mg-1 bacterial dry weight) 3 h after feeding was found to be highest in the bacteria adhering to the rumen wall (13.25 +/- 2.10), lower in the rumen fluid bacteria (8.96 +/- 1.35) and lowest in the bacteria adhering to feed particles in the rumen (5.69 +/- 2.13). The urease activity of bacteria adhering to the rumen wall and of the rumen fluid bacteria of six sheep fed on a high nitrogen diet (21 g N/day) was significantly lower than in sheep with a low N intake and in both cases was roughly the same (3.81 +/- 1.37 and 3.76 +/- 1.02 respectively); it was lowest in bacteria adhering to feed particles in the rumen (1.92 +/- 0.90). It is concluded from the results that the urease activity of rumen fluid bacteria and of bacteria adhering to the rumen wall and to feed particles in the rumen is different and that it falls significantly in the presence of a high nitrogen intake. From the relatively high ureolytic activity of bacteria adhering to the rumen wall in the presence of a low nitrogen intake it is assumed that this is one of the partial mechanisms of the hydrolysis of blood urea entering the rumen across the rumen wall and of its reutilization in the rumen-liver nitrogen cycle in ruminants.     

脱氮除硫菌株的分离鉴定和功能确认

从长期稳定运行的脱氮除硫反应器污泥中,分离获得两株具有脱氮除硫功能的芽孢杆菌。经形态观察、生理试验和16SrDNA序列比对,将两菌株归入芽孢杆菌属,菌株CB归类于Bacillus pseudofirmus,菌株CS则与Bacillus hemicellulosilytus和Bacillus halodurans最为接近。以Biolog板检测,菌株CB的基质多样性不明显,菌株CS则可利用Biolog板中多种碳源。菌株CB和菌株CS都能以硝酸盐氧化硫化物,其中菌株CB对硝酸盐、硫化物的转化能力大于CS,菌株CB对硝酸盐的亲和力也大于菌株CS。     

海带发酵降解中相关海洋功能菌的分离、鉴定与筛选

目的 从海带养殖环境中筛选有效降解海带的海洋功能菌,对海带进行降解处理,提高利用率,增加海带原料价值。方法 采用以果胶或海藻酸钠为唯一碳源的选择性培养基挑选含有果胶酶、褐藻酸裂解酶的菌株;测定海带发酵液中海藻酸含量,复筛降解效果好的单菌及复合菌;16S rDNA测序对菌种进行鉴定。结果 使用含10%鲜海带的富集培养基培养48 h,其中降解效果较好的为1-2和3-10菌种的组合,其降解量为95%。结论 经上述研究选出有效的降解海带功能菌1-2和3-10,其降解海带效果较好,对海带利用率高。     

Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.     

Isolation and identification of bacteria from spent nuclear fuel pools

The aim of the present research was to isolate and identify bacteria from spent nuclear fuel pools of a Spanish nuclear power plant. Water samples were collected and inoculated onto different culture media to isolate the highest number of species. 16S rDNA fragments from colonies growing on solid media were amplified and analysed by denaturing gradient gel electrophoresis. Sequencing revealed the presence of 21 different bacteria belonging to several phylogenetic groups (, , and -Proteobacteria, Actinomycetales, Flavobacterium, and the Bacillus/Staphylococcus group). The isolation of these microorganisms in this particular environment (oligotrophic and radioactive) is highly interesting because of the possibility of their being used for the bioremediation of radionuclide-contaminated waters.