Molecular identification of the microbiota of peeled and unpeeled brown shrimp (Crangon crangon) during storage on ice and at 7.5 °C

The dominant microbiota of brown shrimp (Crangon crangon) were systematically identified during storage under different conditions. Freshly caught shrimp were processed on board the fishing vessel under the best possible hygienic conditions (IDEAL), unpeeled and manually (sterile) peeled, then stored on ice and at 7.5 °C until microbiologically spoiled. Results were compared with industrially processed (INDUSTRIAL) shrimp. Isolates grown on various media were identified by 16S rRNA and gyrB gene sequencing. We examined the total microbiota and microbial population shifts of shrimp under various storage conditions using denaturant gradient gel electrophoresis (DGGE). The microbiota differed somewhat during storage and among the various storage conditions; however, members of the genera Psychrobacter and Pseudoalteromonas were found to dominate the microbiota of all shrimp samples regardless of processing procedures or storage conditions. Most isolates could be identified by gyrB gene sequencing as Psychrobacter immobilis or Psychrobacter cibarius. Also Pseudoalteromonas nigrifaciens, Pseudoalteromonas elyakovii or Pseudoalteromonas paragorgicola dominated the microbiota of brown shrimp during storage. Also species from the genera Planocuccus, Exiguobacterium, Carnobacterium, Pseudomonas, Chryseobacterium and Staphylococcus were detected during storage of brown shrimp.     

Evaluation of DNA extraction methods for freshwater eukaryotic microalgae

The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy^® Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended.     

Investigation of bacterial community in activated sludge with an anaerobic side-stream reactor (ASSR) to decrease the generation of excess sludge

The goal of this study was to investigate the bacterial community in activated sludge with an anaerobic side-stream reactor (ASSR), a process permitting significant decrease in sludge production during wastewater treatment. The study operated five activated sludge systems with different sludge treatment schemes serving as various controls for the activated sludge with ASSR. Bacterial communities were analyzed by denaturing gradient gel electrophoresis (DGGE), sequencing and construction of phylogenetic relationships of the identified bacteria. The DGGE data showed that activated sludge incorporating ASSR contained higher diversity of bacteria, resulting from long solids retention time and recirculation of sludge under aerobic and anaerobic conditions. The similarity of DGGE profiles between ASSR and separate anaerobic digester (control) was high indicating that ASSR is primarily related to conventional anaerobic digesters. Nevertheless, there was also unique bacteria community appearing in ASSR. Interestingly, sludge in the main system and in ASSR showed considerably different bacterial composition indicating that ASSR allowed enriching its own bacterial community different than that from the aeration basin, although two reactors were connected via sludge recirculation. In activated sludge with ASSR, sequences represented by predominant DGGE bands were affiliated with Proteobacteria. The remaining groups were composed of Spirochaetes, Clostridiales, Chloroflexi, and Actinobacteria. Their putative role in the activated sludge with ASSR is also discussed in this study.     

基于PCR-DGGE分析抗性淀粉对高脂饮食HFA小鼠肠道菌群的影响

本文研究了香蕉抗性淀粉对高脂饮食菌群人源化小鼠(HFA)肠道菌群的调控效果。将30只肠道菌群人源化昆明小鼠随机分为3组(10只/组),分别饲喂普通饮食、高脂饮食和高脂饮食+抗性淀粉。提取第0周和第8周新鲜粪便DNA,比较用16S r DNAV6-V8区段特异性引物扩增产物,以PCR-DGGE技术手段检测抗性淀粉组、高脂饮食组和普通饮食组肠道菌群差异。第0周,三组小鼠菌群总菌丰富度,多样性指数和均匀度无显著差异(p0.05),小鼠和粪便供者之间菌群相似性为36%;三组小鼠肠道菌群相似性达到60-93%,PCA分析显示三组小鼠的数据点都聚在一起,说明在干预前无明显差异,适用于分析抗性淀粉对肠道菌群的影响;膳食干涉第8周后小鼠肠道菌群的多样性指数和丰富度数值,抗性淀粉组都极显著低于高脂组和普通饲料组(p0.01),PCA分析也显示三组小鼠肠道菌群各组的数据点都分布在不同区域,说明抗性淀粉对高脂饮食HFA小鼠肠道菌群结构产生显著影响。     

Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis

The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 °C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants.     

DGGE法在盛夏习酒酒醅的微生物菌群结构解析中的应用

利用PCR—DGGE技术分析了酒醅入窖前后的菌群变化。结果表明,发酵前微生物群落结构呈明显多样分布,发酵结束后优势菌明显改变,经同源性比较,Laetobacillus plantarum成为绝对优势菌,Weissella属细菌在酒醅中稳定存在。根据细菌菌群的变化及分布推测,在盛夏习酒（浓香型）酒醅发酵过程中,Lactobacillus和Weisselln细菌为重要菌群。     

用DGGE技术构建白酒酿造微生物指纹图谱的初步研究

利用PCR-DGGE技术及Quantity One软件分析对比了大曲、酒醅不同部位的微生物群落结构.通过从原始样品中直接提取DNA,并对全长16s rDNA及其V3区片断扩增,变性梯度凝胶电泳及软件分析等,发现不同样品之间的微生物群落存在一定联系及差异性.通过图谱与系统发育树的结合,能给不同样品赋予特定的分子标签.显示DGGE技术能为微生物菌落结构提供直观图谱,是研究自然传统发酵食品及其它天然微生态样品的先进方法.     

强化反硝化除磷对A~2O工艺微生物种群变化的影响

强化A2O工艺中的反硝化除磷比例,是提高该工艺处理低C/N比污水时脱氮除磷效率的有效途径。采用52.5 L的A2O反应器处理实际生活污水,研究了系统在不同的反硝化除磷比例情况下微生物的种群变化及关系。试验结果表明,随着水质条件及运行状态的改变,系统反硝化除磷的比例也在变化,同时微生物种群结构表现为一种动态的演替过程,工艺条件与微生物的种群结构具有很强的映射关系。测序结果表明,具有传统除磷功能的Acinetobacter在系统反硝化除磷得到强化的时候会逐渐被淘汰,而Uncultured Chlorobi bacterium会逐渐得到增殖,可能是系统中具有反硝化除磷功能的微生物。     

Bacterial community composition and structure of biofilms developing on nanofiltration membranes applied to wastewater treatment

The structure and microbial communities of biofilms developing on cross-flow nanofiltration (NF) membranes at different temperatures (20, 25 or 34 degrees C) and operation lengths (8h-24days) were studied. Feedwater comprised tertiary quality wastewater effluent or synthetic media mimicking effluents of intermediate quality. After each run, the membranes were autopsied for bacterial enumeration, bacterial community composition and microscopy visualization (SEM, CLSM and AFM/NSOM). Community composition was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) coupled with sequence analysis of 16S rRNA gene fragments from dominant bands. Deposition of polysaccharides and initial bacterial colonization were observed within 8h, whereas developed biofilms markedly affecting membrane permeability were evident from days 2-3 onwards. Regardless of applied conditions, the heterotrophic plate counts in the biofilm were 3-4x10(6)CFU/cm(2) and the thickness of the biofouling layer was 20-30microm. From a total of 22 sequences obtained from 14 independent experiments, most species identified were Gram negative (19 of 22 sequences). Proteobacteria were found to be a prevalent group in all cases (16 of 22 sequences) and among it, the beta-subclass was the most predominant (8 sequences), followed by the gamma-subclass (5 sequences). Pseudomonas/Burkholderia, Ralstonia, Bacteroidetes and Sphingomonas were the dominant groups found in most cases. Even though the microbial population might be important with respect to biofouling patterns, membrane permeability decline seems to be more substantially influenced by the formation and accumulation of exopolymeric substances (EPS).     

Natural organic matter (NOM) removal and structural changes in the bacterial community during artificial groundwater recharge with humic lake water

This study evaluated the removal of natural organic matter (NOM) and structural changes in the microbial community during infiltration of humic lake water at three artificial groundwater recharge (AGR) sites in Finland. The three sites were at waterworks in H?meenlinna, Jyv?skyl? and Tuusula, sites A, B and C, respectively. Site A used groundwater recharge by both basin and sprinkling infiltration, site B used only sprinkling infiltration, and site C used only basin infiltration. Reductions of total organic carbon at sites A, B and C were 91%, 84% and 74%, respectively, in the winter, and 88%, 77% and 73%, respectively, in the summer. The Finnish national recommended value of 2 mg/l for TOC was achieved at all sites and the TOC of natural groundwater at site C was much lower, at 0.6 mg/l. Large molecular fractions of NOM were removed more efficiently than the smaller ones. Total amount of DAPI-stained cells decreased during infiltration at sites A, B and C in winter by 94%, 94% and 75% and in summer by 96%, 97% and 94%, respectively. Bacterial communities in raw waters and extracted groundwaters were diverse with changes occurring during infiltration, which was shown by DNA extraction followed by PCR of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) fingerprinting. While the natural groundwater microbial community was diverse, it was different from that of the extracted groundwater in the AGR area. Simultaneous organic carbon removal and the decrease of bacterial counts during infiltration indicated biodegradation. In addition, the changing DGGE profiles during the process of infiltration, demonstrated that changing environmental conditions were reflected by changes in bacterial community composition.