Adaptations in bacterial catabolic enzyme activity and community structure in membrane-coupled bioreactors fed simple synthetic wastewater

Membrane-coupled bioreactors (MBRs) offer substantial benefits compared to conventional reactor designs for biological wastewater treatment. MBR treatment efficiency, however, has not been optimized because the effects of the MBR on process microbiology are poorly understood. In this study, the structure and function of the microbial communities growing in MBRs fed simple synthetic wastewater were investigated. In four starch-fed MBRs, the bacterial community substantially increased its alpha-glucosidase affinity (>1000-fold), while the leucine aminopeptidase and heptanoate esterase affinities increased slightly (<40-fold) or remained relatively constant. Concomitant to these physiological adaptations, shifts in the bacterial community structure in two of the starch-fed MBRs were detected by PCR-DGGE. Four of the bacterial populations detected by PCR-DGGE were isolated and exhibited specific growth rates in batch culture ranging from 0.009 to 0.22 h(-1). Our results suggest that bacterial communities growing under increasingly stringent nutrient limitation adapt their enzyme activities primarily for the nutrients provided, but that there is also a more subtle response not linked to the substrates included in the feed medium. Our research also demonstrates that MBRs can support relatively complex bacterial communities even on simple feed media.     

内生真菌发酵提取物和植物生长调节剂对大豆根际细菌多样性的影响

为了研究内生真菌发酵提取物和植物生长调节剂对大豆根际细菌多样性的影响,采用PCR-DGGE技术分析了各处理中不同发育期的大豆根际细菌群落变化。结果发现发酵提取液和植物生长调节剂能增加部分优势菌群的数量,但对根际细菌类群结构影响并不明显;生育周期也是影响根际细菌数量的重要因素。另外割胶测序发现优势菌群主要是Proteobacteria(变形菌门)、Acidobacteria(酸杆菌纲)、Nitrospira(硝化螺旋菌属)、Bradyrhizobium(慢生根瘤菌属)等,这些也都是大豆根际比较常见的细菌类群。     

LOW NUTRIENT R2A CULTURE MEDIUM FOR BACTERIAL ENUMERATION FROM POULTRY FEEDS

Poultry feed matrices can be particularly stressful to bacterial populations and limit recovery and accurate enumeration of viable organisms. The objectives in this study were to enumerate bacterial populations with either tryptic soy plate medium or an R2A minimal nutrient medium from commercial poultry and turkey dry feeds and compare enumerations from the two media with feed composition. Total population estimates from tryptic soy plates varied between 3.7 and log₁₀ 5.4 CFU per g feed and depended upon poultry feed source (P < 0.05). R2A plate counts varied between 2.7 and log₁₀ 5.4 CFU per g feed, but were not significantly different among poultry feed sources. Feed R2A plate count populations were significantly correlated (P < 0.01) with tryptic soy plate count populations enumerated from the feed sources, but not protein and fat content of the feed source. It is apparent that bacterial counts recovered by minimal R2A medium are similar to enumerations using tryptic soy plate medium and that R2A medium could be substituted for tryptic soy plate medium for bacterial enumeration in poultry feeds.     

喂养不同饵料对匙吻鲟和鳙消化道微生物区系的影响

为了解消化道的菌群结构及影响因素, 用PCR-DGGE技术和序列分析调查投喂鲜活饵料(BI-L)和配合饲料(BI-C)的鳙肠道菌群, 和投喂鲜活饵料(PS-L, PI-L)和配合饲料(PS-C, PI-C)的匙吻鲟胃和肠道菌群。实验共回收测序16条DGGE条带, 系统发育分析显示鳙和匙吻鲟所有待测样本的优势菌为厚壁菌门(31.25%, 5条)。其他属于-变形菌纲(-Proteobacteria)(25%, 4条)、拟杆菌门(Bacteroidetes)(12.5%, 2条)、梭杆菌门(Fusobacteria)(12.5%, 2条)、蓝藻门(Cyanobacteria)(6.25%, 1条)、放线菌门(Actinobacteria)(6.25%, 1条)、-变形菌门(-Proteobacteria)(6.25%, 1条)。不同饵料喂养的匙吻鲟肠道菌群具有较高的相似性(49%), 不同饵料喂养的鳙肠道菌群也具有较高的相似性(59%), 说明这些生态位中存在着更稳定成型的微生物群落。然而, 两组不同饵料喂养的匙吻鲟胃样本菌群之间存在相当大的差异, 表明匙吻鲟胃部菌群可能更易受到饵料的影响。     

Removal of carbonaceous and nitrogenous pollutants from a synthetic wastewater using a membrane-coupled bioreactor

Two modified Ludzack-Ettinger (MLE)-type membrane-coupled bioreactors (MBRs) were investigated in this study for the purpose of removing both nitrogenous and carbonaceous pollutants from a synthetic wastewater. During the first MBR experiment, removal efficiencies were high (>90%) for chemical oxygen demand (COD) and ammonia, but total nitrogenous pollutant removal efficiency was poor (~25%). Bacterial community analysis of ammonia oxidizing bacteria (AOB) by a nested PCR-DGGE approach detected two Nitrosomonas-like populations and one Nitrosospira-like population. During the initial portion of the second MBR experiment, COD and ammonia removal efficiencies were similar to the first MBR experiment until the COD of the influent wastewater was increased to provide additional electron donors to support denitrification. Total nitrogen removal efficiencies eventually exceeded 90%, with a hydraulic residence time (HRT) of 24 h and a recirculation ratio of 8. When the HRT of the MBR experiment was decreased to 12 h, however, ammonia removal efficiency was adversely affected. A subsequent increase in the HRT to 18 h helped improve removal efficiencies for both ammonia (>85%) and total nitrogenous compounds (~70%). Our research demonstrates that MBRs can be effectively designed to remove both carbonaceous and nitrogenous pollutants. The ability of the microbial community to switch between anoxic (denitrifying) and oxic (nitrifying) conditions, however, represents a critical process constraint for the application of MLE-type MBR systems, such that little benefit is gained compared to conventional designs.     

PCR-DGGE Comparison of Bacterial Community Structure in Fresh and Archived Soils Sampled along a Chihuahuan Desert Elevational Gradient

The polymerase chain reaction coupled with denaturing gradient gel electrophoresis (PCR-DGGE) has been used widely to determine species richness and structure of microbial communities in a variety of environments. Researchers commonly archive soil samples after routine chemical or microbial analyses, and applying PCR-DGGE technology to these historical samples offers evaluation of long-term patterns in bacterial species richness and community structure that was not available with previous technology. However, use of PCR-DGGE to analyze microbial communities of archived soils has been largely unexplored. To evaluate the stability of DGGE patterns in archived soils in comparison with fresh soils, fresh and archived soils from five sites along an elevational gradient in the Chihuahuan Desert were compared using PCR-DGGE of 16S rDNA. DNA from all archived samples was extracted reliably, but DNA in archived soils collected from a closed-canopy oak forest site could not be amplified. DNA extraction yields were lower for most archived soils, but minimal changes in bacterial species richness and structure due to archiving were noted in bacterial community profiles from four sites. Use of archived soils to determine long-term changes in bacterial community structure via PCR-DGGE appears to be a viable option for addressing microbial community dynamics for particular ecosystems or landscapes.     

Eco-physiological Characterization of Soil Bacterial Populations in Different States of Growth

The method based on characterization of microbial populations in terms of their growth rate in agar plates has been used for testing the prediction of the theory of r- and K-selection in a microbial community from a tropical soil. Conditions which could lead bacterial populations to grow exponentially or to enter into a stationary phase were obtained by growing soil microbial populations in a chemostat and in a chemostat with recycle, respectively. Significant differences in population distribution patterns were observed by comparing results from the two growth systems. When soil community was grown in a chemostat and subjected specifically to well-defined r- and K-conditions, stable associations of organisms with r- and K-type characteristics developed as a consequence of environmental pressure. In contrast, when cultivated in chemostat with recycle under the same r- and K-conditions imposed on chemostat cultures, distribution patterns of r- and K-selected populations appeared very little affected by changes in substrate availability.     

Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered.     

Growth rate control of adherent bacterial populations

We report a novel in vitro method which, through application of appropriate nutrient limitations, enables growth rate control of adherent bacterial populations. Exponentially growing cells are collected by pressure filtration onto cellulose acetate membranes. Following inversion into the bases of modified fermentors, membranes and bacteria are perfused with fresh medium. Newly formed and loosely attached cells are eluted with spent medium. Steady-state conditions (dependent upon the medium flow rate) at which the adherent bacterial biomass is constant and proportional to the limiting nutrient concentrations are rapidly achieved, and within limits, the growth rate is proportional to the medium flow rate. Scanning electron microscopic studies showed that such populations consist of individual cells embedded within an extracellular polymer matrix.     

Growth rate control of adherent bacterial populations.

We report a novel in vitro method which, through application of appropriate nutrient limitations, enables growth rate control of adherent bacterial populations. Exponentially growing cells are collected by pressure filtration onto cellulose acetate membranes. Following inversion into the bases of modified fermentors, membranes and bacteria are perfused with fresh medium. Newly formed and loosely attached cells are eluted with spent medium. Steady-state conditions (dependent upon the medium flow rate) at which the adherent bacterial biomass is constant and proportional to the limiting nutrient concentrations are rapidly achieved, and within limits, the growth rate is proportional to the medium flow rate. Scanning electron microscopic studies showed that such populations consist of individual cells embedded within an extracellular polymer matrix.     

Dynamics of bacterial community composition and activity during a mesocosm diatom bloom

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment ( approximately 24 microg of chlorophyll a liter(-1)). At this time bacterial abundance abruptly decreased from 2.8 x 10(6) to 0.75 x 10(6) ml(-1), and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-microm size fraction towards the >1.0-microm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized alpha-Proteobacteria- and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, beta-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.     

Molecular fingerprinting of bacterial populations in groundwater and bottled mineral water

Monitoring the hygienic quality of drinking waters by determining the concentration of fecal indicators with traditional plate count techniques suffers from important drawbacks. In this work, the potential of PCR-DGGE (polymerase chain reaction - denaturing gradient gel electrophoresis) analysis of 16S rDNA genes to fingerprint the bacterial populations of mineral water and groundwater was investigated. A rapid and simple pretreatment to concentrate and release bacterial DNA prior to PCR was explored. This pretreatment was successful for commercially bottled mineral water. For groundwater, an additional resuscitation step was required to obtain a PCR signal. It was clear that the groundwater under scrutiny contained a more diverse bacterial community than the mineral water. A comparison was made between four kinds of mineral waters and one sample of groundwater using the developed procedures. For each kind of water, bacterial populations cultured on R2A plates were also subjected to PCR-DGGE. Comparison of the fingerprints of the plated samples and the original samples suggested the presence of viable but nonculturable bacteria in the waters. The obtained cluster dendrogram indicated that each kind of water was characterized by a specific molecular fingerprint. The sensitivity of the whole of the procedure was between 10(4) and 10(5) cfu ml(-1) as determined using a pure culture of Escherichia coli. The described PCR-DGGE method can constitute the basis of a new and interesting strategy to monitor in a relatively rapid way (less than 24 h) the bacterial quality of waters such as mineral water, groundwater and certain types of reclaimed water.     

Impact of feed efficiency and diet on adaptive variations in the bacterial community in the rumen fluid of cattle

Limited knowledge of the structure and activities of the ruminal bacterial community prevents the understanding of the effect of population dynamics on functional bacterial groups and on host productivity. This study aimed to identify particular bacteria associated with host feed efficiency in steers with differing diets and residual feed intake (RFI) using culture-independent methods: PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis. PCR-DGGE profiles were generated from the ruminal fluid of 55 steers fed a low-energy-density diet and then switched to a high-energy-density diet. Bacterial profile comparisons by multivariate statistical analysis showed a trend only for RFI-related clusters on the high-energy diet. When steers (n = 19) belonging to the same RFI group under both diets were used to identify specific bacterial phylotypes related to feed efficiency traits, correlations were detected between dry matter intake, average daily gain, and copy numbers of the 16S rRNA gene of Succinivibrio sp. in low-RFI (efficient) steers, whereas correlations between Robinsoniella sp. and RFI (P < 0.05) were observed for high-RFI (inefficient) animals. Eubacterium sp. differed significantly (P < 0.05) between RFI groups that were only on the high-energy diet. Our work provides a comprehensive framework to understand how particular bacterial phylotypes contribute to differences in feed efficiency and ultimately influence host productivity, which may either depend on or be independent from diet factors.     

Comparison of PCR-DGGE and selective plating methods for monitoring the dynamics of a mixed culture population in synthetic brewery wastewater

Enrichment of an activated sludge inoculum in synthetic brewery wastewater, which included glucose, maltose, and ethanol, was conducted in batch experiments to identify the dominant microbes present, to determine methodologies capable of monitoring the mixed culture population dynamics, and to determine the consortium's substrate degradation behavior. These results and methodologies were subsequently used in the determination of the population dynamics of suspended and attached microorganisms in a sequencing batch system in the second part of this research work. The three-membered microbial community comprised two bacterial and one fungal species that were identified as Acinetobacter sp., Enterobacter sp., and Candida sp. PCR-DGGE and plating on selective media were used to track the population dynamics of the consortium during the degradation of different substrates in synthetic wastewater containing glucose, maltose, and ethanol. Enterobacter sp. could degrade glucose and maltose but not ethanol, whereas Acinetobacter and Candida could degrade all three carbon sources. In buffered batch mixed culture experiments, Enterobacter was the predominant bacterium until the sugar concentrations decreased to levels that enabled Acinetobacter and Candida to degrade ethanol. PCR-DGGE was effective for detecting the dominant species, but culture-based methods were more accurate for monitoring the population dynamics of these microorganisms during growth in the wastewater medium.     

Community Structure of Actively Growing Bacterial Populations in Plant Pathogen Suppressive Soil

The bacterial community in soil was screened by using various molecular approaches for bacterial populations that were activated upon addition of different supplements. Plasmodiophora brassicae spores, chitin, sodium acetate, and cabbage plants were added to activate specific bacterial populations as an aid in screening for novel antagonists to plant pathogens. DNA from growing bacteria was specifically extracted from the soil by bromodeoxyuridine immunocapture. The captured DNA was fingerprinted by terminal restriction fragment length polymorphism (T-RFLP). The composition of the dominant bacterial community was also analyzed directly by T-RFLP and by denaturing gradient gel electrophoresis (DGGE). After chitin addition to the soil, some bacterial populations increased dramatically and became dominant both in the total and in the actively growing community. Some of the emerging bands on DGGE gels from chitin-amended soil were sequenced and found to be similar to known chitin-degrading genera such as Oerskovia, Kitasatospora, and Streptomyces species. Some of these sequences could be matched to specific terminal restriction fragments on the T-RFLP output. After addition of Plasmodiophora spores, an increase in specific Pseudomonads could be observed with Pseudomonas-specific primers for DGGE. These results demonstrate the utility of microbiomics, or a combination of molecular approaches, for investigating the composition of complex microbial communities in soil.     

Feeding heterogeneity in ciliate populations: Effects of culture age and nutritional state

We have used a novel approach in conjunction with flow cytometry to quantify the biological heterogeneity of populations of the ciliate Tetrahymena pyriformis. It was found that the rate of particle uptake of exponentially growing cells is not uniform among cells and partially correlated with cell size. The physiological state and growth history of the culture was found to affect to a large degree the population's feeding heterogeneity. Stationary phase populations exhibited more uniform feeding behavior, as cell aging affects all cells and effectively reduces their ability to feed. Cells that were removed from the growth medium and resuspended in nonnutritive medium exhibited a more heterogeneous feeding behavior. The starved cells were stimulated to feed at considerably higher rates, and the stimulatory effect was more pronounced for larger cells. It is therefore demonstrated that population heterogeneity has to be evaluated in conjunction with the populations growth state as it is determined by the history of the population's growth and nutritional state. (c) 1994 John Wiley & Sons, Inc.     

集约种植雷竹林土壤细菌群落结构的演变及其影响因素

应用PCR-DGGE技术研究了雷竹林种植过程中土壤细菌群落结构的变化,并运用冗余分析（RDA）手段提取雷竹林土壤细菌群落多样性的干扰控制因子.结果表明：水稻田改种雷竹后,土壤细菌群落的Shannon指数及丰富度指数均显著增加;长期的集约种植过程对雷竹林土壤细菌群落结构产生了影响,主要细菌种群发生了变化,多样性指数表现为先增加、后期大幅降低的趋势.RDA分析表明,土壤pH、总氮、碱解氮以及速效钾这4个变量能够解释高达76.1%的样本总变异,其中土壤pH对细菌群落结构影响最大,但未达到显著水平,表明长期集约种植的雷竹林土壤细菌群落结构的变化是多种环境因子共同作用的结果.     

Biofouling in membrane bioreactors: nexus between polyacrylonitrile surface charge and community composition

The influence of membrane surface charge on biofouling community composition during activated sludge filtration in a membrane bioreactor was investigated in this study using polyacrylonitrile-based membranes. Membranes with different surface properties were synthesized by phase inversion followed by a layer-by-layer modification. Various characterization results showed that the membranes differed only in their surface chemical composition and charge, ie two of them were negative, one neutral and one positive. Membrane fouling experiments were performed for 40 days and the biofouling communities were analyzed. PCR-DGGE fingerprinting indicated selective enrichment of bacterial populations from the sludge suspension within the biofilms at any time point. The biofilm community composition seemed to change with time. However, no difference was observed between the biofilm community of differently charged membranes at specific time points. It could be concluded that membrane charges do not play a decisive role in the long-term selection of the key bacterial foulants.     

PCR-DGGE对长江河口八种野生鱼类肠道菌群多样性的比较研究

目的分析长江河口捕获的8种野生鱼类的肠道菌群多样性的差异并观察这种差异与食性的联系。方法采用PCR-DGGE(denaturing gradient gel electrophoresis)技术,DGGE图谱用PCA(principal component analy-sis)方法进行分析。结果建立了长江口8种鱼野生条件下肠道菌群的DGGE指纹图谱,观察到它们在野生条件下的肠道菌群的差异。其中,营底栖生活的舌鰕虎鱼的肠道菌群和其他7种野生鱼有着明显的差异,其他7种鱼的肠道菌群多样性的差异与它们的食性差异相关。结论PCR-DGGE技术是一种能够快速有效地分析研究鱼类肠道菌群结构的技术。8种野生鱼的肠道菌群的结构有明显的差别,并且食性差异大的鱼类之间肠道菌群差异也     

利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,研究了5种高温和中温白酒大曲细菌群落结构,通过优势条带切胶鉴定确定了大曲中优势细菌种属信息。结果表明,Weissella cibaria、Lactobacillus helveticus、L.fermentum、L.panis等乳酸菌普遍存在于5种大曲中,Ther-moactinomyces sanguinis仅存在于高温曲酱曲中,同时DGGE检测到了传统方法未能分离鉴定的Staphylococcus xylosus、Klebsiella oxytoca。不同工艺大曲细菌群落结构存在明显差异,随着制曲温度的升高,大曲细菌多样性指数有下降趋势。PCR-DGGE技术是一种能够快速有效地研究白酒大曲细菌群落结构的技术。