The Effects of Sample Storage Conditions on the Microbial Community Composition in Hydraulic Fracturing Produced Water

The petroleum industry has an increasing interest in understanding the microbial communities driving biofouling and biocorrosion in reservoirs, wells, and infrastructure. However, sampling of the relevant produced fluids from subsurface environments for microbiological analyses is often challenged by high liquid pressures, workplace regulations, operator liability concerns, and remote sampling locations. These challenges result in infrequent sampling opportunities and the need to store and preserve the collected samples for several days or weeks. Maintaining a representative microbial community structure from produced fluid samples throughout storage and handling is essential for accurate results of downstream microbial analyses. Currently, no sample handling or storage recommendations exist for microbiological analyses of produced fluid samples. We used 16S rRNA gene sequencing to monitor the changes in microbial communities in hypersaline produced water stored at room temperature or at 4?°C for up to 7 days. We also analyzed storage at ?80?°C across a 3-week period. The results suggest ideal handling methods would include placing the collected sample on ice as soon as possible, but at least within 24?h, followed by shipping the samples on ice over 2–3?days, and finally, long-term storage in the ?20?°C or ?80?°C freezer.     

Quantification of Microthrix parvicella in activated sludge bacterial communities by real-time PCR

AIMS: This study was to develop a simple and reliable method for quantifying Microthrix parvicella 16S rRNA gene copies and its application to activated sludge samples collected from wastewater treatment plants (WWTP) with and without foaming problems. METHODS AND RESULTS: The relative frequency of M. parvicella was determined by combining real-time PCR assays for quantification of total bacterial 16S rRNA gene copies and M. parvicella 16S rRNA gene copies. The developed method was applied to analyse 32 activated sludge samples obtained from German WWTP. The level of M. parvicella 16S rRNA gene copies in the 18 nonfoaming samples was below 3% of the total number of 16S rRNA gene copies and in the range of 0-18% for the 14 foaming samples. CONCLUSIONS: The described method allows reliable monitoring of the amount of M. parvicella in activated sludge samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The described method may become an important component of a warning system for forthcoming bulking and foaming episodes.     

Characterization of the Fecal Microbiota Using High-Throughput Sequencing Reveals a Stable Microbial Community during Storage

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at −80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or −80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or −80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.     

rRNA-targeted寡核苷酸探针识别活性污泥微生物群落结构与功能研究进展

活性污泥中微生物群落内部关系非常复杂 ,及时对活性污泥中优势菌群和群落内部关系进行监测是污水处理中采取正确措施的关键。历史研究表明传统培养方法经常导致活性污泥优势菌群检测的失败 ,而r RNA- targeted寡核苷酸探针作为一种快速原位监测活性污泥微生物群落结构和功能的新工具被引入 ,使我们对参与污水净化的微生物群落结构和优势菌群能有较全面的了解。就该方法在识别除磷污泥、脱氮污泥、污泥泡沫和膨胀污泥中微生物群落结构和功能的典型应用进行综述 ,分析了该方法存在的优点和缺点 ,并对目前已建立且应用于活性污泥微生物检测的 r RNA- targeted寡核苷酸探针进行了详细总结     

Sample handling and stability of hepatocyte growth factor in blood samples

As regards clinical studies performed on hepatocyte growth factor (HGF) during recent years, we have aimed in the present study to investigate the eventual differences in sample handling of this cytokine that might influence the results of serum concentrations. Venous blood from patients with current infectious diseases and controls was used in different sub-studies. Compared with samples separated within one hour, no significant changes in serum HGF levels were observed when whole blood stayed 4, or 24h at 6 degrees C before or 6h in room temperature after separation but HGF levels were significantly higher (P<0.01) when whole blood was kept at room temperature 4 and 24h before separation. Serum HGF was stable up to 20 freeze-thaw cycles. The serum concentrations of HGF were significantly higher than levels in the plasma (19%; P<0.05). A significant increase in serum HGF levels (12%, P<0.05) was observed after shaking the whole blood sample to a visible haemolysis, although the HGF concentration in blood cells was around half of that in serum. HGF tolerated storage at -70 degrees C for at least 4 months. We conclude that standardized methods in sample handling are important in the study of HGF concentrations in blood samples.     

Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk

AIMS: To assess the impact of chemical decontamination and refrigerated storage before culture on the recovery of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. METHODS AND RESULTS: Five-millilitre samples of ultra heat-treated (UHT) milk spiked with Myco. paratuberculosis NCTC 8578, B4 or 806R (ca 10(6) CFU ml(-1)) were heated at 63 degrees C for 20 or 30 min by submersion in a water bath. Heat-treated milk (0.5 ml) was cultured immediately into BACTEC 12B medium or refrigerated at 4 degrees C for 48 h before culture. Milk samples that received a 20-min heat treatment were also subjected to decontamination with 0.75% cetylpyridinium chloride (CPC) for 5 h at room temperature before inoculation into BACTEC 12B medium when tested immediately and after 48 h at 4 degrees C. BACTEC vials were monitored for evidence of growth over an 18-week incubation period at 37 degrees C. CPC decontamination resulted in a significant reduction in the number of culture-positive milk samples recovered immediately after heating (P < 0.05) and after refrigerated storage for 48 h (P < 0.01). Refrigerated storage for 48 h before testing did not have any significant effect, beneficial or detrimental, on Myco. paratuberculosis recovery rates. CONCLUSIONS: CPC decontamination applied to milk immediately or 48 h after heating will adversely affect the recovery of viable Myco. paratuberculosis, possibly leading to nonrecovery of the organism although viable cells are present in the original milk sample. SIGNIFICANCE AND IMPACT OF THE STUDY: Published pasteurization studies in which milk samples were decontaminated before culture will have underestimated the survival capability of Myco. paratuberculosis after high-temperature, short-time pasteurization. CPC decontamination should not be applied to pasteurized milk in future studies.     

Evaluation of group-specific, 16S rRNA-targeted scissor probes for quantitative detection of predominant bacterial populations in dairy cattle rumen

AIMS: To develop a suite of group-specific, rRNA-targeted oligonucleotide scissor probes for the quantitative detection of the predominant bacterial groups within the ruminal microbial community with the rRNA cleavage reaction-mediated microbial quantification method. METHODS AND RESULTS: Oligonucleotides that complement the conserved sites of the 16S rRNA of phylogenetically defined groups of bacteria that significantly contribute to the anaerobic fermentation of carbohydrates in ruminal ecosystems were selected from among published probes or were newly designed. For each probe, target-specific rRNA cleavage was achieved by optimizing the formamide concentration in the reaction mixture. The set of scissor probes was then used to analyse the bacterial community in the rumen fluids of four healthy dairy cows. In the rumen fluid samples, the genera Bacteroides/Prevotella and Fibrobacter and the Clostridium coccoides-Eubacterium rectale group were detected in abundance, accounting for 44-48%, 2.9-10%, and 9.1-10% of the total 16S rRNA, respectively. The coverage with the probe set was 71-78% of the total bacterial 16S rRNA. CONCLUSIONS: The probe set coupled with the sequence-specific small-subunit rRNA cleavage method can be used to analyse the structure of a ruminal bacterial community. SIGNIFICANCE AND IMPACT OF THE STUDY: The probe set developed in this study provides a tool for comprehensive rRNA-based monitoring of the community members that dominate ruminal ecosystems. As the ruminal microbial community can be perturbed, it is important to track its dynamics by analysing microbiological profiles under specific conditions. The method described here will provide a convenient approach for such tracking.     

Power analysis for real-time PCR quantification of genes in activated sludge and analysis of the variability introduced by DNA extraction

The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.     

Viruses in sewage: effect of phosphate removal with calcium hydroxide (lime).

During calcium hydroxide (lime) treatment (pH 9.6 to 10.5) of wastewaters for phosphate removal there was also a two-log removal of added poliovirus (type I, Sabin) from effluents. A similar virus reduction was seen in the sludge generated in these experiments. However, in view of the limitations of techniques for virus recovery from sludge, only a small portion of the infectious virus present in lime sludge may have been detected. Storage of lime sludge at 28 degrees C for up to 48 h produced no appreciable reduction in the virus titre. Five sets of field samples of sewage, effluents, and sludge from a sewage treatment plant (Kemptville, Ont.) which utilizes lime for phosphate removal were also examined for indigenous viruses being BS-C-1 cells. All of the sample of lime sludge and 80% of the samples of both sewage and lime-treated effluent revealed virus; after chlorination only 20% of the lime-treated effluent samples were positive for virus. In contrast, in an earlier study with essentially the same experimental set up, 76% of the sample of chlorinated primary effluent were found to contain virus. Because of the easily detectable quantities of infectious virus in lime sludge and due to the lack of virus inactivation during storage of such sludge, caution must be exercised in its handling and disposal.     

活性污泥微生物菌群研究方法进展

活性污泥是活性污泥法处理污水系统的功能主体。人类对活性污泥微生物菌群的认识随着其研究方法的发展而逐步深入。传统培养方法只能检测到活性污泥中1％～15％的微生物。随着一系列基于免培养的分子生物学技术的出现,活性污泥中菌群的复杂性和多样性以惊人的速度被人们认识,大量依靠传统检测方法未能发现却在活性污泥中起关键作用的微生物逐渐被发现。许多模拟活性污泥菌群生存环境条件的现代培养技术开始发展,且已成功培养了一部分传统培养方法不能培养的细菌类群,这为研究基于免培养方法发现的大量新的微生物菌群的生理特性和作用机制提供了可能,也无疑将把人们对活性污泥菌群的认识推向一个新的层次．主要介绍活性污泥微生物菌群研究的一系列方法,从传统培养方法到基于免培养的现代分子生物学技术,再到现代培养技术,着重论述了现代分子生物学技术及其在活性污泥微生物菌群研究中的进展。     

Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis

Aim: The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF). Methods: Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT‐PCR‐DGGE and qPCR analysis of the bacterial and fungal communities was carried out. Results: Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae. Conclusions: The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4°C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.     

Diversity and dynamics of Archaea in an activated sludge wastewater treatment plant

ABSTRACT: BACKGROUND: The activated sludge process is one of the most widely used methods for treatment of wastewater and the microbial community composition in the sludge is important for the process operation. While the bacterial communities have been characterized in various activated sludge systems little is known about archaeal communities in activated sludge. The diversity and dynamics of the Archaea community in a full-scale activated sludge wastewater treatment plant were investigated by fluorescence in situ hybridization, terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes. RESULTS: The Archaea community was specialized and dominated by Methanosaeta-like species. During a 15 month period major changes in the community composition were only observed twice despite seasonal variations in environmental and operating conditions. Water temperature appeared to be the process parameter that affected the community composition the most. Several terminal restriction fragments also showed strong correlations with sludge properties and effluent water properties. The Archaea were estimated to make up 1.6-% of total cell numbers in the activated sludge and were present both as single cells and colonies of varying sizes. CONCLUSIONS: The results presented here show that Archaea can constitute a constant and integral part of the activated sludge and that it can therefore be useful to include Archaea in future studies of microbial communities in activated sludge.     

Characterization of Thiobacillus thioparus isolated from an activated sludge bioreactor used for hydrogen sulfide treatment

AIMS: To compare Thiobacillus thioparus population dynamics in a control and a test activated sludge (AS) bioreactor, used for hydrogen sulfide (H(2)S) degradation. METHODS AND RESULTS: Denaturing gradient gel electrophoresis (DGGE) was used to confirm the presence of T. thioparus, and real-time PCR was used to quantify the level of this bacterium in the AS samples. The DGGE analysis showed a band for T. thioparus in all samples, with the band being more prominent in the test sample with H(2)S diffusion. It also showed that although a change occurred in the diversity of the microbial population in the test sludge after 6 weeks of H(2)S diffusion, the microbial community structure of the test and control was still similar. Thiobacillus thioparus-specific PCR primers confirmed that 50% of the isolates from both the test and control bioreactors were T. thioparus. The thiobacilli population became more efficient at degrading the diffused H(2)S. This increase in efficiency was confirmed by a significant increase in the number of isolates from the test sludge compared with those from the control sludge, when they were grown in a thiosulfate-rich liquid medium. CONCLUSIONS: It was concluded that the use of AS process for H(2)S removal encourages the population of T. thioparus to increase even at times when the total biomass concentration shows a decrease. SIGNIFICANCE AND IMPACT OF THE STUDY: The research results give an insight into the dynamics of the microbial population in an AS pilot plant used in a dual role, to treat the wastewater and H(2)S.     

Eco-physiological characterization of fluorescence in situ hybridization probe-targeted denitrifiers in activated sludge using culture-independent methods

AIMS: This study proposes the application of a culture-independent method [fluorescence in situ hybridization (FISH)] and a bioreactor operation control strategy to characterize environmental micro-organisms according to their survival strategies in a mixed suspension culture. Eco-physiological characteristics of two 16S rRNA probe-targeted denitrifiers (DEN581 and DEN124) were investigated against the availability of two resources. METHODS AND RESULTS: Four sequencing batch reactors were operated with manipulation of the sludge retention times to enforce limited and excess availability of two nutrients, namely acetate and nitrite, to the biomass. DEN581 FISH probe-targeted denitrifiers demonstrated dominance when the ratio of either acetate or nitrite to biomass was in excess, while DEN124-targeted organisms dominated when the above were limited. CONCLUSIONS: The study demonstrated that microbial populations in mixed cultures can be selected by changing the substrate availability (Rs) to biomass (X) ratio. The manipulation of the specific resource availability (Rs/X) determined which one of the studied probe-targeted denitrifiers (DEN124 or DEN581) became dominant. Rs/X provides a basis to study the physiology of micro-organisms that cannot be isolated in pure culture from activated sludge. SIGNIFICANCE AND IMPACT OF THE STUDY: The eco-physiological characterization of micro-organisms responsible for biological nutrient removal is anticipated to assist process designers and operators to optimize a specific biological process, such as denitrification.     

Fourier transform infrared spectroscopy as a metabolite fingerprinting tool for monitoring the phenotypic changes in complex bacterial communities capable of degrading phenol

The coking process produces great volumes of wastewater contaminated with pollutants such as cyanides, sulfides and phenolics. Chemical and physical remediation of this wastewater removes the majority of these pollutants; however, these processes do not remove phenol and thiocyanate. The removal of these compounds has been effected during bioremediation with activated sludge containing a complex microbial community. In this investigation we acquired activated sludge from an industrial bioreactor capable of degrading phenol. The sludge was incubated in our laboratory and monitored for its ability to degrade phenol over a 48 h period. Multiple samples were taken across the time‐course and analysed by Fourier transform infrared (FT‐IR) spectroscopy. FT‐IR was used as a whole‐organism fingerprinting approach to monitor biochemical changes in the bacterial cells during the degradation of phenol. We also investigated the ability of the activated sludge to degrade phenol following extended periods (2–131 days) of storage in the absence of phenol. A reduction was observed in the ability of the microbial community to degrade phenol and this was accompanied by a detectable biochemical change in the FT‐IR fingerprint related to cellular phenotype of the microbial community. In the absence of phenol a decrease in thiocyanate vibrations was observed, reflecting the ability of these communities to degrade this substrate. Actively degrading communities showed an additional new band in their FT‐IR spectra that could be attributed to phenol degradation products from the ortho‐ and meta‐cleavage of the aromatic ring. This study demonstrates that FT‐IR spectroscopy when combined with chemometric analysis is a very powerful high throughput screening approach for assessing the metabolic capability of complex microbial communities.     

Managing the excessive proliferation of glycogen accumulating organisms in industrial activated sludge by nitrogen supplementation: A FISH-NanoSIMS approach

Defluviicoccus vanus-related glycogen accumulating organisms (GAO) regularly proliferate in industrial wastewater treatment plants handling high carbon but nitrogen deficient wastes. When GAO dominate, they are associated with poor performance, characterised by slow settling biomass and turbid effluents. Although their ecophysiology has been studied thoroughly in domestic waste treatment plants, little attention has been paid to them in aerobic industrial systems.In this study, the effect of nitrogen addition on GAO carbon metabolism was investigated during an 8 h cycle. Activated sludge dominated by GAO from a winery wastewater sequencing batch reactor was incubated under different carbon to nitrogen (COD:N) ratios (100:1, 60:1 and 20:1) using ¹³C — acetate and ¹⁵N — urea. GAO cell assimilation was quantified using FISH-NanoSIMS. The activated sludge community was assessed by 16S rRNA gene profiling, DNA and storage polymer production. Carbon and nitrogen quantification at the cellular level by NanoSIMS revealed that low (COD:N of 100:1) or null nitrogen concentrations enhanced GAO carbon uptake. COD:N ratios of 60:1 and 20:1 reduced GAO carbon uptake and promoted whole microbial community DNA production. Nitrogen dosing at COD:N ratios of 60:1 or higher was demonstrated as feasible strategy for controlling the excessive GAO growth in high COD waste treatment plants.     

In situ visualization of high genetic diversity in a natural microbial community.

Simultaneous in situ visualization of seven distinct bacterial genotypes, all affiliated with the phylogenetically narrow group of beta-1 Proteobacteria, was achieved in activated sludge. This finding indicates that the high diversity found in the same sample by direct rRNA sequence retrieval was indeed present in this complex community. By the combination of comparative rRNA sequence analysis, in situ hybridization with fluorescently labeled, rRNA-targeted oligonucleotides and confocal laser scanning microscopy several microbial populations can be analyzed for abundance, relative spatial distribution and phylogeny directly at their site of action without prior cultivation.     

Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacterium complex, Gordona spp., and Gordona (Nocardia) amarae, respectively. The use of a universal base analog, 5-nitroindole, in oligonucleotide probe design was evaluated by comparing the characteristics of two different versions of the Mycobacterium complex probe. The temperature of dissociation of each probe was determined. Probe specificity studies with a diverse collection of 67 target and nontarget rRNAs demonstrated the specificity of the probes to the target groups. Whole-cell hybridizations with fluorescein- and rhodamine-labeled probes were performed with pure cultures of various members of the Mycobacterium complex as well as with environmental samples from a full-scale activated sludge plant which experienced foaming. Quantitative membrane hybridizations with activated sludge and anaerobic digester foam showed that 15.0 to 18.3% of the total small-subunit rRNAs could be attributed to members of the Mycobacterium complex, of which a vast majority consisted of Gordona rRNA. Several G. amarae strains made up only a very small percentage of the Gordona strains present. We demonstrated that group-specific rRNA probes are useful tools for the in situ monitoring and identification of filamentous bacteria in activated sludge systems.     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.