厌氧生境体系中产氢产乙酸细菌的FISH定量解析

产氢产乙酸细菌是一类在有机物厌氧降解过程中起重要作用的细菌。以基于16S rRNA序列设计的特异性寡核苷酸探针为基础,优化FISH实验条件,确定该技术检测产氢产乙酸细菌的实验条件为样品固定19h、乙醇脱水5min,杂交缓冲液中甲酰胺浓度55%。运用建立的FISH技术检测了几种厌氧消化体系中产氢产乙酸细菌的数量,并与用传统MPN方法的结果进行了比较。结果表明,产氢产乙酸细菌分布广泛,废水处理UASB反应器和动物消化道,特别是反刍动物瘤胃中的产氢产乙酸细菌数量较高,其丰度分别为1.70×109 cells/mL样品,6.50×108 cells/mL样品。湖底沉积物中产氢产乙酸细菌数量较少,仅占整个微生物群落的0.4%,含量为1.20×108 cells/mL样品。     

Layered structure of granules in upflow anaerobic sludge blanket reactor gives microbial populations resistance to metal ions

Metal ions (Cd²⁺, Cu²⁺, Ni²⁺, Zn²⁺ and Cr³⁺) did not affect glucose degradation or the production of methane during anaerobic digestion with intact and disintegrated granules from a UASB (Upflow Anaerobic Sludge Blanket) reactor. However, when Cu²⁺ was at 500 mg g^–1 VSS (volatile suspended solids) in the media, the glucose degradation rates and methane production rates decreased by 14% and 32% in disintegrated granules, respectively, whereas, in intact granules, decreases were 3% and 14%, respectively. When various electroplating metal ions were tested, 50% inhibition of acetate degradation and methane production were produced by 210–770 mg g^–1 VSS and 120–630 mg g^–1 VSS, respectively. The relative toxicity of the electroplating metals on methane production was in the order of Zn²⁺ (most toxic) > Ni²⁺ > Cu²⁺ > Cr³⁺ > Cd²⁺ (least toxic).     

Characterization of microbial communities in anaerobic bioreactors using molecular probes

The microbial community structure of twenty-one single-phase and one two-phase full-scale anaerobic sewage sludge digesters was evaluated using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens and sulfate-reducing bacteria. These probe results were interpreted in combination with results from traditional chemical analyses and metabolic activity assays. It was determined that methanogens in healthy mesophilic, single-phase sewage sludge digesters accounted for approximately 8–12% of the total community and thatMethanosarcinales andMethanomicrobiales constituted the majority of the total methanogen population.Methanobacteriales andMethanococcales played a relatively minor role in the digesters. Phylogenetic groups of mesophilic, Gram-negative sulfate-reducing bacteria were consistently present at significant levels:Desulfovibrio andDesulfobulbus spp. were the dominant sulfate-reducing populations,Desulfobacter andDesulfobacterium spp. were present at lower levels, andDesulfosarcina, Desulfococcus, andDesulfobotulus spp. were absent. Sulfate reduction by one or more of these populations played a significant role in all digesters evaluated in this study. In addition, sulfate-reducing bacteria played a role in favoring methanogenesis by providing their substrates. The analysis of the two-phase digester indicated that true phase separation was not accomplished: significant levels of active methanogens were present in the first phase. It was determined that the dominant populations in the second phase were different from those in the single-phase digesters.     

Biological hydrogen production in a UASB reactor with granules. I: Physicochemical characteristics of hydrogen-producing granules

Hydrogen-producing granules with an excellent settling ability were cultivated in an upflow anaerobic sludge blanket reactor treating a sucrose-rich synthetic wastewater. The physicochemical characteristics of granules were evaluated in this study. The mature granules had a diameter ranging from 1.0 to 3.5 mm and an average density of 1.036 +/- 0.005 g/mL, whereas they had good settling ability and a high settling velocity of 32-75 m/h. The low ratio of proteins/carbohydrates for the extracellular polymeric substances (EPS) in the granules suggests that carbohydrates rather than proteins, might play a more important role in the formation of the H(2)-producing granules. The contact angle of the mature granules, 54 +/- 2 degrees , was larger than that of the seed sludge (38 +/- 2 degrees ), indicating that the microbial cells in the H(2)-producing granules had higher hydrophobicity. The granules had fractal nature with a fractal dimension of 1.78. Their porosities were in the range of 0-0.70, and increased with increasing granule size. The ratios between the observed and predicted settling velocities by Stokes' law were in a range of 1.00-1.50, and the fluid collection efficiency of the granules ranged from 0 to 0.19, indicating that their permeabilities were lower and that there was little advective flow through their interior. Experimental results also suggest that molecular diffusion appeared to play an important role in the mass transfer through the H(2)-producing granules.     

Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-targeted oligonucleotide probes

The increasing significance of members of the genus Sphingomonas in biotechnological applications has led to an increased interest in the diversity, abundance and ecophysiological potential of this group of Gram-negative bacteria. This general focus provides a challenge to improve means for identification of sphingomonads; eg molecular genetic methods for rapid and specific detection could facilitate screening of new isolates. Here, fluorescently labeled oligonucleotide probes targeted against 16S rRNA were used to typify strains previously assigned to the genus. All 46 sphingomonads tested including type strains of 21 Sphingomonasspecies could be detected with a probe originally designed for the genus and all but one with a probe designed for the alpha-4 subgroup of the Proteobacteria. The two probes are suitable for direct detection of sphingomonads in pure and mixed cultures as well as in environmental samples of unknown composition. The probes were used to identify sphingomonads in situ in activated sludge samples. Sphingomonads were rather abundant accounting for about 5–10% of the total cells in municipal sludges. Distinct patterns in aggregation of the cells suggest that these organisms could be involved in the formation process of sludge flocs. Received 27 May 1999/ Accepted in revised form 22 August 1999     

Biosorption and biodegradation of pentachlorophenol (PCP) in an upflow anaerobic sludge blanket (UASB) reactor

In order to understand the fate of PCP in upflow anaerobic sludge blanket reactor (UASB) more completely, the sorption and biodegradation of pentachlorophenol (PCP) by anaerobic sludge granules were investigated. The anaerobic granular sludge degrading PCP was formed in UASB reactor, which was seeded with anaerobic sludge acclimated by chlorophenols. At the hydraulic retention time (HRT) of 20–22 h, and PCP loading rate of 200–220 mg l⁻¹ d⁻¹, UASB reactor exhibited good performance in treating wastewater which containing 170–180 mg l⁻¹ PCP and the PCP removal rate of 99.5% was achieved. Sequential appearance of tetra-, tri-, di-, and mono-chlorophenol was observed in the reactor effluent after 20 mg l⁻¹ PCP introduction. Sorption and desorption of PCP on the anaerobic sludge granules were all fitted to the Freundlich isotherm equation. Sorption of PCP was partly irreversible. The Freundlich equation could describe the behavior of PCP amount sorbed by granular sludge in anaerobic reactor reasonably well. The results demonstrated that the main mechanism leading to removal of PCP on anaerobic granular sludge was biodegradation, not sorption or volatization.     

Application of redox mediators to accelerate the transformation of reactive azo dyes in anaerobic bioreactors.

Azo dyes are nonspecifically reduced under anaerobic conditions but the slow rates at which reactive azo dyes are converted presents a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds. As quinones have been found to catalyze reductive transfers by acting as redox mediators, the application of anthraquinone-2,6-disulfonic acid (AQDS) during continuous anaerobic treatment of the reactive azo dye, Reactive Red 2 (RR2), was evaluated. A mixture of volatile fatty acids was used as the electron-donating primary substrate. Batch experiments demonstrated that AQDS could increase the first-order rate constant of RR2 reductive cleavage by one order of magnitude. In the continuous experiment, treatment of RR2 containing synthetic wastewater in a lab-scale upflow anaerobic sludge blanket (UASB) reactor yielded low dye removal efficiencies (<30%). Consequently, severe toxicity problems occurred, eventually resulting in almost complete inhibition of the methanogenic activity. Addition of catalytic concentrations of AQDS (19 microM) to the reactor influent caused an immediate increase in the dye removal efficiency and recovery of biological activity. Ultimately, RR2 removal efficiency stabilized at 88%, and higher AQDS loads resulted in higher RR2 removal efficiencies (up to 98% at 155 microM AQDS). Examination of the RR2 decolorizing properties of dye-adapted reactor sludge and of nonadapted reactor seed sludge revealed that RR2 decolorization was principally a biologically driven transfer of reducing equivalents from endogenous and added substrates to the dye. Hydrogen, added in bulk, was clearly the preferred electron donor. Bacteria that couple dye decolorization to hydrogen oxidation were naturally present in seed sludge. However, enrichment was required for the utilization of electrons from volatile fatty acids for dye reduction. The stimulatory effect of AQDS on RR2 decolorization by AQDS-unadapted sludge was mainly due to assisting the electron transfer from endogenous substrates in the sludge to the dye. The stimulatory effect of AQDS on RR2 decolorization by sludge from the AQDS-exposed reactor was, in addition, strongly associated with the transfer of electrons from hydrogen and acetate to the dye, probably due to enrichment of specialized AQDS-reducing bacteria.     

Anammox bacteria enrichment in upflow anaerobic sludge blanket (UASB) reactor

We investigated the anaerobic ammonium oxidation (anammox) reaction in a labscale upflow anaerobic sludge blanket (UASB) reactor. Our aim was to detect and enrich the organisms responsible for the anammox reaction using a synthetic medium that contained low concentrations of substrates (ammonium and nitrite). The reactor was inoculated with granular sludge collected from a full-scale anaerobic digestor used for treating brewery wastewater. The experiment was performed during 260 days under conditions of constant ammonium concentration (50 mg NH₄/⁺-N/L) and different nitrite concentrations (50∼150 mg NO₂-N/L). After 200 days, anammox activity was observed in the system. The microorganisms involved in this anammox reaction were identified as CandidatusB. Anammoxidans andK. Stuttgartiensis using fluorescencein situ hybridization (FISH) method.     

Anaerobic methanethiol degradation in upflow anaerobic sludge bed reactors at high salinity (> or =0.5 M Na(+))

The feasibility of anaerobic methanethiol (MT) degradation at elevated sodium concentrations was investigated in a mesophilic (30 degrees C) lab-scale upflow anaerobic sludge bed (UASB) reactor, inoculated with estuarine sediment originating from the Wadden Sea (The Netherlands). MT was almost completely degraded (>95%) to sulfide, methane and carbon dioxide at volumetric loading rates up to 37 mmol MT x L(-1) x day(-1), 0.5 M sodium (NaCl or NaHCO(3)) and between pH 7.3 and 8.4. Batch experiments revealed that inhibition of MT degradation started at sodium (both NaCl and NaHCO(3)) concentrations exceeding 0.8 M. Sulfide inhibited MT degradation already around 3 mM (pH 8.3).     

Biological hydrogen production in a UASB reactor with granules. II: Reactor performance in 3-year operation

The experiment was conducted to evaluate the performance of an upflow anaerobic sludge blanket (UASB) with granules for H(2) production from a sucrose-rich synthetic wastewater at various substrate concentrations (5.33-28.07 g-COD/L) and hydraulic retention times (HRTs) (3-30 h) for over 3 years. The kinetics of H(2) production was evaluated, and the sludge yield and endogenous decay coefficient of the H(2)-producing granules were estimated to be 0.334 g-VSS/g-COD and 0.004/h, respectively. Based on Gibbs free energy calculations, the formation thermodynamics of caproate, an important aqueous product, were analyzed. Experimental results show that the H(2) partial pressure in biogas decreased with increasing substrate concentration, but was not sensitive to the variation of HRT in a range of 6-22 h. The H(2) production rate increased with increasing substrate concentration, but decreased with increasing HRT. The H(2) yield was in the range of 0.49-1.44 mol-H(2)/mol-glucose. Acetate, butyrate, caporate, and ethanol were the main aqueous products in the reactor, and their concentrations were dependent on both substrate concentration and HRT. An elevated substrate concentration resulted in a shift of fermentation from butyrate- to caporate-type in the reactor and the formation of caproate was dependent on the H(2) partial pressure. The 3-year experimental results demonstrate that H(2) could be produced continuously and stably from the acidogenic-granule-based UASB reactor.     

Quantification of whole-cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells

The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis.     

A survey of the relative abundance of specific groups of cellulose degrading bacteria in anaerobic environments using fluorescence in situ hybridization

AIMS: The utility of fluorescence in situ hybridization (FISH) for detecting uncultured micro-organisms in environmental samples has been shown in numerous habitats. In this study a suite of three FISH probes for cellulolytic bacteria is described and their efficacy is demonstrated by quantifying the relative abundance of the target micro-organisms in a range of industrial biomass samples. METHODS AND RESULTS: The probes were designed from data derived from an artificial landfill leachate reactor study and 16S rRNA gene databases. The original biomass sample proved to be well described by the three probes targeting a total of 51% of the bacterial (EUBMIX targeted) cells in quantitative FISH experiments. CONCLUSIONS: Three probes were developed and applied to samples from a range of industrial digesters. The CSTG1244 probe, specific for organisms closely related to Clostridium stercorarium, were observed in the widest range of samples (7 of the 19 samples tested). The CTH216a FISH probe, specific for organisms closely related to Clostridium thermocellum, described the highest proportion of the bacterial population within any one sample (46% in an anaerobically digested sludge sample). Finally, the BCE216a probe, specific for organisms closely related to Bacteroides cellulosolvens, achieved the lowest level of hybridisation of the three probes tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the three groups of anaerobic cellulolytic micro-organisms were present in different bioreactors but at variable abundances ranging from low (where other organisms would have been responsible for cellulolysis) to high. We showed the potential of using group specific FISH probes and quantitative FISH in environmental studies. The utility of using newly designed FISH probes was demonstrated by their ability to detect and quantify the target bacterial groups in samples from a range of industrial wastewater digesters.     

Chromosomal domains of chimpanzee are diverged from human as revealed by in situ hybridization using human genomic probe

Utilization of repetitive DNA probes to assess the taxonomic affinity between related species has become the most powerful tool in evolutionary biology today. Consequently, tremendous strides have recently been made towards establishing the phylogenetic relationship of humans with chimpanzee. We employed human genomic proe (P5080 B.5) to identify the degree of divergence of chimpanzee genome from humans. A small protion of structurally distinct genomic areas in chimpanzee could be identified by fluorescencein situ hybridization (FISH) technique when compared to human DNA. The genomic divergence is confined mainly to the chromosomal ends in chimpanzee and may be an important phylogenetic characteristic in human evolution.     

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.     

应用荧光原位杂交技术检测康柏西普基因在CHO细胞染色体上的整合状态

CHO细胞是常用的哺乳动物表达工程细胞。外源基因整合至CHO细胞染色体后,在大规模蛋白质生产过程中,由于相关压力撤除,外源基因存在丢失的可能,因此有必要对其整合稳定性进行检测。康柏西普(conbercept)是一个能够特异性结合VEGF-A的各种异构体、VEGF-B以及PlGF,从而发挥抗血管生成活性的融合蛋白。康柏西普目前已在美国进入Ⅲ期临床试验。本文运用荧光原位杂交对康柏西普基因在CHO细胞的整合状态进行了检测,发现经过4和19次传代后,康柏西普基因依然能稳定整合在基因组上,并且呈现出3个特点：(1)分布在一条染色体上,而不是多条染色体上;(2)分布在较长的染色体上;(3)在同一染色体上有较多拷贝数。同时,荧光定量PCR结果证明基因拷贝数无明显改变,ELISA检测证明蛋白表达水平亦无明显改变。上述实验证明在经过19次传代以后,康柏西普基因仍然稳定整合在基因组中,并可活跃表达,为康柏西普大规模生产及产品质控提供了有力依据。     

Detection of Helicobacter pylori and determination of clarithromycin susceptibility using formalin-fixed, paraffin-embedded gastric biopsy specimens by fluorescence in situ hybridization

BACKGROUND: Clarithromycin resistance and poor compliance to therapy are often responsible for Helicobacter pylori eradication therapy failure. AIM: To evaluate fluorescence in situ hybridization (FISH) as a nonculture method to simultaneously detect H. pylori and to identify clarithromycin resistance. METHODS: Fifty-four patients with dyspepsia (17 male, 37 female subjects; mean age, 46.5; range, 21-78 years) were studied. Two antrum and corpus biopsies were taken from each patient. Positive rapid urease test (RUT) and histopathologic examinations defined H. pylori positivity. A total of 108 formalin-fixed paraffin-embedded gastric mucosal biopsies were examined retrospectively by the FISH (seaFAST H. pylori Combi-Kit) method. RESULTS: Forty-five patients (83.3%) were H. pylori positive and 43 (95.5%) were also positive by FISH. There were two false-positive FISH results. Fourteen patients (31.1%) had clarithromycin-susceptible strains, 4 (8.9%) resistant strains, and 27 (60%) both susceptible and resistant strains. CONCLUSION: FISH results correlated well with H. pylori infection and were able to identify clarithromycin-susceptible and -resistant strains. This technique will be helpful in determining the bacterial density and the success of treatment where clarithromycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.     

Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis

Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.