Methanogenic profiles by denaturing gradient gel electrophoresis using order-specific primers in anaerobic sludge digestion

In the present study, the diversity of methanogenic populations was monitored for 25 days, together with the process data for an anaerobic batch reactor treating waste-activated sludge. To understand this microbial diversity and dynamics, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted at two different taxonomic levels: the domain and order levels. The DGGE profiles of the domain Archaea and the three orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales were comparatively analyzed after each DGGE band was sequenced to enable identification. The DGGE profiles of the three orders showed methanogens belonging to each order that were not detected in the DGGE profile of the Archaea. This discrepancy may have resulted from PCR bias or differences in the abundances of the three microbial orders in the anaerobic bioreactor. In conclusion, to fully understand the detailed methanogenic diversity and dynamics in an anaerobic bioreactor, it is necessary to conduct DGGE analysis with 16S rRNA gene primers that target lower taxonomic groups.     

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.     

Biotransformation of nitrophenols in upflow anaerobic sludge blanket reactors

Four identical bench-scale upflow anaerobic sludge blanket (UASB) reactors, R1, R2, R3 and R4, were used to assess nitrophenols degradation at four different hydraulic retention times (HRT). Reactor R1 was used as control, whereas R2, R3, and R4 were fed with 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP), respectively. The concentration of each nitrophenol was gradually varied from 2 to 30 mg/l during acclimation. After acclimation reactors were operated under steady-state conditions at four different HRTs – 30, 24, 18, and 12 h, to study its effect on the removal of nitrophenols. Overall removal of 2-NP and 4-NP was always more than 99% but 2,4-DNP removal decreased from 96% to 89.7% as HRT was lowered from 30 to 12 h. 2-Aminophenol (2-AP), 4-aminophenol (4-AP) and 2-amino,4-nitrophenol (2-A,4-NP) were found to be the major intermediates during the degradation of 2-NP, 4-NP and 2,4-DNP, respectively. Out of the total input of nitrophenolic concentration (30 mg/l), on molar basis, about 41.2–48.4% of 2-NP, 59.4–68% of 4-NP, 30–26.6% of 2,4-DNP was recovered in the form of their respective amino derivatives at 30–12 h HRT. COD removal was 98–89%, 97–56%, 97–52%, and 94–46% at 30–12 h HRT for R1, R2, R3 and R4, respectively. Average cell growth was observed to be 0.15 g volatile suspended solid (VSS) per g COD consumed. Methanogenic inhibition was observed at lower HRTs (18 and 12 h), however denitrification was always more than 99% with non-detectable level of nitrite. The granules developed inside the reactors were black in color and their average size varied between 1.9 and 2.1 mm.     

Continuous dechlorination of tetrachloroethene in an upflow anaerobic sludge blanket reactor

Influences of hydraulic retention time (HRT) on dechlorination of tetrachloroethene (PCE) were investigated in an upflow anaerobic sludge blanket (UASB) reactor inoculated with anaerobic granular sludge non-pre-exposed to chlorinated compounds. PCE was introduced into the reactor at a loading rate of 3 mg/l d. PCE removal increased from 51 +/- 5% to 87 +/- 3% when HRT increased from 1 to 4 d, corresponding to an increase in the PCE biotransformation rate from 10.5 +/- 2.3 to 21.3 +/- 3.7 mumol/d. A higher ethene production rate, 0.9 +/- 0.2 mumol/d, was attained without accumulation of dichloroethenes at the HRT of 4 d. Dehalococcoides-like species were detected in sludge granules by fluorescence in situ hybridization, with signal strength in proportion to the extent of PCE dechlorination.     

Phototrophic bacterial production of oleic acid in an illuminated upflow anaerobic sludge blanket reactor

Phototrophic bacterial cells in the effluent from a lighted upflow anaerobic sludge blanket reactor supplied with a medium containing 142 mg S (as SO₄ ^2–) l^–1 accumulated a 6.8% w/w oleic acid content in cells and 19 mg cell-bound oleic acid l^–1 in the effluent. Pure cultures of Rhodopseudomonas palustris and Blastochloris sulfoviridis isolated from the effluent also accumulated 5.1 and 6.4% w/w oleic acid contents in cells, respectively. The oleic acid content in the cells recovered from the LUASB reactor effluent was related to the phototrophic bacterial population in the LUASB reactor. The inverse relationship was observed in the LUASB reactor between phototrophic bacterial growth and sulfate concentration in the influent.     

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).     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/ COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82% and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82%and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.     

应用DGGE研究微生物群落时的常见问题分析

变性梯度凝胶电泳(DGGE)是通过核酸片段对微生物群落进行研究,可以监测未培养细菌及其功能基因,被广泛地应用于微生物群落多样性和动态分析,并成为微生物分子生态学研究中的重要手段之一。文中论述了DGGE操作过程中遇到的常见问题,并提出了相应的解决方法。全面分析了样品预处理过程和PCR扩增效果对DGGE分析的影响,探讨了DGGE图谱的优化过程和图谱分析方法,并对DGGE的应用前景进行了综述。     

Anaerobic biodegradation of linear alkylbenzene sulfonate (LAS) in upflow anaerobic sludge blanket (UASB) reactors

The anaerobic biodegradation of Linear Alkylbenzene Sulfonate (LAS) was studied in Upflow Anaerobic Sludge Blanket Reactors (UASB). One reactor was fed with easily degradable substrates and commercial LAS solution during a period of 3 months (Reactor 1), meanwhile a second reactor was fed with a commercial LAS solution without co-substrate (Reactor 2) during 4 months. Both reactors were operated with an organic loading rate of 4–5 mg-LAS/l*day and a hydraulic retention time of one day.The LAS biodegradation was determined by full mass balance. LAS was analysed by HPLC in the liquid phase (influent and effluent streams of the reactors) as well as in the solid phase (granular sludge used as biomass). The results indicate a high level of removal (primary biodegradation: 64–85%). Biodegradation was higher in the absence of external co-substrates than in the presence of additional sources of carbon. This indicates that the surfactant can be partially used as carbon and energy source by anaerobic bacteria. Under the operating conditions used, inhibition of the methanogenic activity or any other negative effects on the biomass due to the presence of LAS were not observed. The methanogenic activity remained high and stable throughout the experiment.     

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.     

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.     

Characterisation of the bacterial community associated with early stages of Great Scallop (Pecten maximus), using denaturing gradient gel electrophoresis (DGGE)

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA was used to characterise and compare bacterial communities associated with scallop larvae (Pecten maximus), in different production units in a shellfish hatchery. Water and larvae samples were collected from three different aquaculture systems; stagnant, flow-through and a flow- through system with seawater treated with ozone. Samples were also collected from different algal cultures, inlet tanks and water pipes leading to the different aquaculture systems. Clear differences were seen between the bacterial community associated with the larvae and in the water from the different aquaculture systems. However, there were high similarities in the community composition between different water samples and between larvae samples collected at different time periods, indicating a high stability in the bacterial communities. Fifty three percent of the sequences from these samples were similar to 16S rRNA gene sequences of members of the gamma-subclass of the Proteobacteria. The different algal cultures had different bacterial communities, however 73 percent of the sequences were similar to 16S rRNA gene sequences of members of the alpha-subclass of the Proteobacteria. Differences in the DGGE profiles were also seen between the samples taken from the inlet tanks and water pipes, indicating a change in the bacterial community composition as the water passed through the pipes. To our knowledge this is the first study investigating bacterial communities associated with Great Scallop larvae in different aquaculture systems including noncultured components.     

Survival of a bacterial/archael consortium on building materials as revealed by molecular methods

The analysis of microbial communities using molecular techniques has become a common method to describe their components. In this study some building materials were inoculated with a microbial consortium of 14 microorganisms, subjected to alternate wetting/drying cycles for six months, and left to rest for eight years. At the end of the resting period, most members of the consortium were detected by molecular methods, which indicate a remarkable preservation of the DNA, although only one bacterium was able to grow in a culture medium. The experimental approach we carried out indicates that the list of microorganisms obtained from DNA analyses reflects not only the actual composition of the microbial communities, but is also a reflection of the microorganisms that were once active on the building materials.     

Effects of hydrogen and formate on the degradation of propionate and butyrate in thermophilic granules from an upflow anaerobic sludge blanket reactor.

Degradation of propionate and butyrate in whole and disintegrated granules from a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor fed with acetate, propionate, and butyrate as substrates was examined. The propionate and butyrate degradation rates in whole granules were 1.16 and 4.0 mumol/min/g of volatile solids, respectively, and the rates decreased 35 and 25%, respectively, after disintegration of the granules. The effect of adding different hydrogen-oxidizing bacteria (both sulfate reducers and methanogens), some of which used formate in addition to hydrogen, to disintegrated granules was tested. Addition of either Methanobacterium thermoautotrophicum delta H, a hydrogen-utilizing methanogen that does not use formate, or Methanobacterium sp. strain CB12, a hydrogen- and formate-utilizing methanogen, to disintegrated granules increased the degradation rate of both propionate and butyrate. Furthermore, addition of a thermophilic sulfate-reducing bacterium (a Desulfotomaculum sp. isolated in our laboratory) to disintegrated granules improved the degradation of both substrates even more than the addition of methanogens. By monitoring the hydrogen partial pressure in the cultures, a correlation between the hydrogen partial pressure and the degradation rate of propionate and butyrate was observed, showing a decrease in the degradation rate with increased hydrogen partial pressure. No significant differences in the stimulation of the degradation rates were observed when the disintegrated granules were supplied with methanogens that utilized hydrogen only or hydrogen and formate. This indicated that interspecies formate transfer was not important for stimulation of propionate and butyrate degradation.     

Bacterial population changes in a membrane bioreactor for graywater treatment monitored by denaturing gradient gel electrophoretic analysis of 16S rRNA gene fragments

The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD(5)), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time.     

Phylogenetic relationships ofThiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genusThiomicrospira. To identify these ‘phylotypes’ in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three describedThiomicrospira species, viz.,Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate,Thiomicrospira sp. strain MA2-6. AllThiomicrospira species exceptTms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria.Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated withThiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivisionThiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence ofTms. crunogena. Identifying a dominant molecular isolate asTms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another ‘phylotype’ represented a newThiomicrospira species, phylogenetically in an intermediate position betweenTms. crunogena andTms. pelophila. The third ‘phylotype’ was identified as aDesulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.     

PCR-based dENATURING GRADIENT GEL ELECTROPHORETICtypification and identification of the microbial consortium present in Kefir grains

Kefir grains have a complex microbiological composition that makes it difficult to obtain an optimal and constant starter culture necessary for the production of a quality Kefir beverage. The microbes present in the grains have in the past been identified using traditional methods such as growth on selective media and morphological and physiological characteristics. The aim of this study was to typify and identify the complex microbial community present in mass cultured, traditionally cultured and Irish Kefir grains by PCR amplification of a variable part of the ribosomal RNA (rRNA) genes in Eubacteria and yeasts and resolving these PCR fragments by denaturing gradient gel electrophoresis (DGGE). Unique PCR-based DGGE fingerprints were obtained for the Eubacterial and yeast species present in the three different grain types. A part of the Eubacterial and yeast rRNA genes were sequenced and compared to sequences available on NCBI. The phylogenetic relatedness of the amplified and sequenced lactobacilli was determined. Different bands in the Eubacterial and the yeast DGGE profiles of the mass cultured grains were identified to species level. A DGGE marker was constructed providing a quick reference for the identification of the members of the Eubacterial microbial population in mass cultured Kefir grains.