Identification of important microbial populations in the mesophilic and thermophilic phenol-degrading methanogenic consortia

Active mesophilic and thermophilic phenol-degrading methanogenic consortia were obtained after an 18-month acclimation and enriching process in the serum bottles, and characterized using the rRNA-based molecular approach. As revealed by cloning, fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP), these two enrichments differed greatly in the community structures. The results for the first time suggest that group TA in the Deltaproteobacteria (88.0% of EUBmix FISH-detectable bacterial cell area) and Pelotomaculum spp. in the Desulfotomaculum family (81.2%) were the predominant fermentative bacteria under mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions, respectively. These populations closely associated with mesophilic and thermophilic members of Methanosaetaceae, Methanobacteriaceae and Methanomicrobiales to mineralize phenol as the sole carbon substrate to carbon dioxide and methane. Moreover, these two enrichments could mineralize terephthalate and benzoate. During benzoate degradation in the mesophilic enrichment, a shift in the predominant bacterial population from Deltaproteobacteria group TA to Syntrophus spp. was observed, suggesting Syntrophus-related spp. could have a higher substrate affinity for benzoate. FISH further revealed that member of the Deltaproteobacteria group TA represented more than 68.3% of EUBmix FISH-detectable bacterial cell area in a full-scale mesophilic bioreactor treating phenol-containing wastewaters.     

The impacts of cypermethrin pesticide application on the non-target microbial community of the pepper plant phyllosphere

Although pesticides have been extensively used for controlling insects and disease pathogens of plants, little is known regarding the impacts of applying these pesticides on the microbial community in the plant phyllosphere. Here, we report the effects of cypermethrin pesticide application upon the microbial community of the pepper plant phyllosphere. Assessments were made using culture-independent techniques including phospholipid fatty acid analysis (PLFA) and 16S rRNA gene directed Polymerase Chain Reaction with Denaturing Gradient Gel Electrophoresis (PCR-DGGE). During the 21 day greenhouse study, PLFA results indicated that both total and bacterial biomass increased after application of the pesticide. PLFA profiles also indicated that Gram-negative bacteria became predominant. DGGE analysis confirmed a significant change in bacterial community structure within the phyllosphere following the pesticide application where different dendrogram clusters were observed between control and treated samples. Phylogenetic analysis also suggested a change in bacterial phyla following treatment, where bands sequenced within control cultures were predominantly of the Firmicutes phylum, but those bands sequenced in the treated samples were predominantly members of the Bacteroidetes and γ-Proteobacteria phyla. In conclusion, this study revealed an increase in bacterial abundance and a shift in community composition within the pepper plant phyllosphere following the pesticide application, and highlighted the effective use of PLFA and PCR-DGGE for studying the effect of pesticides upon indigenous phyllosphere microbes.     

Bacterial community structures in MBRs treating municipal wastewater: relationship between community stability and reactor performance

Bacterial community structures in pilot-scale conventional membrane bioreactors (CMBRs) and hybrid MBRs (HMBRs) which were combined with pre-coagulation/sedimentation were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescence in situ hybridization (FISH) techniques. The results were compared with the community structure in a full-scale activated sludge (AS) process treating the same municipal wastewater. The Dice index (Cs) of similarity analysis of DGGE banding patterns demonstrated that the microbial community in AS was more similar to those in CMBR1 and CMBR2 than HMBR1 and HMBR2. This suggested that influent wastewater composition had a larger impact on bacterial community structures. Long-term community structure changes in the HMBRs and CMBRs were monitored and analyzed over 240 days by Non-metric multidimensional scaling (NMDS) analysis of DGGE banding patterns. The NMDS analysis revealed that both HMBRs and CMBRs had marked changes in community structures during the first about 100 days. Thereafter the perpetual fluctuations of bacterial community structures were observed in both HMBRs and CMBRs, even though the stable MBR performances (the performance was measured as membrane permeability and removal of dissolved organic carbon, DOC) were achieved. These results suggest that not only the stability, but also the adequate dynamics ("flexibility") of the bacterial community structure are important for the stable performance of the MBRs treating complex municipal wastewater.     

Microbial community dynamics during start-up of acidogenic anaerobic reactors

Start-up of two acidogenic reactors under mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions was carried out with methanogenic granular sludge as an inoculum and dairy wastewater as feed. During these 71 days of the start-up period, microbial community dynamics in these two acidogenic reactors, as monitored by denaturing gradient gel electrophoresis (DGGE) and dot-blot hybridization with group-specific oligonucleotide probes, was correlated to reactor performance. Due to pH drop to 5.5, DGGE community fingerprints for domains Bacteria and Archaea populations showed significant shifts after 13 days of operation, and this change was accompanied with an increase in volatile fatty acid production, a decrease in methane formation, and rapid sludge disintegration. Dot-blot hybridization results further indicated that the decrease in methane production was related to the decrease in Archaea population in particular with methanogens from 34.1% of total 16S-rRNA in the seed sludge to 8% within the first 13 days, and to 2-5% at day 71. Among the methanogens monitored, the class Methanomicrobiales was the most abundant followed up by Methanobacteriales and Methanococcales. Due to an elevated temperature, the microbial community change was more significant and rapid in the thermophilic reactor than in the mesophilic reactor. Significant microbial population changes took place at the first 13 days for both reactors, but a longer period up to 71 days was required to establish a microbial community with a stable metabolic activity.     

Microbial population dynamics during aerobic sludge granulation at different organic loading rates

Laboratory experiments were carried out to investigate the evolution of the bacterial community during aerobic sludge granulation. The experiments were conducted in three 2.4L sequencing batch reactors (SBRs) that were seeded with activated sludge and fed with glucose-based synthetic wastewater. Three different influent organic concentrations were introduced into the three SBRs, R1, R2 and R3, resulting in chemical oxygen demand (COD) loading rates of 1.5 (R1), 3.0 (R2) and 4.5 (R3)kg/m(3)d, respectively. Changes in bacterial diversity throughout the granulation process were monitored and analysed using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) techniques. The experimental results demonstrate that glucose-fed aerobic granules could be formed without significant presence of filamentous bacteria. Granules formed at different loading rates had different morphology, structural properties and bacterial species. A higher loading rate resulted in faster formation of larger and loose granules, while a lower loading rate resulted in slower formation of smaller and more tightly packed granules. The biomass underwent a dynamic transformation in terms of bacterial species richness and dominance during the granulation process. The reactor with the highest substrate loading rate had the lowest species diversity, while the reactor with the lowest substrate loading rate had the highest species diversity. Different dominant species of beta- and gamma-Proteobacteria and Flavobacterium within the granule communities from the three different SBRs were confirmed by analysis of 16S rDNA sequences of the PCR products separated by DGGE. It is apparent that a few common bacterial species play an important role in the formation and growth of aerobic granules and help sustain the granular sludge structure in the bioreactors.     

Phylogenetic analysis of the bacterial community in a full scale autothermal thermophilic aerobic digester (ATAD) treating mixed domestic wastewater sludge for land spread

The bacterial community associated with a full scale autothermal thermophilic aerobic digester (ATAD) treating sludge, originating from domestic wastewater and destined for land spread, was analysed using a number of molecular approaches optimised specifically for this high temperature environment. 16S rDNA genes were amplified directly from sludge with universally conserved and Bacteria-specific rDNA gene primers and a clone library constructed that corresponded to the late thermophilic stage (t = 23 h) of the ATAD process. Sequence analyses revealed various 16S rDNA gene sequence types reflective of high bacterial community diversity. Members of the bacterial community included α- and β-Proteobacteria, Actinobacteria with High G + C content and Gram-Positive bacteria with a prevalence of the Firmicutes (Low G + C) division (class Clostridia and Bacillus). Most of the ATAD clones showed affiliation with bacterial species previously isolated or detected in other elevated temperature environments, at alkaline pH, or in cellulose rich environments. Several phylotypes associated with Fe(III)- and Mn(IV)-reducing anaerobes were also detected. The presence of anaerobes was of interest in such large scale systems where sub-optimal aeration and mixing is often the norm while the presence of large amounts of capnophiles suggest the possibility of limited convection and entrapment of CO₂ within the sludge matrix during digestion. Comparative analysis with organism identified in other ATAD systems revealed significant differences based on optimised techniques. The abundance of thermophilic, alkalophilic and cellulose-degrading phylotypes suggests that these organisms are responsible for maintaining the elevated temperature at the later stages of the ATAD process.     

Isolation, identification and utilization of thermophilic strains in aerobic digestion of sewage sludge

Two representative thermophilic bacterial strains (T1 and T2) were isolated from a one-stage autothermal thermophilic aerobic digestion pilot-scale reactor. 16S rRNA gene analysis indicated that they were Hydrogenophilaceae and Xanthomonodaceae. These isolated strains were inoculated separately and/or jointly in sewage sludge, to investigate their effects on sludge stabilization under thermophilic aerobic digestion condition. Four digestion conditions were tested for 480 h. Digestion without inoculation and inoculation with strain T2, as well as joint- inoculation with strains T1 and T2, achieved 32.6%, 43.0%, and 38.2% volatile solids (VS) removal, respectively. Removal in a digester inoculated with stain T1 only reached 27.2%. For the first 144 h, the three inoculated digesters all experienced higher VS removal than the digester without inoculations. Both specific thermophilic strains and micro-environment significantly affected the VS removal. DGGE profiles revealed that the isolated strains T1 and T2 can successfully establish in the thermophilic digesters. Other viable bacteria (including anaerobic or facultative microbes) also appeared in the digestion system, enhancing the microbial activity.     

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

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

Characterization of microbial populations in pilot-scale fluidized-bed reactors treating perchlorate- and nitrate-laden brine

A salt-tolerant, perchlorate- and nitrate-reducing bacterial culture developed previously was used to inoculate two acetate-fed fluidized bed reactors (FBRs) which treated a 6% ion-exchange regenerant brine containing 500 ± 84 mg-N/L nitrate and 4.6 ± 0.6 mg/L perchlorate. The reactors were operated in series in continuous flow mode for 107 days after an acclimation period of 65 days. Pilot operation data suggest that complete denitrification was achieved after 70 days of operation, but significant perchlorate removal was not observed. Molecular analysis of the inoculum culture and biomass from the pilot plant samples using denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH) revealed that the composition of the biomass in the pilot-plant was evolving with time in each FBR. The total number of Azoarcus/Denitromonas decreased in the first reactor with time and position in the bioreactor during acclimation and operation. FISH analysis clearly revealed that the number of Halomonas which was the dominant nitrate-reducing organism increased in the first reactor. This indicates a shift towards nitrate reduction which corresponds to the operation data. Both DGGE and FISH demonstrated that the Azoarcus/Denitromonas was still present in the second bioreactor, which indicated that the removal of nitrate in the first reactor was allowing the perchlorate-reducing organisms to establish themselves in the second reactor. The study also suggests that FISH was more effective for analysis of the composition of these cultures and it would be a better tool for the routine monitoring of cultures.     

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

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

The inoculum effect on the ammonia-oxidizing bacterial communities in parallel sequential batch reactors

Three identical sequential batch reactors (SBRs) were each inoculated with sludge from a full-scale wastewater treatment plant (WWTP) treating a waste stream of different origin, i.e. a hospital, a meat processing company, and a municipal WWTP. The SBRs were run in parallel for 84 consecutive days to investigate whether the reactors would become more phylogenetically similar or stay separated concerning their functionality and microbial communities. Overall, the nitrification functionality was high throughout the experiment, and the size and structure of the sludge flocs were very similar. The total bacterial and ammonia-oxidizing bacterial (AOB) communities were analyzed by PCR-DGGE. Cluster analysis demonstrated very distinct bacterial communities in the three SBRs, not showing any trend becoming more similar. The carrying capacity, dynamics and functional organization of the communities were assessed by DGGE analysis and based on these patterns the range-weighted richness, moving window analysis, and constructing Pareto-Lorenz evenness distribution curves were calculated. Between the SBRs, highly comparable internal structure and dynamics of the AOB communities were observed, although they had only one AOB DGGE band in common. These observations indicate that community characteristics such as the extent of biodiversity and dynamics are more important indicators of good microbial functionality than the presence of certain specific species.     

Identification of microorganisms involved in reductive dehalogenation of chlorinated ethenes in an anaerobic microbial community

In this study, we report on phylogenetic and physiological characterization of an anaerobic culture capable of reductive dehalogenation of tetrachloroethene (PCE) obtained from a PCE-contaminated site. The culture was enriched using different combinations of electron donors (hydrogen and acetate) and electron acceptors (PCE, cis-1,2-dichloroethene (cDCE) and controls without chlorinated ethenes). The resulting subcultures were analyzed using three different approaches: chemical analysis to document conversion of chlorinated ethenes; polymerase chain reaction (PCR) of 16S rRNA gene fragments and denaturing gradient gel electrophoresis (DGGE) to compare community compositions; fluorescence in situ hybridization (FISH) to quantify specific groups of microorganisms using oligonucleotide probes previously designed or newly designed based on the sequences retrieved from sequence analysis of specific DGGE bands. Members of two genera which contain bacteria capable of reductive dehalogenation were detected in the culture: Dehalococcoides and Desulfitobacterium. The combined analyses suggested that Dehalococcoides-like bacteria are associated with complete dehalogenation of chlorinated ethenes to ethene with hydrogen as electron donor; and Desulfitobacterium-like bacteria, in contrast, are associated with incomplete PCE dehalogenation to cDCE and appear to be able to use acetate as electron donor. In addition, Sporomusa-like bacteria were identified, which most likely act as homoacetogens. The results demonstrated that combination of culture enrichment with different substrates, DGGE, and FISH allowed a detailed qualitative and quantitative characterization of the dominant microorganisms associated with reductive dehalogenation.     

Long-term effects of operating temperature and sulphate addition on the methanogenic community structure of anaerobic hybrid reactors

The diversity, population dynamics, and activity profiles of methanogens in anaerobic granular sludges from two anaerobic hybrid reactors treating a molasses wastewater both mesophilically (37 degrees C) and thermophilically (55 degrees C) during a 1081 day trial were determined. The influent to one of the reactors was supplemented with sulphate, after an acclimation period of 112 days, to determine the effect of competition with sulphate-reducing bacteria on the methanogenic community structure. Sludge samples were removed from the reactors at intervals throughout the operational period and examined by amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) and partial sequencing of 16S rRNA genes. In total, 18 operational taxonomic units (OTUs) were identified, 12 of which were sequenced. The methanogenic communities in both reactors changed during the operational period. The seed sludge and the reactor biomass sampled during mesophilic operation, both in the presence and absence of sulphate, was characterised by a predominance of Methanosaeta spp. Following temperature elevation, the dominant methanogenic sequences detected in the non-sulphate supplemented reactor were closely related to Methanocorpusculum parvum. By contrast, the dominant OTUs detected in the sulphate-supplemented reactor upon temperature increase were related to the hydrogen-utilising methanogen, Methanobacterium thermoautotrophicum. The observed methanogenic community structure in the reactors correlated with the operational performance of the reactors during the trial and with physiological measurements of the reactor biomass. Both reactors achieved chemical oxygen demand (COD) removal efficiencies of over 90% during mesophilic operation, with or without sulphate supplementation. During thermophilic operation, the presence of sulphate resulted in decreased reactor performance (effluent acetate concentrations of >3000 mg/l and biogas methane content of <25%). It was demonstrated that methanogenic conversion of acetate at 55 degrees C was extremely sensitive to inhibition by sulphide (50% inhibition at 8-17 mg/l unionised sulphide at pH 7.6-8.0), while the conversion of H(2)/CO(2) methanogenically was favoured. The combination of experiments carried out demonstrated the presence of specific methanogenic populations during periods of successful operational performance.     

Mesophilic and thermophilic activated sludge post-treatment of paper mill process water

Increasing system closure in paper mills and higher process water temperatures make the applicability of thermophilic treatment systems increasingly important. The use of activated sludge as a suitable thermophilic post-treatment system for anaerobically pre-treated paper process water from a paper mill using recycled wastepaper was studied. Two lab-scale plug flow activated sludge reactors were run in parallel for 6 months; a thermophilic reactor at 55 degrees C and a reference reactor at 30 degrees C. Both reactors were operated simultaneously at 20, 15 and 10 days SRT. The effects of temperature and SRT on sludge settleability and chemical oxygen demand (COD) removal efficiencies of different fractions were studied. Total COD removal percentages over the whole experimental period were 58+/-5% at 30 degrees C and 48 +/- 10% at 55 degrees C. The effect of the SRT on the total COD removal was negligible. Differences in total COD removal between both systems were due to a lesser removal of soluble and colloidal COD at 55 degrees C compared to the reference system. At 30 degrees C, colloidal COD removal percentages were 65+/-25%, 75+/-17% and 86+/-22% at 20, 15 and 10 days SRT, respectively. At 55 degrees C, these percentages were 48+/-34%, 40+/-28% and 70+/-25%, respectively. The effluent concentrations of colloidal COD in both systems were related to the influent concentration of colloidal material. The thermophilic sludge was not able to retain influent colloidal material as well as the mesophilic sludge causing a higher thermophilic effluent turbidity. Sludge settling properties were excellent in both reactor systems. These were neither temperature nor SRT dependent but were rather caused by extensive calcium precipitation in the aeration tanks creating a very dense sludge. For application in the board industry, a thermophilic in line treatment system seems feasible. The higher effluent turbidity is most likely offset by the energy gains of treatment under thermophilic conditions.     

Semi-continuous L-lactate fermentation of garbage without sterile condition and analysis of the microbial structure

In order to develop a simple L-lactate fermentation of organic wastes, acidogenic fermentation was investigated in semi-continuous culture at 55 degrees C with using unsterilized artificial garbage as feedstock. And, sludge from a thermophilic acidification reactor was inoculated at the start of the fermentation. The effects of pH and hydraulic retention time (HRT) on performance of L-lactate production were discussed in a series of operational conditions with regard to long-term stability. The best operational conditions for L-lactate fermentation are proposed to be 55 degrees C, pH 6 and HRT of 10 days to get the highest yield of lactate based on initial carbohydrate in the feedstock. The yield of 0.74 and produced L-lactate optical purity of 96.7% were obtained on these operational conditions. Another L-lactate fermentation, which were semi-continuous and batch mode operations, were conducted to present reproducibility of the fermentation. Microbial structures in the semi-continuous fermentations were analyzed with using 16S rDNA libraries, and Bacillus coagulans was shown to be the most predominant species in the L-lactate fermented cultural broth.     

Dynamic and distribution of ammonia-oxidizing bacteria communities during sludge granulation in an anaerobic-aerobic sequencing batch reactor

The structure dynamic of ammonia-oxidizing bacteria (AOB) community and the distribution of AOB and nitrite-oxidizing bacteria (NOB) in granular sludge from an anaerobic-aerobic sequencing batch reactor (SBR) were investigated. A combination of process studies, molecular biotechniques and microscale techniques were employed to identify and characterize these organisms. The AOB community structure in granules was substantially different from that of the initial pattern of the inoculants sludge. Along with granules formation, the AOB diversity declined due to the selection pressure imposed by process conditions. Denaturing gradient gel electrophoresis (DGGE) and sequencing results demonstrated that most of Nitrosomonas in the inoculating sludge were remained because of their ability to rapidly adapt to the settling-washing out action. Furthermore, DGGE analysis revealed that larger granules benefit more AOB species surviving in the reactor. In the SBR were various size granules coexisted, granule diameter affected the distribution range of AOB and NOB. Small and medium granules (d < 0.6 mm) cannot restrict oxygen mass transfer in all spaces of the sludge. Larger granules (d > 0.9 mm) can result in smaller aerobic volume fraction and inhibition of NOB growth. All these observations provide support to future studies on the mechanisms responsible for the AOB in granules systems.     

Evaluation of the impact of bioaugmentation and biostimulation by in situ hybridization and microelectrode

Three rotating disk biofilm reactors were operated to evaluate whether bioaugmentation and biostimulation can be used to improve the start-up of microbial nitrification. The first reactor was bioaugmented during start-up period with an enrichment culture of nitrifying bacteria, the second reactor received a synthetic medium containing NH(4)(+) and NO(2)(-) to facilitate concomitant proliferation of ammonia- and nitrite-oxidizing bacteria, and the third reactor was used as a control. To evaluate the effectiveness of bioaugmentation and biostimulation approaches, time-dependent developments of nitrifying bacterial community and in situ nitrifying activity in biofilms were monitored by fluorescence in situ hybridization (FISH) technique and microelectrode measurements of NH(4)(+), NO(2)(-), NO(3)(-), and O(2). In situ hybridization results revealed that addition of the enrichment culture of nitrifying bacteria significantly facilitated development of dense nitrifying bacterial populations in the biofilm shortly after, which led to a rapid start-up and enhancement of in situ nitrification activity. The inoculated bacteria could proliferate and/or survive in the biofilm. In addition, the addition of nitrifying bacteria increased the abundance of nitrifying bacteria in the surface of the biofilm, resulting in the higher nitrification rate. On the other hand, the addition of 2.1mM NO(2)(-) did not stimulate the growth of nitrite-oxidizing bacteria and did inhibit the proliferation of ammonia-oxidizing bacteria instead. Thus, the start-up of NO(2)(-) oxidation was unchanged, and the start-up of NH(4)(+) oxidation was delayed. In all the three biofilm reactors, data sets of time series analyses on population dynamics of nitrifying bacteria determined by FISH, in situ nitrifying activities determined by microelectrode measurements, and the reactor performances revealed an approximate agreement between the appearance of nitrifying bacteria and the initiation of nitrification activity, suggesting that the combination of these techniques was a very powerful monitoring tool to evaluate the effectiveness of bioaugmentation and biostimulation strategies.     

Monitoring thiocyanate-degrading microbial community in relation to changes in process performance in mixed culture systems near washout

Changes in microbial community structure, associated with changes in process performance, were investigated with respect to the sludge retention time (SRT) in bioreactors treating thiocyanate. Among the seven reactors operated at 0.8-3.0 d SRTs, respectively, the reactor at 2.0 d SRT displayed the maximal thiocyanate removal rate of 240.2mg/L/d. However, the thiocyanate removal efficiency suddenly decreased from 96.1% to 43.1% when the SRT was reduced from 2.0 to 1.8d, corresponding to a 50.1% drop in the removal rate. Microbial communities in the reactors operated at short SRTs, near washout, were analyzed by denaturing gradient gel electrophoresis (DGGE) based on bacterial 16S rRNA genes. All band sequences recovered were assigned to two phyla, Proteobacteria and Bacteriodetes. A Thiobacillus-like microorganism was commonly detected in all the reactors and is suggested to be the main organism responsible for thiocyanate decomposition. Several DGGE band sequences were closely related to the environmental clones detected in environments rich in sulfur and/or nitrogen compounds. Statistical analysis of the DGGE profiles demonstrated that the structure of thiocyanate-degrading communities, as well as the process performance, changed with change in SRT. The microbial community profiles were not always more closely related to those at similar SRT than those at less similar SRT on the non-metric multidimensional scaling (NMDS) map. This was also supported by clustering analysis. These results were contrary to the general notion that the community structures in continuous systems will be controlled by the washout of microbial populations. Our experimental results suggest that the structure of a microbial thiocyanate-degrading community at a given SRT would not be determined only by the washout effect.     

Microbiota in anaerobic digestion of sewage sludge with and without co‐substrates

In this study, seven full‐scale anaerobic digesters, with or without co‐substrate regime, were analysed by physicochemical and molecular biological methods. A combination of robust community fingerprinting and Illumina MiSeq sequencing revealed a core bacterial community dominated by Chloroflexi, Firmicutes, Bacteroidetes and Proteobacteria, with variations in the profiles because of differences in the co‐substrate feeding regime. Despite these differences, physicochemical properties revealed a stable performance of all reactors, indicating a resilient bacterial microbiota in all full‐scale reactors. A rich bacterial core community ensured reactor functionality, whilst feeding regime and reactor type impacted the overall and the core bacterial diversity. Within the Archaea, Methanosaeta dominated in all reactors. Results indicated no relationship between archaeal community structure and the type of co‐substrate digested. Methanogens rely on the metabolic end products of bacterial activity and are thus less dependent on differences in the initial co‐substrate regime.