Nitrogen removal characteristics analyzed with gas and microbial community in thermophilic aerobic digestion for piggery waste treatment.

In order to characterize the nitrogen conversion characteristics in a thermophilic aerobic digestion (TAD) system, a laboratory study has been conducted with the analysis of effluent gas and microbial community in the sludge samples. The lab TAD system was operated with HRT of 3 days and 60 degrees C. Based on the nitrogen mass balance, it has been found that about 2/3 of the daily load of nitrogen was converted to the gaseous form of nitrogen whereas cellular transformation and unmetabolized nitrogen accounted for about 1/3. Among the gaseous nitrogen transformation, significant amount of influent nitrogen had been converted to N2 gas (29% of influent N) and N2O (9% of influent N). Ammonia conversion was only 28% of influent N. The detection of N2O gas is a clear indication of the biological nitrogen reduction process in the thermophilic aerobic digester. No conclusive evidence for the existence of aerobic deammonification has been found. The microbial community analysis showed that thermophilic bacteria such as Bacillus thermocloacae, Bacillus sp. and Clostridial groups dominated in this TAD reactor. The diverse microbial community in TAD sludge may play an important role in removing both strong organics and nitrogen from piggery waste.     

Changes of microbial characteristics of retained sludge during low-temperature operation of an EGSB reactor for low-strength wastewater treatment.

In this study, a lab scale EGSB reactor was operated for 400 days to investigate the influence of temperature-decrease on the microbial characteristic of retained sludge. The EGSB reactor was started-up at 15 degrees C seeding with 20 degrees C-grown granular sludge. The influent COD of synthetic wastewater was set at 0.6-0.8 gCOD/L. The process-temperature was stepwise reduced from 15 degrees C to 5 degrees C during 400 days operation. Decrease of temperature of the reactor from 15 degrees C to 10 degrees C caused the decline of COD removal efficiency. However, continuous operation of the EGSB reactor led the efficient treatment of wastewater (70% of COD removal, 50% of methane recovery) at 10 degrees C. We confirmed that the both acetate-fed and hydrogen-fed methanogenic activities of retained sludge clearly increased under 15 to 20 degrees C. Changes of microbial profiles of methanogenic bacteria were analyzed by 16S rDNA-targeted DGGE analysis and cloning. It shows that genus Methanospirillum as hydrogen-utilizing methanogen proliferated due to low temperature operation of the reactor. On the other hand, genus Methanosaeta presented in abundance as acetoclastic-methanogen throughout the experiment.     

Microbial communities and their interactions in biofilm systems: an overview.

Several important advances have been made in the study of biofilm microbial populations relating to their spatial structure (or architecture), their community structure, and their dependence on physicochemical parameters. With the knowledge that hydrodynamic forces influence biofilm architecture came the realization that metabolic processes may be enhanced if certain spatial structures can be forced. An example is the extent of plasmid-mediated horizontal gene transfer in biofilms. Recent in situ work in defined model systems has shown that the biofilm architecture plays a role for genetic transfer by bacterial conjugation in determining how far the donor cells can penetrate the biofilm. Open channels and pores allow for more efficient donor transport and hence more frequent cell collisions leading to rapid spread of the genes by horizontal gene transfer. Such insight into the physical environment of biofilms can be utilized for bioenhancement of catabolic processes by introduction of mobile genetic elements into an existing microbial community. If the donor organisms themselves persist, bioaugmentation can lead to successful establishment of newly introduced species and may be a more successful strategy than biostimulation (the addition of nutrients or specific carbon sources to stimulate the authochthonous population) as shown for an enrichment culture of nitrifying bacteria added to rotating disk biofilm reactors using fluorescent in situ hybridization (FISH) and microelectrode measurements of NH4+, NO2-, NO3-, and O2. However, few studies have been carried out on full-scale systems. Bioaugmentation and bioenhancement are most successful if a constant selective pressure can be maintained favoring the promulgation of the added enrichment culture. Overall, knowledge gain about microbial community interactions in biofilms continues to be driven by the availability of methods for the rapid analysis of microbial communities and their activities. Molecular tools can be grouped into those suitable for ex situ and in situ community analysis. Non-spatial community analysis, in the sense of assessing changes in microbial populations as a function of time or environmental conditions, relies on general fingerprinting methods, like DGGE and T-RFLP, performed on nucleic acids extracted from biofilm. These approaches have been most useful when combined with gene amplification, cloning and sequencing to assemble a phylogenetic inventory of microbial species. It is expected that the use of oligonucleotide microarrays will greatly facilitate the analysis of microbial communities and their activities in biofilms. Structure-activity relationships can be explored using incorporation of 13C-labeled substrates into microbial DNA and RNA to identify metabolically active community members. Finally, based on the DNA sequences in a biofilm, FISH probes can be designed to verify the abundance and spatial location of microbial community members. This in turn allows for in situ structure/function analysis when FISH is combined with microsensors, microautoradiography, and confocal laser scanning microscopy with advanced image analysis.     

Variations in microbial community during nitrogen removal by in situ oxygen-enhanced indigenous nitrogen-removal bacteria

In this study, the enclosure system exhibited perfect nitrogen removal performance with in situ oxygen-enhanced indigenous aerobic denitrifying bacteria in an enclosure experiment. We explored changes in the microbial community during the nitrogen removal process using the MiSeq high-throughput sequencing technology. The results revealed a total of 7974 and 33653 operational taxonomic units (OTUs) for water and sediment systems, respectively, with 97% similarity. The OTUs were found to be affiliated with eight main phyla (Proteobacteria, Actinobacteria, Cyanobacteria, Bacteroidetes, Planctomycetes, Chloroflexi, Firmicutes, and Actinobacteria). The diversity of the enhanced system was found to be higher than that of the control system. Principal component analysis (PCA) revealed that significant spatial and temporal differences were exhibited in the microbial community during nitrogen removal in the enclosure experiment. Redundancy analysis (RDA) indicated that physical parameters (temperature, dissolved oxygen, and pH), nitrogen (total nitrogen and nitrate), functional genes (nirK and nirS), and dissolved organic carbon (DOC) were the most important factors affecting bacterial community function and composition. Lastly, the results suggested that the variation in the microbial community could be analyzed through the MiSeq high-throughput sequencing technology, which may provide technical support for future field tests.     

Ecological engineering of bioaugmentation from side-stream nitrification

Wastewater treatment relies on careful integration of environmental engineering with microbial ecology. This would seem to be particularly the case when attempting to enhance survivability of organisms introduced from outside the main-stream reactor, i.e. bioaugmentation. Molecular biology tools were utilised in this study to assist in understanding the mechanisms of successful bioaugmentation. Molecular fingerprinting showed that side-stream reactor configuration strongly influenced ammonia-oxidising bacteria (AOB) community structure. In both lab-scale and full-scale systems, AOB communities in the side-stream and main-stream were very similar. The experimental systems revealed that a PFR side-stream produced greater diversity of AOB than a CSTR side-stream in a PFR main-stream system, whereas the full-scale side-stream resulted in essentially an AOB monoculture. Phylogenetic analysis revealed less diversity than molecular fingerprinting perhaps due to biases in the cloning/transformation procedure.     

Anaerobic co-digestion of winery wastewater.

The operational performance of anaerobic batch reactors treating winery wastewater (WW) combined with waste activated sludge (WAS) in different proportions was investigated under mesophilic conditions. In these experiments it was shown that for anaerobic digestion of WW alone, methane production rate was lower than the rates achieved when WW and WAS were treated together. When WW was mixed with WAS at a concentration of 50% WW resulted in the highest methane production rates. A simplified anaerobic model was used to determine the main kinetic parameters; maximum COD reduction rate (q(DA)) and maximum methane generation rate (k(max)). The maximum values of q(DA) and k(max) were 16.50 kgCOD COD(-1) d(-1) and 14.34 kgCOD kgCOD(-1) d(-1), respectively.     

Analysis of community structures in anaerobic processes using a quantitative real-time PCR method.

The methanogenic community structures of four different anaerobic processes were characterized using a quantitative real-time PCR with group-specific primer and probe sets targeting the 16S rRNA gene (rDNA). The group specific primer and probe sets were developed and used to detect the orders Methanosarcinales, and the families Methanosarcinaceae and Methanosaetaceae. Two separate sets targeting the domains Archaea and Bacteria were also used. Each microbial population in different anaerobic processes was determined and the relative abundance in the system was compared with each other. Dominant methanogenic populations and the community structures in the processes were varied by hydraulic retention time and acetate concentration. This indicates that the real-time PCR method with the primer and probe sets is a promising tool to analyze community structures in anaerobic processes.     

Influence of dye type and salinity on aerobic decolorization of azo dyes by microbial consortium and the community dynamics

In this research, aerobic decolorization of different azo dyes by a microbial community was studied. The results showed that more than 80% of four azo dyes (100 mg/L) could be aerobically decolorized by the microbial consortium, however, the time needed was obviously different. Kinetic data indicated that the processes were well described by zero-order kinetics, and the chemical structures of dyes had obvious influence on decolorization rates. On the other hand, effects of salinity on decolorization were also investigated. There was still 40% dye removal for Acid Brilliant Red GR when the salinity increased to 250 g/L. And the microbial community structures with different salinity were detected by PCR-DGGE. It was shown that the same two bacteria were dominant in all decolorization systems, and some typical halophilic microorganisms were found under higher-salt conditions.     

Physical characteristics of a waste activated sludge: conditioning responses and correlations with a synthetic surrogate.

The efficient and economic management of waste activated sludge (WAS) requires a proper understanding of the sludge's material properties. Though there has been much study of WAS, an adequate linkage between its physical and chemical properties has been elusive. In particular, the conditioning and dewatering of WAS are expensive operations, and the addition of polymer to WAS leads to even more complex material behavior that is difficult to optimize. This paper reports on an extensive characterization of WAS, both with and without polymer conditioning. We combined the classical "jar test" approach with less conventional rheometric and electrokinetic measurements. In addition to the use of sampled WAS, a synthetic surrogate sludge was formulated, attempting to duplicate properties of WAS and allow more extensive characterization of a reproducible surrogate to WAS. Results with both the synthetic surrogate and WAS indicated that the traditional, electrokinetic, and rheological properties were related. However, the dose optima by rheometry were somewhat higher in both cases.     

Analysis of nitrifying bacterial communities in aerobic biofilm reactors with different DO conditions using molecular techniques

In order to assess the relationship between the dissolved oxygen (DO) concentration and the characteristics of nitrifying bacterial communities in an aerobic biofilm reactor, molecular techniques including denaturing gradient gel electrophoresis (DGGE)/cloning based on PCR targeting 16S rRNA and the amoA gene and fluorescence in situ hybridisation (FISH) were conducted. The D-1, D-2, D-3 and D-4 reactors with different DO concentrations (1, 3, 5 and 7 mg/L, respectively) were set up in the thermostat and acclimated. The optimal DO concentration with stable nitrification efficiency was above 5.0 mg/L. As was shown by the results of DGGE and cloning, the community of ammonia-oxidising bacteria (AOB) and the ratio of Nitrosomonas sp. changed only slightly despite their differing nitrification efficiencies. The results of FISH indicated that higher DO concentrations resulted in an increase in AOB and nitrite-oxidising bacteria (NOB), and a reduction in heterotrophic microorganisms. The INT-dehydrogenase activity (DHA) test demonstrated that the activity of AOB decreased with reductions in the DO concentration. This means that the DO concentration does not influence the community of AOB, but rather the activity of AOB. In the relationship between the attached biomass and the nitrification efficiency, only the active biomass affected the nitrification efficiencies.     

Microbial community characterization of an UASB treating increased organic loading rates of vitamin C biosynthesis wastewater

The microbial community of a mesophilic lab-scale upflow anaerobic sludge blanket (UASB) reactor treating vitamin C biosynthesis wastewater at gradually elevated organic loading rates (OLRs) was characterized using 16S rDNA-based polymerase chain reaction-DGGE (denatured gradient gel electrophoresis) analysis. The DGGE fingerprints suggested that the elevated OLRs did not cause any significant changes in the microbial community. The predominant bacterial bands were affiliated with the Firmicutes (Clostridiales, four bands), Proteobacteria (Deltaproteobacteria, six bands), Bacteroidetes, and Synergistetes, respectively. All the archaeal bands were very similar to already known methanogenic species: Methanobacterium formicicum (two bands), Methanomethylovorans hollandica (one band) and Methanosaeta concilli (two bands), which belonged to the divisions Methanobacteria and Methanomicrobia, respectively.     

Structure and microbial composition of nitrifying microbial aggregates and their relation to internal mass transfer effects.

This paper presents an analysis of the structure and microbial composition of nitrifying aggregates, formed as either flocs or granules, in sequencing batch reactors (SBR) operated with a high ammonium load. The structure and microbial community of the aggregates was determined by fluorescence in situ hybridisation (FISH). The aggregate structure and size was related to mass transfer limitations observed by measurements of OURs measured by either a titrimetric and off-gas analysis sensor (TOGA) or by microsensors. The FISH analysis showed that the spatial arrangement of the microbial consortia correlated well with the oxygen gradients inside the aggregates. In the larger aggregates, the ammonium- and nitrite-oxidising bacteria were mainly concentrated to the outer 100-200 microm, whereas in the floc system, the bacteria were distributed throughout the entire aggregate. This indicates that the internal mass transfer resistance is considerably larger when the aggregate size increases which is directly supported by TOGA measurements.     

Modelling the competition of planktonic and sessile aerobic heterotrophs for complementary nutrients in biofilm reactor.

A comprehensive, simplified microbial biofilm model was developed to evaluate the impact of bioreactor operating parameters on changes in microbial population abundance. Biofilm simulations were conducted using three special cases: fully penetrated, internal mass transfer resistance and external mass transfer resistance. The results of model simulations showed that for certain operating conditions, competition for growth limiting nutrients generated oscillations in the abundance of planktonic and sessile microbial populations. These oscillations resulted in the violation of the competitive exclusion principle where the number of microbial populations was greater than the number of growth limiting nutrients. However, the operating conditions which impacted microbial community diversity were different for the three special cases. Comparing the results of model simulations for dispersed-growth, biofilms and bioflocs showed that oscillations and microbial community diversity were a function of competition as well as other key features of the ecosystem. The significance of the current study is that it is the first to examine competition as a mechanism for controlling microbial community diversity in biofilm reactors.     

Dynamic changes in environment condition and microbial community structure in trench and flat seabed sediments of Tokyo Bay, Japan.

Dynamic changes in the chemical environment in the bottom of overlying water and microbial community structure in trench and flat seabed sediments were evaluated during summer and autumn in Tokyo Bay, Japan, to elucidate the response of microbial community changes as a consequence of dredging activity. Quinone profile analysis was performed to evaluate the changes in microbial community structure in the sediments. Bottom shape and location of each station affected the chemical environment of the overlying water. The trench bottom shape had longer anoxic conditions than the flat bottom shape. Nitrogen and phosphorus concentrations affected the microbial density in the sediment. During anoxic conditions, the ubiquinone/menaquinone ratio (UQ/MK) was less than unity and increased with rising dissolved oxygen (DO) concentrations. The dominant quinone species in the trench and flat seabed sediments were MK with 6 and 7 isoprene units (MK-6 and MK-7) and UQ with 8 and 9 isoprene units (UQ-8 and UQ-9). MK-6 and UQ-8 containing bacteria might have a great influence on the sulfur cycle of the aquatic ecosystem. While, MK-7 and UQ-9 containing bacteria correlated with the deposition of phototropic bacteria cells onto the seabed sediment. The trench bottom shape contained higher concentrations of MK-6, MK-7, UQ-8 and UQ-9, especially during summer.     

Syntrophic acetate oxidation in two-phase (acid-methane) anaerobic digesters

The microbial processes involved in two-phase anaerobic digestion were investigated by operating a laboratory-scale acid-phase (AP) reactor and analyzing two full-scale, two-phase anaerobic digesters operated under mesophilic (35 °C) conditions. The digesters received a blend of primary sludge and waste activated sludge (WAS). Methane levels of 20% in the laboratory-scale reactor indicated the presence of methanogenic activity in the AP. A phylogenetic analysis of an archaeal 16S rRNA gene clone library of one of the full-scale AP digesters showed that 82% and 5% of the clones were affiliated with the orders Methanobacteriales and Methanosarcinales, respectively. These results indicate that substantial levels of aceticlastic methanogens (order Methanosarcinales) were not maintained at the low solids retention times and acidic conditions (pH 5.2-5.5) of the AP, and that methanogenesis was carried out by hydrogen-utilizing methanogens of the order Methanobacteriales. Approximately 43, 31, and 9% of the archaeal clones from the methanogenic phase (MP) digester were affiliated with the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales, respectively. A phylogenetic analysis of a bacterial 16S rRNA gene clone library suggested the presence of acetate-oxidizing bacteria (close relatives of Thermacetogenium phaeum, 'Syntrophaceticus schinkii,' and Clostridium ultunense). The high abundance of hydrogen consuming methanogens and the presence of known acetate-oxidizing bacteria suggest that acetate utilization by acetate oxidizing bacteria in syntrophic interaction with hydrogen-utilizing methanogens was an important pathway in the second-stage of the two-phase digestion, which was operated at high ammonium-N concentrations (1.0 and 1.4 g/L). A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for syntrophic acetate oxidation and weak-acid inhibition adequately described the dynamic profiles of volatile acid production/degradation and methane generation observed in the laboratory-scale AP reactor. The model was validated with historical data from the full-scale digesters.     

The performance of the sludge pretreatment system with venturi tubes.

This research investigates the feasibility of the venturi cavitation system (VCS) for the sludge pretreatment to increase biodegradability. The performances of the VCS depended on the inclination angle of the venturi outlet, and better results obtained with 12 degrees than with 8 degrees or 15 degrees . Although it is energy efficient to use several venturies in series, the number of the venturies should be determined with detailed fluid dynamic calculations. The linear relationship between total solid (TS) concentration and the increases in soluble chemical oxygen demand (Delta SCOD) was observed for both wasted activated sludge (WAS) and primary sludge, which might be related to the better conditions for cavitation development at high TS concentrations even with higher viscosity. The VCS achieved better energy efficiency in terms of Delta SCOD/kJ compared to high-speed homogenizer (HSH). On the other hand, the VCS showed a similar energy efficiency for mixed sludge with 1.8% TS, but lower efficiency for WAS with 4% TS when compared to ultrasonic disintegration.     

Towards exposure of elusive metabolic mixed-culture processes: the application of metaproteomic analyses to activated sludge.

Protein expression is a direct reflection of specific microbial activities in any ecosystem. In order to assess protein expression in mixed microbial communities, the feasibility of applying proteomic techniques to activated sludge samples has recently been demonstrated. We report the application of metaproteomics to two activated sludges from a laboratory-scale sequencing batch reactor with dissimilar phosphorus removal performances. Fluorescence in situ hybridization (FISH) revealed that the sludge with good enhanced biological phosphorus removal performance (EBPR) was dominated by Betaproteobacteria (65% of EUBMIX binding cells) and gave positive signals for the Rhodocyclus-type PAO specific probe (59%). The non-EBPR sludge was dominated by tetrad-forming Alphaproteobacteria (75%). With regard to the proteomic investigation, 630 individual protein spots were matched across the replicate groups of the anaerobic and aerobic phases of the EBPR sludge with 9.4% of all spots being statistically different between the two phases. The non-EBPR metaproteomic maps exhibited 590 matched spots with 14.7% statistical differences between the two phases. Overall, the non-EBPR sludge expressed around 30% more significant differences than the EBPR sludge. The comparison of protein expression in the two sludges showed that their metaproteomes were substantially different and this was reflected in their microbial community structures and metabolic transformations.     

Determination of the microbial diversity of anaerobic-aerobic activated sludge by a novel molecular biological technique

Sequential anaerobic-aerobic batch reactors were maintained on acetate/peptone and two different P/total organic carbon ratios that select for microbial communities enriched for either glycogen-accumulating organisms (GAO) or polyphosphate-accumulating organism (PAO). The community profiles of the eubacterial population and gram-positive high G-C bacteria (HGC) were characterized and compared by determining the terminal restriction fragment length polymorphisms (T-RFLP) of 16S rDNA. The Hhal+Rsal digested 5′ T-RFLP patterns of the eubacterial 16S rDNA amplified from the GAO- and PAOenriched communities were made up with 12 and 14 rank-abundant fragments (i.e., ribotypes), respectively. Among those ribotypes detected in the GAO-enriched community, only seven were found in the PAO-enriched community. The HGC group could only account for no more than 6% and 17% of the eubacterial 16S rDNA amplified from the GAO- and PAO-enriched communities, respectively. Within the HGC community, at least 16 and 10 rank-abundant ribotypes were observed m the Mspl digested T-RFLP patterns of GAO- and PAO-enriched communities, respectively. Among those HGC ribotypes observed in both communities, only five were in common. These indicate that the enrichment processes leading to the establishment of GAO- and PAO-specific communities caused the dramatic difference and complexity in the microbial population.     

Analysis of effect of nicotine on microbial community structure in sediment using PCR-DGGE fingerprinting

Solid or liquid waste containing a high concentration of nicotine can pollute sediment in rivers and lakes, and may destroy the ecological balance if it is directly discharged into the environment without any treatment. In this study, the polymerase chain reaction(PCR) and denaturing gradient gel electrophoresis(DGGE) method was used to analyze the variation of the microbial community structure in the control and nicotinecontaminated sediment samples with nicotine concentration and time of exposure. The results demonstrated that the growth of some bacterial species in the nicotine-contaminated sediment samples was inhibited during the exposure. Some bacteria decreased in species diversity and in quantity with the increase of nicotine concentration or time of exposure, while other bacteria were enriched under the effect of nicotine, and their DGGE bands changed from undertones to deep colors. The microbial community structure, however, showed a wide variation in the nicotinecontaminated sediment samples, especially in the sediment samples treated with high-concentration nicotine. The Jaccard index was only 35.1%between the initial sediment sample and the sediment sample with a nicotine concentration of 0.030 mg/g after 28 d of exposure. Diversity indices showed that the contaminated groups had a similar trend over time. The diversity indices of contaminated groups all decreased in the first7 d after exposure, then increased until day 42. It has been found that nicotine decreased the diversity of the microbial community in the sediment.     

Population dynamics of chromate reducing bacteria in a bioreactor system developed for the treatment of chromate wastewater

The population dynamics of microbes was investigated in a bioreactor system for the treatment of chromate wastewater supplemented with chromate reducing bacteria Enterobacter cloacae strain HO-1. In this study, respiratory quinone profile and PCR techniques were used to analyse the microbial population. The change in microbial community structure and density was observed on the basis of the respiratory quinone profile since the predominant quinone of HO-1 is ubiquinone-8 (Q-8) and the amount of quinone is proportional to the cell mass the quantity of microbes and the Q-8 fraction of the total quinones were increased after cultivation with aeration, while long-term operation brought about a decrease in the fraction of Q-8. In addition, the amount of HO-1 was determined by the PCR amplification rate under conditions where the amplification was exponential to the reaction cycles. The cell density of HO-1 and total microbes determined from these procedures compared well with the numbers of HO-1 cells counted selectively using agar plates containing chromate.