Cold adaptation and replicable microbial community development during long-term low-temperature anaerobic digestion treatment of synthetic sewage.
ABSTRACT: The development and activity of a cold-adapting microbial community was monitored during low-temperature anaerobic digestion (LtAD) treatment of wastewater. Two replicate hybrid anaerobic sludge bed-fixed-film reactors treated a synthetic sewage wastewater at 12°C, at organic loading rates of 0.25-1.0 kg chemical oxygen demand (COD) m-3 d-1, over 889 days. The inoculum was obtained from a full-scale anaerobic digestion reactor, which was operated at 37°C. Both LtAD reactors readily degraded the influent with COD removal efficiencies regularly exceeding 78% for both the total and soluble COD fractions. The biomass from both reactors was sampled temporally and tested for activity against hydrolytic and methanogenic substrates at 12°C and 37°C. Data indicated that significantly enhanced low-temperature hydrolytic and methanogenic activity developed in both systems. For example, the hydrolysis rate constant (k) at 12°C had increased 20-30-fold by comparison to the inoculum by day 500. Substrate affinity also increased for hydrolytic substrates at low temperature. Next generation sequencing demonstrated that a shift in a community structure occurred over the trial, involving a 1-log-fold change in 25 SEQS (OTU-free approach) from the inoculum. Microbial community structure changes and process performance were replicable in the LtAD reactors.
Project description:The anaerobic packed-bed (AP) and hybrid packed-bed (HP) reactors containing methanogenic microbial consortia were applied to treat synthetic soft drink wastewater, which contains polyethylene glycol (PEG) and fructose as the primary constituents. The AP and HP reactors achieved high COD removal efficiency (>95%) after 80 and 33 days of the operation, respectively, and operated stably over 2 years. 16S rRNA gene pyrotag analyses on a total of 25 biofilm samples generated 98,057 reads, which were clustered into 2,882 operational taxonomic units (OTUs). Both AP and HP communities were predominated by Bacteroidetes, Chloroflexi, Firmicutes, and candidate phylum KSB3 that may degrade organic compound in wastewater treatment processes. Other OTUs related to uncharacterized Geobacter and Spirochaetes clades and candidate phylum GN04 were also detected at high abundance; however, their relationship to wastewater treatment has remained unclear. In particular, KSB3, GN04, Bacteroidetes, and Chloroflexi are consistently associated with the organic loading rate (OLR) increase to 1.5 g COD/L-d. Interestingly, KSB3 and GN04 dramatically decrease in both reactors after further OLR increase to 2.0 g COD/L-d. These results indicate that OLR strongly influences microbial community composition. This suggests that specific uncultivated taxa may take central roles in COD removal from soft drink wastewater depending on OLR.
Project description:Various anaerobic starter seeds from different sources were investigated for their efficacies in treatment of different types of wastewater. Six combinations of starter seeds and wastewaters were selected out of 25 combination batch experiments and operated in semicontinuous reactors. It was noticed that the efficacies of various anaerobic starter seeds for biogas production from different types of wastewater in terms of reactor performance and stability were depended on wastewater characteristics and F/M ratio affecting microbial community and their microbial activities. However, exogenous starter seed can be used across different types of wastewater with or without acclimatization. Four reactors reached the targeted OLR of 2?kg COD/m3·d with high performance and stability except for concentrated rubber wastewater (RBw), even using high active starter seeds of cassava starch (CSs) and palm oil (POs). The toxic compounds in RBw such as ammonia and sulfate might also adversely affect methanogenic activity in CSsRBw and POsRBw reactors. DGGE analysis showed that propionate utilizers, Smithella propionica strain LYP and Syntrophus sp., were detected in all samples. For Archaea domain, methylotrophic, hydrogenotrophic, and acetoclastic methanogens were also detected. Syntrophic relationships were assumed between propionate utilizers and methanogens as acetate/H2 producers and utilizers, respectively.
Project description:Background:Food waste is a large bio-resource that may be converted to biogas that can be used for heat and power production, or as transport fuel. We studied the anaerobic digestion of food waste in a staged digestion system consisting of separate acidogenic and methanogenic reactor vessels. Two anaerobic digestion parameters were investigated. First, we tested the effect of 55 vs. 65 °C acidogenic reactor temperature, and second, we examined the effect of reducing the hydraulic retention time (HRT) from 17 to 10 days in the methanogenic reactor. Process parameters including biogas production were monitored, and the microbial community composition was characterized by 16S amplicon sequencing. Results:Neither organic matter removal nor methane production were significantly different for the 55 and 65 °C systems, despite the higher acetate and butyrate concentrations observed in the 65 °C acidogenic reactor. Ammonium levels in the methanogenic reactors were about 950 mg/L NH4+ when HRT was 17 days but were reduced to 550 mg/L NH4+ at 10 days HRT. Methane production increased from ~ 3600 mL/day to ~ 7800 when the HRT was decreased. Each reactor had unique environmental parameters and a correspondingly unique microbial community. In fact, the distinct values in each reactor for just two parameters, pH and ammonium concentration, recapitulate the separation seen in microbial community composition. The thermophilic and mesophilic digesters were particularly distinct from one another. The 55 °C acidogenic reactor was mainly dominated by Thermoanaerobacterium and Ruminococcus, whereas the 65 °C acidogenic reactor was initially dominated by Thermoanaerobacterium but later was overtaken by Coprothermobacter. The acidogenic reactors were lower in diversity (34-101 observed OTU0.97, 1.3-2.5 Shannon) compared to the methanogenic reactors (472-513 observed OTU0.97, 5.1-5.6 Shannon). The microbial communities in the acidogenic reactors were > 90% Firmicutes, and the Euryarchaeota were higher in relative abundance in the methanogenic reactors. Conclusions:The digestion systems had similar biogas production and COD removal rates, and hence differences in temperature, NH4+ concentration, and pH in the reactors resulted in distinct but similarly functioning microbial communities over this range of operating parameters. Consequently, one could reduce operational costs by lowering both the hydrolysis temperature from 65 to 55 °C and the HRT from 17 to 10 days.
Project description:We report, for the first time, extensive biologically mediated phosphate removal from wastewater during high-rate anaerobic digestion (AD). A hybrid sludge bed/fixed-film (packed pumice stone) reactor was employed for low-temperature (12°C) anaerobic treatment of synthetic sewage wastewater. Successful phosphate removal from the wastewater (up to 78% of influent phosphate) was observed, mediated by biofilms in the reactor. Scanning electron microscopy and energy dispersive X-ray analysis revealed the accumulation of elemental phosphorus (?2%) within the sludge bed and fixed-film biofilms. 4', 6-diamidino-2-phenylindole (DAPI) staining indicated phosphorus accumulation was biological in nature and mediated through the formation of intracellular inorganic polyphosphate (polyP) granules within these biofilms. DAPI staining further indicated that polyP accumulation was rarely associated with free cells. Efficient and consistent chemical oxygen demand (COD) removal was recorded, throughout the 732-day trial, at applied organic loading rates between 0.4 and 1.5 kg COD m(-3) d(-1) and hydraulic retention times of 8-24 h, while phosphate removal efficiency ranged from 28 to 78% on average per phase. Analysis of protein hydrolysis kinetics and the methanogenic activity profiles of the biomass revealed the development, at 12°C, of active hydrolytic and methanogenic populations. Temporal microbial changes were monitored using Illumina MiSeq analysis of bacterial and archaeal 16S rRNA gene sequences. The dominant bacterial phyla present in the biomass at the conclusion of the trial were the Proteobacteria and Firmicutes and the dominant archaeal genus was Methanosaeta. Trichococcus and Flavobacterium populations, previously associated with low temperature protein degradation, developed in the reactor biomass. The presence of previously characterized polyphosphate accumulating organisms (PAOs) such as Rhodocyclus, Chromatiales, Actinobacter, and Acinetobacter was recorded at low numbers. However, it is unknown as yet if these were responsible for the luxury polyP uptake observed in this system. The possibility of efficient phosphate removal and recovery from wastewater during AD would represent a major advance in the scope for widespread application of anaerobic wastewater treatment technologies.
Project description:Methanogenic community structure and dynamics were investigated in two different, replicated anaerobic wastewater treatment reactor configurations [inverted fluidized bed (IFB) and expanded granular sludge bed (EGSB)] treating synthetic dairy wastewater, during operating temperature transitions from 37°C to 25°C, and from 25°C to 15°C, over a 430-day trial. Non-metric multidimensional scaling (NMS) and moving-window analyses, based on quantitative real-time PCR data, along with denaturing gradient gel electrophoresis (DGGE) profiling, demonstrated that the methanogenic communities developed in a different manner in these reactor configurations. A comparable level of performance was recorded for both systems at 37°C and 25°C, but a more dynamic and diverse microbial community in the IFB reactors supported better stability and adaptative capacity towards low temperature operation. The emergence and maintenance of particular bacterial genotypes (phylum Firmicutes and Bacteroidetes) was associated with efficient protein hydrolysis in the IFB, while protein hydrolysis was inefficient in the EGSB. A significant community shift from a Methanobacteriales and Methanosaetaceae towards a Methanomicrobiales-predominated community was demonstrated during operation at 15°C in both reactor configurations.
Project description:Implementation of partial nitritation anammox (PNA) in the mainstream (municipal wastewater treatment) is still under investigation. Microbial community structure and reactor type can influence the performance of PNA reactor; yet, little is known about the role of the community composition of the inoculum and the reactor configuration under mainstream conditions. Therefore, this study investigated the community structure of inocula of different origin and their consecutive community dynamics in four different lab-scale PNA reactors with 16S rRNA gene amplicon sequencing. These reactors were operated for almost 1 year and subjected to realistic seasonal temperature fluctuations as in moderate climate regions, that is, from 20°C in summer to 10°C in winter. The sequencing analysis revealed that the bacterial community in the reactors comprised: (1) a nitrifying community (consisting of anaerobic ammonium-oxidizing bacteria (AnAOB), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB)); (2) different heterotrophic denitrifying bacteria and other putative heterotrophic bacteria (HB). The nitrifying community was the same in all four reactors at the genus level, although the biomasses were of different origin. Community dynamics revealed a stable community in the moving bed biofilm reactors (MBBR) in contrast to the sequencing batch reactors (SBR) at the genus level. Moreover, the reactor design seemed to influence the community dynamics, and reactor operation significantly influenced the overall community composition. The MBBR seems to be the reactor type of choice for mainstream wastewater treatment.
Project description:BACKGROUND:Oleaginous microalgae contain a high level of lipids, which can be extracted and converted to biofuel. The lipid-extracted residue can then be further utilized through anaerobic digestion to produce biogas. However, long-chain fatty acids (LCFAs) have been identified as the main inhibitory factor on microbial activity of anaerobic consortium. In this study, the mechanism of LCFA inhibition on anaerobic digestion of whole and lipid-extracted algal biomass was investigated with a range of calcium concentrations against various inoculum to substrate ratios as a means to alleviate the LCFA inhibition. RESULTS:Whole algal biomass of Nannochloropsis salina represents high lipid content algal biomass while lipid-extracted residue represents its low lipid counterpart. The anaerobic digestion experiments were conducted in a series of serum bottles at 35 °C for 20 days. A kinetic model, considering LCFA inhibition on hydrolysis, acidogenesis as well as methanogenesis steps, was developed from the observed phenomenon of inhibition factors as a function of the LCFA concentration and specific biomass content or calcium concentration. The results showed that inoculum to substrate ratio had a stronger effect on biogas production than calcium, and calcium had no effect on biogas production when inoculum concentration was extremely low. The microbial community analysis by high-throughput Illumina Miseq sequencing indicated that diversity of both bacterial and methanogenic communities decreased with elevation of lipid concentration. Hydrolytic bacteria and aceticlastic methanogens dominated bacterial and archaea communities, respectively, in both high and low LCFA concentration digesters. CONCLUSIONS:This study demonstrated that inoculum concentration has a more significant effect on alleviating LCFA inhibition than calcium concentration, while calcium only played a role when inoculum concentration met a threshold level. The model revealed that each functional microbial group was subject to different levels of LCFA inhibition. Although methanogens were the most susceptible microbes to LCFA inhibition, the inhibition factor for hydrolytic bacteria was more highly affected by inoculum concentration. The microbial community analysis indicated that the bacterial community was affected more than the methanogenic community by high LCFAs concentration. Syntrophic acetogens were sensitive to high LCFA concentrations and thus showed a decreased abundance in such an environment. Graphical abstractProposed mechanism of calcium mitigated LCFA inhibition.
Project description:Gibberellin wastewater cannot be directly discharged without treatment due to its high concentrations of sulfate and organic compounds and strong acidity. Therefore, multi-stage anaerobic bioreactor + micro-aerobic+ anoxic/aeration (A/O) + biological contact oxidation combined processes are used to treat gibberellin wastewater. However, knowledge of the treatment effects of the A/O process and bacterial community structure in the aeration tank reactors of such systems is sparse. Therefore, this study was conducted to investigate the treatment effects and operation of the A/O process on gibberellin wastewater, as well as changes in the bacterial community structure of activated sludge in the aeration tank during treatment. Moreover, removal was examined based on evaluation of effluent after A/O treatment. Although influent chemical oxygen demand (COD), NH3-N and total phosphorus (TP) fluctuated, effluent COD, NH3-N and TP remained stable. Moreover, average COD, NH3-N and TP removal efficiency were 68.41%, 93.67% and 45.82%, respectively, during the A/O process. At the phylum level, Proteobacteria was the dominant phylum in all samples, followed by Chloroflexi, Bacteroidetes and Actinobacteria. Proteobacteria played an important role in the removal of organic matter. Chloroflexi was found to be responsible for the degradation of carbohydrates and Bacteroidetes also had been found to be responsible for the degradation of complex organic matters. Actinobacteria are able to degrade a variety of environmental chemicals. Additionally, Anaerolineaceae_uncultured was the major genus in samples collected on May 25, 2015, while Novosphingobium and Nitrospira were dominant in most samples. Nitrosomonas are regarded as the dominant ammonia-oxidizing bacteria, while Nitrospira are the main nitrite-oxidizing bacteria. Bacterial community structure varied considerably with time, and a partial Mantel test showed a highly significant positive correlation between bacterial community structure and DO. The bacterial community structure was also positively correlated with temperature and SO42-.
Project description:Anaerobic granulation at elevated salinities has been discussed in several analytical and engineering based studies. They report either enhanced or decreased efficiencies in relation to different Na+ levels. To evaluate this discrepancy, we focused on the microbial and structural dynamics of granules formed in two upflow anaerobic sludge blanket (UASB) reactors treating synthetic wastewater at low (5 g/L Na+) and high (20 g/L Na+) salinity conditions. Granules were successfully formed in both conditions, but at high salinity, the start-up inoculum quickly formed larger granules having a thicker gel layer in comparison to granules developed at low salinity. Granules retained high concentrations of sodium without any negative effect on biomass activity and structure. 16S rRNA gene analysis and Fluorescence in Situ Hybridization (FISH) identified the acetotrophic Methanosaeta harundinacea as the dominant microorganism at both salinities. Fluorescence lectin bar coding (FLBC) screening highlighted a significant shift in the glycoconjugate pattern between granules grown at 5 and 20 g/L of Na+, and the presence of different extracellular domains. The excretion of a Mannose-rich cloud-like glycoconjugate matrix, which seems to form a protective layer for some methanogenic cells clusters, was found to be the main distinctive feature of the microbial community grown at high salinity conditions.
Project description:We studied the peptide-degrading anaerobic communities of methanogenic reactors from two mesophilic full-scale modified upflow anaerobic sludge blanket (UASB) reactors treating brewery wastewater in Colombia. Most probable number (MPN) counts varied between 7.1 x 10(8) and 6.6 × 10(9) bacteria/g volatile suspended solids VSS (Methanogenic Reactor 1) and 7.2 × 10(6) and 6.4 × 10(7) bacteria/g (VSS) (Methanogenic Reactor 2). Metabolites detected in the highest positive MPN dilutions in both reactors were mostly acetate, propionate, isovalerate and, in some cases, negligible concentrations of butyrate. Using the highest positive dilutions of MPN counts, 50 dominant strains were isolated from both reactors, and 12 strains were selected for sequencing their 16S rRNA gene based on their phenotypic characteristics. The small-subunit rRNA gene sequences indicated that these strains were affiliated to the families Propionibacteriaceae, Clostridiaceae and Syntrophomonadaceae in the low G + C gram-positive group and Desulfovibrio spp. in the class ?-Proteobacteria. The main metabolites detected in the highest positive dilutions of MPN and the presence of Syntrophomonadaceae indicate the effect of the syntrophic associations on the bioconversion of these substrates in methanogenic reactors. Additionally, the potential utilization of external electron acceptors for the complete degradation of amino acids by Clostridium strains confirms the relevance of these acceptors in the transformation of peptides and amino acids in these systems.