Stability and reproducibility of low-temperature anaerobic biological wastewater treatment

The reproducibility of low-temperature anaerobic biological wastewater treatment trials was evaluated. Two identical anaerobic expanded granular sludge bed bioreactors were used to treat synthetic volatile fatty acid-based industrial wastewater under ambient conditions (18-20 degrees C) and to investigate the effect of various environmental perturbations on reactor performance and microbial community dynamics, which were assessed by chemical oxygen demand removal or effluent volatile fatty acid determination and terminal restriction fragment length polymorphism analysis, respectively. Methanogenic activity was monitored using specific methanogenic activity assays. Reactor performance and microbial community dynamics were each well replicated between Reactor 1 and Reactor 2. Archaeal dynamics, in particular, were associated with reactor operating parameters. Terminal restriction fragment length polymorphism data suggested dynamic acetoclastic and hydrogenophilic methanogenic populations and were in agreement with temporal specific methanogenic activity data. Putative psychrophilic populations were observed in anaerobic bioreactor sludge for the first time.     

Cultivation of low-temperature (15°C), anaerobic, wastewater treatment granules

Aims: Anaerobic sludge granules underpin high‐rate waste‐to‐energy bioreactors. Granulation is a microbiological phenomenon involving the self‐immobilization of several trophic groups. Low‐temperature anaerobic digestion of wastes is of intense interest because of the economic advantages of unheated bioenergy production technologies. However, low‐temperature granulation of anaerobic sludge has not yet been demonstrated. The aims of this study were to (i) investigate the feasibility of anaerobic sludge granulation in cold (15°C) bioreactors and (ii) observe the development of methanogenic activity and microbial community structure in developing cold granules. Methods and Results: One mesophilic (R1; 37°C) and two low‐temperature (R2 and R3, 15°C) laboratory‐scale, expanded granular sludge bed bioreactors were seeded with crushed (diameter <0·4 mm) granules and were fed a glucose‐based wastewater for 194 days. Bioreactor performance was assessed by chemical oxygen demand removal, biogas production, granule growth and temporal methanogenic activity. Granulation was observed in R2 and R3 (up to 33% of the sludge). Elevated hydrogenotrophic methanogenesis was observed in psychrophilically cultivated biomass, but acetoclastic methanogenic activity was also retained. Denaturing gradient gel electrophoresis of archaeal 16S rRNA gene fragments indicated that a distinct community was associated with developing and mature granules in the low‐temperature (LT) bioreactors. Conclusions: Granulation was observed at 15°C in anaerobic bioreactors and was associated with H₂/CO₂‐mediated methanogenesis and distinct community structure development. Significance and Impact of the Study: Granulation underpins high‐rate anaerobic waste treatment bioreactors. Most LT bioreactor trials have employed mesophilic seed sludge, and granulation <20°C was not previously documented.     

Microbial community structure and dynamics in anaerobic fluidized‐bed and granular sludge‐bed reactors: influence of operational temperature and reactor configuration

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.     

Assessment of anaerobic wastewater treatment failure using terminal restriction fragment length polymorphism analysis

AIMS: The suitability of genetic fingerprinting to study the microbiological basis of anaerobic bioreactor failure is investigated. METHODS AND RESULTS: Two laboratory-scale anaerobic expanded granular sludge bed bioreactors, R1 and R2, were used for the mesophilic (37 degrees C) treatment of high-strength [10 g chemical oxygen demand (COD) l(-1)] synthetic industrial-like wastewater over a 100-day trial period. A successful start up was achieved by both bioreactors with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading during the trial induced a reduction in the COD removal of R1, while R2 maintained satisfactory performance (COD removal >90%) throughout the experiment. Specific methanogenic activity measurements of biomass from both reactors indicated that the main route of methane production was hydrogenotrophic methanogenesis. Terminal restriction fragment length polymorphism (TRFLP) analysis was applied to the characterization of microbial community dynamics within the system during the trial. The principal differences between the two consortia analysed included an increased abundance of Thiovulum- and Methanococcus-like organisms and uncultured Crenarchaeota in R1. CONCLUSIONS: The results indicated that there was a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput fingerprinting techniques, such as TRFLP, have been demonstrated as practically relevant for biomonitoring of anaerobic reactor communities.     

Selective enrichment of Geobacter sulfurreducens from anaerobic granular sludge with quinones as terminal electron acceptors

A quinone-respiring, enrichment culture derived from methanogenic granular sludge was phylogenetically characterized by using a combined cloning-denaturing gradient gel electrophoresis (DGGE) method, which revealed that the consortium developed was dominated by a single microorganism: 97% related, in a sequence of 1520 base pairs, to Geobacter sulfurreducens. The enrichment culture could grow with acetate, formate or H₂ when humic acids, the humic model compound, anthraquinone-2,6-disulfonate (AQDS), or chelated Fe(III) was provided as a terminal electron acceptor. The occurrence of a humic acid- or quinone-respiring microorganism in the microbial community of a wastewater treatment system suggests that this type of microorganisms may play a potential role in anaerobic bioreactors treating humus-containing wastewaters.     

Performance and diversity of polyvinyl alcohol-degrading bacteria under aerobic and anaerobic conditions

Objectives

To compare the degradation performance and biodiversity of a polyvinyl alcohol-degrading microbial community under aerobic and anaerobic conditions.

Results

An anaerobic–aerobic bioreactor was operated to degrade polyvinyl alcohol (PVA) in simulated wastewater. The degradation performance of the bioreactor during sludge cultivation and the microbial communities in each reactor were compared. Both anaerobic and aerobic bioreactors demonstrated high chemical oxygen demand removal efficiencies of 87.5 and 83.6 %, respectively. Results of 16S rDNA sequencing indicated that Proteobacteria dominated in both reactors and that the microbial community structures varied significantly under different operating conditions. Both reactors obviously differed in bacterial diversity from the phyla Planctomycetes, Chlamydiae, Bacteroidetes, and Chloroflexi. Betaproteobacteria and Alphaproteobacteria dominated, respectively, in the anaerobic and aerobic reactors.

Conclusions

The anaerobic–aerobic system is suitable for PVA wastewater treatment, and the microbial genetic analysis may serve as a reference for PVA biodegradation.

     

Temporal microbial diversity changes in solvent-degrading anaerobic granular sludge from low-temperature (15 degrees C) wastewater treatment bioreactors

Anaerobic sludge granules were obtained from laboratory-scale anaerobic bioreactors used to treat pharmaceutical-like (methanol-, acetone- and propanol-contaminated) wastewater under low-temperature conditions (15 degrees C). The microbial diversity and diversity changes of the sludge samples were ascertained by applying 16S rRNA gene cloning and terminal restriction fragment length polymorphism (TRFLP) analyses, respectively, and using sludge samples from the inoculum, throughout and at the conclusion of the bioreactor trial. Data from genetic fingerprinting correlated well with those from physiological activity assays of the reactor biomass. Specifically, for example, TRFLP profiles indicated the dominance of hydrogenotrophic methanogens within the archaeal community, thus supporting the findings of specific methanogenic activity measurements. TRFLP data supported the hypothesis that the deviation between the replicated reactors, in terms of treatment efficiency, was associated with succession within the microbial communities present, and indicated that community development was linked to both operating temperature and wastewater composition. Fluorescence in situ hybridization (FISH) was also applied, to quantitatively assess the abundance of selected microbial groups, and revealed the underestimation of the abundance Methanosarcina by gene cloning analysis and demonstrated the spatial arrangement of these organisms within the architecture of the low-temperature solvent-degrading anaerobic biofilms.     

Reactor performance and microbial community of an EGSB reactor operated at 20 and 15°C

Aims:  To investigate the effects of low temperatures on the performance and microbial community of anaerobic wastewater treatment.
Methods and Results:  An expanded granular sludge bed (EGSB) reactor was employed to treat synthetic brewery wastewater at 20 and 15°C. Reactor performance was represented by chemical oxygen demand (COD) removal efficiency, while the microbial community was analysed using denaturing gradient gel electrophoresis (DGGE) and clone technology. When the hydraulic retention time (HRT) was maintained at 18 h, COD removal efficiencies above 85% were obtained at both 20 and 15°C, with influent COD concentrations up to 7300 and 4100 mg l⁻¹, respectively. At 15°C, the COD removal efficiency was more easily manipulated by increasing the influent COD concentration. DGGE and clone results for both temperatures revealed that Methanosaeta and Methanobacterium were two dominant methanogens, and that the majority of the eubacterial clones were represented by Firmicutes . When the temperature decreased from 20 to 15°C, both archaeal and eubacterial communities had higher diversity, and the proportion of Methanosaeta (acetate-utilizing methanogens) decreased markedly from 60·0% to 49·3%, together with an increase in proportions of hydrogen-utilizing methanogens (especially Methanospirillum ).
Conclusions:  The feasibility of psychrophilic anaerobic treatment of low and medium strength organic wastewaters was demonstrated, although lower temperature could significantly affect both reactor performance and the anaerobic microbial community.
Significance and Impact of the Study:  The findings enrich the theory involving the microbial community and the application of anaerobic treatment in a psychrophilic environment.     

Microbial activity and community structure in a lake sediment used for psychrophilic anaerobic wastewater treatment

Aims: The aim of the study was to investigate the feasibility of a continuous reactor for psychrophilic anaerobic wastewater treatment by using the sludge from cold natural environment. Methods and Results: Six sludge samples (S1–S6) were collected from different cold natural locations to select sludge with high anaerobic microbial activity under low temperatures. After a 225‐day incubation, the maximum specific methane production rate of a waterfowl lake sediment (S1) at 15°C (70·5 mLCH₄ gVSS^?1 day^?1) was much higher than all other samples. S1 was thus chosen as the seed sludge for the reactor treating synthetic brewery wastewater at 15°C, by immobilizing the micro‐organisms on polyurethane foam carriers. The chemical oxygen demand (COD) removal efficiency reached over 80% after 240‐day operation at an organic loading rate of 5·3 kg m^?3 day^?1, and significant enrichment of biomass was observed. Clone libraries of the microbial communities in the inoculum had high diversities for both archaea and bacteria. Along with a decrease in microbial community diversities, the dominant bacteria (79·5%) at the end of the operation represented the phylum Firmicutes, while the dominant archaeon (41·5%) showed a similarity of 98% with the psychrotolerant methanogen Methanosarcina lacustris. Conclusions: The possibility of using anaerobic micro‐organisms from cold environments in anaerobic wastewater treatment under psychrophilic conditions is supported by these findings. Significance and Impact of the Study: This study enriches the theory on microbial community and the application on anaerobic treatment of sludge from cold natural environments.     

Comparison of bacterial diversity in full scale anammox bioreactors operated under different conditions

Bacterial community structure of full‐scale anammox bioreactor is still mainly unknown. It has never been analyzed whether different anammox bioreactor configurations might result in the development of different bacterial community structures among these systems. In this work, the bacterial community structure of six full‐scale autotrophic nitrogen removal bioreactors located in The Netherlands and China operating under three different technologies and with different influent wastewater characteristics was studied by the means of pyrotag sequencing evaluation of the bacterial assemblage yielded a great diversity in all systems. The most represented phyla were the Bacteroidetes and the Proteobacteria, followed by the Planctomycetes. 14 OTUs were shared by all bioreactors, but none of them belonged to the Brocadiales order. Statistical analysis at OTU level showed that differences in the microbial communities were high, and that the main driver of the bacterial assemblage composition was different for the distinct phyla identified in the six bioreactors, depending on bioreactor technology or influent wastewater characteristics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1464–1472, 2015     

Microbial response to single‐cell protein production and brewery wastewater treatment

As global fisheries decline, microbial single‐cell protein (SCP) produced from brewery process water has been highlighted as a potential source of protein for sustainable animal feed. However, biotechnological investigation of SCP is difficult because of the natural variation and complexity of microbial ecology in wastewater bioreactors. In this study, we investigate microbial response across a full‐scale brewery wastewater treatment plant and a parallel pilot bioreactor modified to produce an SCP product. A pyrosequencing survey of the brewery treatment plant showed that each unit process selected for a unique microbial community. Notably, flow equalization basins were dominated by Prevotella, methanogenesis effluent had the highest levels of diversity, and clarifier wet‐well samples were sources of sequences for the candidate bacterial phyla of TM7 and BD1‐5. Next, the microbial response of a pilot bioreactor producing SCP was tracked over 1 year, showing that two different production trials produced two different communities originating from the same starting influent. However, SCP production resulted generally in enrichment of several clades of rhizospheric diazotrophs of Alphaproteobacteria and Betaproteobacteria in the bioreactor and even more so in the final product. These diazotrophs are potentially useful as the basis of a SCP product for commercial feed production.     

COD removal from expanded granular sludge bed effluent using a moving bed biofilm reactor and their microbial community analysis

The bioreactor performance of a moving bed biofilm reactor (MBBR) as post-treatment of expanded granular sludge bed (EGSB) effluent was investigated. Moreover, the microbial communities of the two bioreactors during different operation periods were studied. The MBBR was efficient for COD removal with the mean efficiency of 82.4%, and produced an effluent with high and stable quality against shock loading resulting from the low temperature applied to EGSB. The study indicates that the microbial community in the reactors could adapt to perturbations such as influent wastewater characteristics and operation temperature, which is beneficial to maintain efficient and stable COD removal in the combined EGSB-MBBR system. Archaeal 16S rRNA gene sequence analysis indicated the presence of Methanomethylovorans, Methanolinea, Methanoregula boonei, Methanosarcina barkeri, and Methanospirillum hungatei in the EGSB. Bacterial 16S rRNA gene sequence analysis indicated the presence of Runella limosa, Dokdonella, Sphaerotilus, Hydrogenophaga, and Pseudomonas in the MBBR. The EGSB-MBBR system established here could be used as an efficient option for organic matter removal, which holds a great potential in practical applications for nutrients (N and P) removal.     

Microbial diversity differences within aerobic granular sludge and activated sludge flocs

In this study, we investigated during 400 days the microbial community variations as observed from 16S DNA gene DGGE banding patterns from an aerobic granular sludge pilot plant as well as the from a full-scale activated sludge treatment plant in Epe, the Netherlands. Both plants obtained the same wastewater and had the same relative hydraulic variations and run stable over time. For the total bacterial population, a similarity analysis was conducted showing that the community composition of both sludge types was very dissimilar. Despite this difference, general bacterial population of both systems had on average comparable species richness, entropy, and evenness, suggesting that different bacteria were sharing the same functionality. Moreover, multi-dimensional scaling analysis revealed that the microbial populations of the flocculent sludge system moved closely around the initial population, whereas the bacterial population in the aerobic granular sludge moved away from its initial population representing a permanent change. In addition, the ammonium-oxidizing community of both sludge systems was studied in detail showing more unevenness than the general bacterial community. Nitrosomonas was the dominant AOB in flocculent sludge, whereas in granular sludge, Nitrosomonas and Nitrosospira were present in equal amounts. A correlation analysis of process data and microbial data from DGGE gels showed that the microbial diversity shift in ammonium-oxidizing bacteria clearly correlated with fluctuations in temperature.     

Microbial community analysis of a full-scale DEMON bioreactor

Full-scale applications of autotrophic nitrogen removal technologies for the treatment of digested sludge liquor have proliferated during the last decade. Among these technologies, the aerobic/anoxic deammonification process (DEMON) is one of the major applied processes. This technology achieves nitrogen removal from wastewater through anammox metabolism inside a single bioreactor due to alternating cycles of aeration. To date, microbial community composition of full-scale DEMON bioreactors have never been reported. In this study, bacterial community structure of a full-scale DEMON bioreactor located at the Apeldoorn wastewater treatment plant was analyzed using pyrosequencing. This technique provided a higher-resolution study of the bacterial assemblage of the system compared to other techniques used in lab-scale DEMON bioreactors. Results showed that the DEMON bioreactor was a complex ecosystem where ammonium oxidizing bacteria, anammox bacteria and many other bacterial phylotypes coexist. The potential ecological role of all phylotypes found was discussed. Thus, metagenomic analysis through pyrosequencing offered new perspectives over the functioning of the DEMON bioreactor by exhaustive identification of microorganisms, which play a key role in the performance of bioreactors. In this way, pyrosequencing has been proven as a helpful tool for the in-depth investigation of the functioning of bioreactors at microbiological scale.     

PCR-based denaturing gradient gel electrophoretic evaluation of changes in the non-methanogenic population of stressed upflow anaerobic sludge blanket granules

Summary The performance of upflow anaerobic sludge blanket (UASB) bioreactors is influenced by the composition of the substrate and the microbial species present in the granules. The aim of this study was to determine if a change in the structure of the non-methanogenic microbial community takes place when UASB brewery granules are subjected to the sudden addition of different carbon sources at different concentrations. A shift in the microbial community did occur when the granules were subjected to lactate medium. The granules that were stressed with glucose medium did not show changes in the microbial consortium regardless of the increase in the glucose concentrations. The polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) method was successfully applied to show changes in the structure of the microbes present in UASB granules that were cultivated under different environmental conditions.     

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.     

Bacterial community dynamics in long‐term operation of a pilot plant using aerobic granular sludge to treat pig slurry

Aerobic granular sludge represents an interesting approach for simultaneous organic matter and nitrogen removal in wastewater treatment plants. However, the information about microbial communities in aerobic granular systems dealing with industrial wastewater like pig slurry is limited. Herein, bacterial diversity and dynamics were assessed in a pilot scale plant using aerobic granular sludge for organic matter and nitrogen elimination from swine slurry during more than 300 days. Results indicated that bacterial composition evolved throughout the operational period from flocculent activated sludge, used as inoculum, to mature aerobic granules. Bacterial diversity increased at the beginning of the granulation process and then declined due to the application of transient organic matter and nitrogen loads. The operational conditions of the pilot plant and the degree of granulation determined the microbial community of the aerobic granules. Brachymonas, Zoogloea and Thauera were attributed with structural function as they are able to produce extracellular polymeric substances to maintain the granular structure. Nitrogen removal was justified by partial nitrification (Nitrosomonas) and denitrification (Thauera and Zoogloea), while Comamonas was identified as the main organic matter oxidizing bacteria. Overall, clear links between bacterial dynamics and composition with process performance were found and will help to predict their biological functions in wastewater ecosystems improving the future control of the process. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1212–1221, 2016     

中试厌氧氨氧化反应器的启动与调控

研究了中试厌氧氨氧化(Anaerobic ammonium oxidation,Anammox)反应器的启动性能。结果表明,以硝化反硝化污泥、短程硝化污泥、厌氧絮体污泥和厌氧颗粒污泥混合接种,经过255d的运行,可在常温下(5oC～27oC)成功启动中试Anammox反应器,反应器的基质氮去除速率可达1.30kg/(m3·d)。厌氧氨氧化是致碱反应,厌氧氨氧化成为反应器内的主导反应后,进水pH宜控制在厌氧氨氧化适宜范围的偏低水平(6.8左右)。亚硝酸盐既是Anammox菌的基质,也是抑制剂,控制进水亚硝酸盐浓度(13～36mg/L)有助于厌氧氨氧化反应。菌种是生物反应器的功能之源,向中试装置投加少量厌氧氨氧化污泥(投加比2%),可大大加速中试Anammox反应器的启动进程。     

Temperature dependent (37-15°C) anaerobic digestion of a trichloroethylene-contaminated wastewater

The impact of a trichloroethylene (TCE) contaminated wastewater on the microbial community structure of an anaerobic granular biomass at 15 °C compared to 37 °C was investigated. Four expanded granular sludge bed (EGSB) bioreactors (R1-R4) were employed in pairs at 37 and 15 °C. The influents of one of each pair were supplemented with increasing concentrations of TCE (max. 60 mg l⁻¹). At 37 °C, stable operation was maintained with 88% COD removal and >99% TCE removal at maximum influent TCE concentrations. R3 performance decreased at influent TCE concentration of 60 mg l⁻¹, although TCE removal rates of >97% were recorded. Archaeal community analysis via clone library and quantitative polymerase chain reaction (qPCR) analysis, and bacterial community analysis via denaturing gradient gel electrophoresis (DGGE), indicated that temperature resulted in a greater change in community structure than the presence of TCE, and clones related to cold adaptation of biomass were identified at 15 °C.     

Nitrification in hybrid bioreactors treating simulated domestic wastewater

Aim

To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors).

Methods and Results

Three bench‐scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5‐day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (>95%). However, when HRT was reduced to 2·5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia‐oxidizing bacteria, AOB; nitrite‐oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB–NOB activity were less apparent, resulting in incomplete nitrification.

Conclusions

Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification.

Significance and Impact of the Study

This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.