David G. Weissbrodt

Mainstream partial nitritation and anammox: long-term process stability and effluent quality at low temperatures

The implementation of autotrophic anaerobic ammonium oxidation processes for the removal of nitrogen from municipal wastewater (known as “mainstream anammox”) bears the potential to bring wastewater treatment plants close to energy autarky. The aim of the present work was to assess the long-term stability of partial nitritation/anammox (PN/A) processes operating at low temperatures and their reliability in meeting nitrogen concentrations in the range of typical discharge limits below 2 mgNH4-N·L−1 and 10 mgNtot·L−1. Two main 12-L sequencing batch reactors were operated in parallel for PN/A on aerobically pre-treated municipal wastewater (21 ± 5 mgNH4-N·L−1 and residual 69 ± 19 mgCODtot·L−1) for more than one year, including over 5 months at 15 °C. The two systems consisted of a moving bed biofilm reactor (MBBR) and a hybrid MBBR (H-MBBR) with flocculent biomass. Operation at limiting oxygen concentrations (0.15–0.18 mgO2·L−1) allowed stable suppression of the activity of nitrite-oxidizing bacteria at 15 °C with a production of nitrate over ammonium consumed as low as 16% in the MBBR. Promising nitrogen removal rates of 20–40 mgN·L−1·d−1 were maintained at hydraulic retention times of 14 h. Stable ammonium and total nitrogen removal efficiencies over 90% and 70% respectively were achieved. Both reactors reached average concentrations of total nitrogen below 10 mgN·L−1 in their effluents, even down to 6 mgN·L−1 for the MBBR, with an ammonium concentration of 2 mgN·L−1 (set as operational threshold to stop aeration). Furthermore, the two PN/A systems performed almost identically with respect to the biological removal of organic micropollutants and, importantly, to a similar extent as conventional treatments. A sudden temperature drop to 11 °C resulted in significant suppression of anammox activity, although this was rapidly recovered after the temperature was increased back to 15 °C. Analyses of 16S rRNA gene-targeted amplicon sequencing revealed that the anammox guild of the bacterial communities of the two systems was composed of the genus “Candidatus Brocadia”. The potential of PN/A systems to compete with conventional treatments for biological nutrients removal both in terms of removal rates and overall effluent quality was proven.

Activity and growth of anammox biomass on aerobically pre-treated municipal wastewater.

Direct treatment of municipal wastewater (MWW) based on anaerobic ammonium oxidizing (anammox) bacteria holds promise to turn the energy balance of wastewater treatment neutral or even positive. Currently, anammox processes are successfully implemented at full scale for the treatment of high-strength wastewaters, whereas the possibility of their mainstream application still needs to be confirmed. In this study, the growth of anammox organisms on aerobically pre-treated municipal wastewater (MWWpre-treated), amended with nitrite, was proven in three parallel reactors. The reactors were operated at total N concentrations in the range 5–20 mgN∙L−1, as expected for MWW. Anammox activities up to 465 mgN∙L−1∙d−1 were reached at 29 °C, with minimum doubling times of 18 d. Lowering the temperature to 12.5 °C resulted in a marked decrease in activity to 46 mgN∙L−1∙d−1 (79 days doubling time), still in a reasonable range for autotrophic nitrogen removal from MWW. During the experiment, the biomass evolved from a suspended growth inoculum to a hybrid system with suspended flocs and wall-attached biofilm. At the same time, MWWpre-treated had a direct impact on process performance. Changing the influent from synthetic medium to MWWpre-treated resulted in a two-month delay in net anammox growth and a two to three-fold increase in the estimated doubling times of the anammox organisms. Interestingly, anammox remained the primary nitrogen consumption route, and high-throughput 16S rRNA gene-targeted amplicon sequencing analyses revealed that the shift in performance was not associated with a shift in dominant anammox bacteria (“Candidatus Brocadia fulgida”). Furthermore, only limited heterotrophic denitrification was observed in the presence of easily biodegradable organics (acetate, glucose). The observed delays in net anammox growth were thus ascribed to the acclimatization of the initial anammox population or/and the development of a side population beneficial for them. Additionally, by combining microautoradiography and fluorescence in situ hybridization it was confirmed that the anammox organisms involved in the process did not directly incorporate or store the amended acetate and glucose. In conclusion, these investigations strongly support the feasibility of MWW treatment via anammox.

The feasibility of automated online flow cytometry for in-situ monitoring of microbial dynamics in aquatic ecosystems.

Fluorescent staining coupled with flow cytometry (FCM) is often used for the monitoring, quantification and characterization of bacteria in engineered and environmental aquatic ecosystems including seawater, freshwater, drinking water, wastewater, and industrial bioreactors. However, infrequent grab sampling hampers accurate characterization and subsequent understanding of microbial dynamics in all of these ecosystems. A logic technological progression is high throughput and full automation of the sampling, staining, measurement, and data analysis steps. Here we assess the feasibility and applicability of automated FCM by means of actual data sets produced with prototype instrumentation. As proof-of-concept we demonstrate examples of microbial dynamics in (i) flowing tap water from a municipal drinking water supply network and (ii) river water from a small creek subject to two rainfall events. In both cases, automated measurements were done at 15-min intervals during 12–14 consecutive days, yielding more than 1000 individual data points for each ecosystem. The extensive data sets derived from the automated measurements allowed for the establishment of baseline data for each ecosystem, as well as for the recognition of daily variations and specific events that would most likely be missed (or miss-characterized) by infrequent sampling. In addition, the online FCM data from the river water was combined and correlated with online measurements of abiotic parameters, showing considerable potential for a better understanding of cause-and-effect relationships in aquatic ecosystems. Although several challenges remain, the successful operation of an automated online FCM system and the basic interpretation of the resulting data sets represent a breakthrough toward the eventual establishment of fully automated online microbiological monitoring technologies.

Assessment of bacterial and structural dynamics in aerobic granular biofilms

Aerobic granular sludge (AGS) is based on self-granulated flocs forming mobile biofilms with a gel-like consistence. Bacterial and structural dynamics from flocs to granules were followed in anaerobic-aerobic sequencing batch reactors (SBR) fed with synthetic wastewater, namely a bubble column (BC-SBR) operated under wash-out conditions for fast granulation, and two stirred-tank enrichments of Accumulibacter (PAO-SBR) and Competibacter (GAO-SBR) operated at steady-state. In the BC-SBR, granules formed within 2 weeks by swelling of Zoogloea colonies around flocs, developing subsequently smooth zoogloeal biofilms. However, Zoogloea predominance (37–79%) led to deteriorated nutrient removal during the first months of reactor operation. Upon maturation, improved nitrification (80–100%), nitrogen removal (43–83%), and high but unstable dephosphatation (75–100%) were obtained. Proliferation of dense clusters of nitrifiers, Accumulibacter, and Competibacter from granule cores outwards resulted in heterogeneous bioaggregates, inside which only low abundance Zoogloea (<5%) were detected in biofilm interstices. The presence of different extracellular glycoconjugates detected by fluorescence lectin-binding analysis showed the complex nature of the intracellular matrix of these granules. In the PAO-SBR, granulation occurred within two months with abundant and active Accumulibacter populations (56 ± 10%) that were selected under full anaerobic uptake of volatile fatty acids and that aggregated as dense clusters within heterogeneous granules. Flocs self-granulated in the GAO-SBR after 480 days during a period of over-aeration caused by biofilm growth on the oxygen sensor. Granules were dominated by heterogeneous clusters of Competibacter (37 ± 11%). Zoogloea were never abundant in biomass of both PAO- and GAO-SBRs. This study showed that Zoogloea, Accumulibacter, and Competibacter affiliates can form granules, and that the granulation mechanisms rely on the dominant population involved.

Linking bacterial population dynamics and nutrient removal in the granular sludge biofilm ecosystem engineered for wastewater treatment

Intensive nutrient removal from wastewater in anaerobic-aerobic systems using granular sludge should rely on optimal balances at biofilm and microbial ecology levels. This study targets the impacts of reactor characteristics and fluctuations in operation conditions on nutrient removal and bacterial community structures by means of microbial and numerical ecology methods. The dynamics of both predominant and accompanying populations were investigated with high resolution on temporal and phylogenetic scales in two reactors operated during 5 months with synthetic wastewater. Multivariate analyses highlighted significant correlations from process to microbial scales in the first reactor, whereas nitrification and phosphorus removal might have been affected by oxygen mass transfer limitations with no impact at population level in the second system. The bacterial community continuum of the first reactor was composed of two major antagonistic Accumulibacter-Nitrosomonas-Nitrospira and Competibacter-Cytophaga-Intrasporangiaceae clusters that prevailed under conditions leading to efficient P- (> 95%) and N-removal (> 65%) and altered P- (< 90%) and N-removal (< 60%), respectively. A third cluster independent of performances was dominated by Xanthomonadaceae affiliates that were on average more abundant at 25 °C (31 ± 5%) than at 20 °C (22 ± 4%). Starting from the physiological traits of the numerous phylotypes identified, a conceptual model is proposed as a base for functional analysis in the granular sludge microbiome and for future investigations with complex real wastewater.

Identification of trigger factors selecting for polyphosphate- and glycogen-accumulating organisms in aerobic granular sludge sequencing batch reactors

Nutrient removal performances of sequencing batch reactors using granular sludge for intensified biological wastewater treatment rely on optimal underlying microbial selection. Trigger factors of bacterial selection and nutrient removal were investigated in these novel biofilm systems with specific emphasis on polyphosphate- (PAO) and glycogen-accumulating organisms (GAO) mainly affiliated with Accumulibacter and Competibacter, respectively. In a first dynamic reactor operated with stepwise changes in concentration and ratio of acetate and propionate (Ac/Pr) under anaerobic feeding and aerobic starvation conditions and without wasting sludge periodically, propionate favorably selected for Accumulibacter (35% relative abundance) and stable production of granular biomass. A Plackett-Burman multifactorial experimental design was then used to screen in eight runs of 50 days at stable sludge retention time of 15 days for the main effects of COD concentration, Ac/Pr ratio, COD/P ratio, pH, temperature, and redox conditions during starvation. At 95% confidence level, pH was mainly triggering direct Accumulibacter selection and nutrient removal. The overall PAO/GAO competition in granular sludge was statistically equally impacted by pH, temperature, and redox factors. High Accumulibacter abundances (30-47%), PAO/GAO ratios (2.8-8.4), and phosphorus removal (80-100%) were selected by slightly alkaline (pH > 7.3) and lower mesophilic (<20 °C) conditions, and under full aeration during fixed 2-h starvation. Nitrogen removal by nitrification and denitrification (84-97%) was positively correlated to pH and temperature. In addition to alkalinity, non-limited organic conditions, 3-carbon propionate substrate, sludge age control, and phase length adaptation under alternating aerobic-anoxic conditions during starvation can lead to efficient nutrient-removing granular sludge biofilm systems.

Bacterial Selection during the Formation of Early-Stage Aerobic Granules in Wastewater Treatment Systems Operated Under Wash-Out Dynamics

Aerobic granular sludge is attractive for high-rate biological wastewater treatment. Biomass wash-out conditions stimulate the formation of aerobic granules. Deteriorated performances in biomass settling and nutrient removal during start-up have however often been reported. The effect of wash-out dynamics was investigated on bacterial selection, biomass settling behavior, and metabolic activities during the formation of early-stage granules from activated sludge of two wastewater treatment plants (WWTP) over start-up periods of maximum 60 days. Five bubble-column sequencing batch reactors were operated with feast-famine regimes consisting of rapid pulse or slow anaerobic feeding followed by aerobic starvation. Slow-settling fluffy granules were formed when an insufficient superficial air velocity (SAV; 1.8 cm s−1) was applied, when the inoculation sludge was taken from a WWTP removing organic matter only, or when reactors were operated at 30°C. Fast-settling dense granules were obtained with 4.0 cm s−1 SAV, or when the inoculation sludge was taken from a WWTP removing all nutrients biologically. However, only carbon was aerobically removed during start-up. Fluffy granules and dense granules were displaying distinct predominant phylotypes, namely filamentous Burkholderiales affiliates and Zoogloea relatives, respectively. The latter were predominant in dense granules independently from the feeding regime. A combination of insufficient solid retention time and of leakage of acetate into the aeration phase during intensive biomass wash-out was the cause for the proliferation of Zoogloea spp. in dense granules, and for the deterioration of BNR performances. It is however not certain that Zoogloea-like organisms are essential in granule formation. Optimal operation conditions should be elucidated for maintaining a balance between organisms with granulation propensity and nutrient removing organisms in order to form granules with BNR activities in short start-up periods.

Biological control of biofilms on membranes by metazoans.

Traditionally, chemical and physical methods have been used to control biofouling on membranes by inactivating and removing the biofouling layer. Alternatively, the permeability can be increased using biological methods while accepting the presence of the biofouling layer. We have investigated two different types of metazoans for this purpose, the oligochaete Aelosoma hemprichi and the nematode Plectus aquatilis. The addition of these grazing metazoans in biofilm-controlled membrane systems resulted in a flux increase of 50% in presence of the oligochaetes (Aelosoma hemprichi), and a flux increase of 119-164% in presence of the nematodes (Plectus aquatilis) in comparison to the control system operated without metazoans. The change in flux resulted from (1) a change in the biofilm structure, from a homogeneous, cake-like biofilm to a more heterogeneous, porous structure and (2) a significant reduction in the thickness of the basal layer. Pyrosequencing data showed that due to the addition of the predators, also the community composition of the biofilm in terms of protists and bacteria was strongly affected. The results have implications for a range of membrane processes, including ultrafiltration for potable water production, membrane bioreactors and reverse osmosis.

Effect of particulate organic substrate on aerobic granulation and operating conditions of sequencing batch reactors

The formation and application of aerobic granules for the treatment of real wastewaters still remains challenging. The high fraction of particulate organic matter (XS) present in real wastewaters can affect the granulation process. The present study aims at understanding to what extent the presence of XS affects the granule formation and the quality of the treated effluent. A second objective was to evaluate how the operating conditions of an aerobic granular sludge (AGS) reactor must be adapted to overcome the effects of the presence of XS. Two reactors fed with synthetic wastewaters were operated in absence (R1) or presence (R2) of starch as proxy for XS. Different operating conditions were evaluated. Our results indicated that the presence of XS in the wastewater reduces the kinetic of granule formation. After 52 d of operation, the fraction of granules reached only 21% in R2, while in R1 this fraction was of 54%. The granules grown in presence of XS had irregular and filamentous outgrowths in the surface, which affected the settleability of the biomass and therefore the quality of the effluent. An extension of the anaerobic phase in R2 led to the formation of more compact granules with a better settling ability. A high fraction of granules was obtained in both reactors after an increase of the selection pressure for fast-settling biomass, but the quality of the effluent remained low. Operating the reactors in a simultaneous fill-and-draw mode at a low selection pressure for fast-settling biomass showed to be beneficial for substrate removal efficiency and for suppressing filamentous overgrowth. Average removal efficiencies for total COD, soluble COD, ammonium, and phosphate were 87 ± 4%, 95 ± 1%, 92 ± 10%, and 87 ± 12% for R1, and 72 ± 12%, 86 ± 5%, 71 ± 12%, and 77 ± 11% for R2, respectively. Overall our study demonstrates that the operating conditions of AGS reactors must be adapted according to the wastewater composition. When treating effluents that contain XS, the selection pressure should be significantly reduced.

PyroTRF-ID: a novel bioinformatics methodology for the affiliation of terminal-restriction fragments using 16S rRNA gene pyrosequencing data

BackgroundIn molecular microbial ecology, massive sequencing is gradually replacing classical fingerprinting techniques such as terminal-restriction fragment length polymorphism (T-RFLP) combined with cloning-sequencing for the characterization of microbiomes. Here, a bioinformatics methodology for pyrosequencing-based T-RF identification (PyroTRF-ID) was developed to combine pyrosequencing and T-RFLP approaches for the description of microbial communities. The strength of this methodology relies on the identification of T-RFs by comparison of experimental and digital T-RFLP profiles obtained from the same samples. DNA extracts were subjected to amplification of the 16S rRNA gene pool, T-RFLP with the HaeIII restriction enzyme, 454 tag encoded FLX amplicon pyrosequencing, and PyroTRF-ID analysis. Digital T-RFLP profiles were generated from the denoised full pyrosequencing datasets, and the sequences contributing to each digital T-RF were classified to taxonomic bins using the Greengenes reference database. The method was tested both on bacterial communities found in chloroethene-contaminated groundwater samples and in aerobic granular sludge biofilms originating from wastewater treatment systems.ResultsPyroTRF-ID was efficient for high-throughput mapping and digital T-RFLP profiling of pyrosequencing datasets. After denoising, a dataset comprising ca. 10′000 reads of 300 to 500 bp was typically processed within ca. 20 minutes on a high-performance computing cluster, running on a Linux-related CentOS 5.5 operating system, enabling parallel processing of multiple samples. Both digital and experimental T-RFLP profiles were aligned with maximum cross-correlation coefficients of 0.71 and 0.92 for high- and low-complexity environments, respectively. On average, 63±18% of all experimental T-RFs (30 to 93 peaks per sample) were affiliated to phylotypes.ConclusionsPyroTRF-ID profits from complementary advantages of pyrosequencing and T-RFLP and is particularly adapted for optimizing laboratory and computational efforts to describe microbial communities and their dynamics in any biological system. The high resolution of the microbial community composition is provided by pyrosequencing, which can be performed on a restricted set of selected samples, whereas T-RFLP enables simultaneous fingerprinting of numerous samples at relatively low cost and is especially adapted for routine analysis and follow-up of microbial communities on the long run.