Enriched Nitrifying Research Articles

Genome-resolved metagenomics reveals the nitrifiers enrichment and species succession in activated sludge under extremely low dissolved oxygen

Nitrification, a process carried out by aerobic microorganisms that oxidizes ammonia to nitrate via nitrite, is an indispensable step in wastewater nitrogen removal. To facilitate energy and carbon savings, applying low dissolved oxygen (DO) is suggested to shortcut the conventional biological nitrogen removal pathway, however, the impact of low DO on nitrifying communities within activated sludge is not fully understood. This study used genome-resolved metagenomics to compare nitrifying communities under extremely low- and high-DO. Two bioreactors were parallelly operated to perform nitrification and DO was respectively provided by limited gas-liquid mass transfer from the atmosphere (AN reactor, DO < 0.1 mg/L) and by sufficient aeration (AE reactor, DO > 5.0 mg/L). Low DO was thought to limit nitrifiers growth; however, we demonstrated that complete nitrification could still be achieved under the extremely low-DO conditions, but with no nitrite accumulation observed. Kinetic analysis showed that after long-term exposure to low DO, nitrifiers had a higher oxygen affinity constant and could maintain a relatively high nitrification rate, particularly at low levels of DO (<0.2 mg/L). Community-level gene analysis indicated that low DO promoted enrichment of nitrifiers (the genera Nitrosomonas and Nitrospira, increased by 2.3- to 4.3-fold), and also harbored with 2.3 to 5.3 times higher of nitrification functional genes. Moreover, 46 high-quality (>90 % completeness and <5 % contamination) with 3 most abundant medium-quality metagenome-assembled genomes (MAGs) were retrieved using binning methods. Genome-level phylogenetic analysis revealed the species succession within nitrifying populations. Surprisingly, compared to DO-rich conditions, low-DO conditions were found to efficiently suppressed the ordinary heterotrophic microorganisms (e.g., the families Anaerolineales, Phycisphaerales, and Chitinophagales), but selected for the specific candidate denitrifiers (within phylum Bacteroidota). This study provides new microbial insights to demonstrate that low-DO favors the enrichment of autotrophic nitrifiers over heterotrophs with species-level successions, which would facilitate the optimization of energy and carbon management in wastewater treatment.

Treatment of Anaerobic Digester Liquids via Membrane Biofilm Reactors: Simultaneous Aerobic Methanotrophy and Nitrogen Removal.

Anaerobic digestion (AD) produces useful biogas and waste streams with high levels of dissolved methane (CH4) and ammonium (NH4+), among other nutrients. Membrane biofilm reactors (MBfRs), which support dissolved methane oxidation in the same reactor as simultaneous nitrification and denitrification (ME-SND), are a potential bubble-less treatment method. Here, we demonstrate ME-SND taking place in single-stage, AD digestate liquid-fed MBfRs, where oxygen (O2) and supplemental CH4 were delivered via pressurized membranes. The effects of two O2 pressures, leading to different O2 fluxes, on CH4 and N removal were examined. MBfRs achieved up to 98% and 67% CH4 and N removal efficiencies, respectively. The maximum N removal rates ranged from 57 to 94 mg N L-1 d-1, with higher overall rates observed in reactors with lower O2 pressures. The higher-O2-flux condition showed NO2- as a partial nitrification endpoint, with a lower total N removal rate due to low N2 gas production compared to lower-O2-pressure reactors, which favored complete nitrification and denitrification. Membrane biofilm 16S rRNA amplicon sequencing showed an abundance of aerobic methanotrophs (especially Methylobacter, Methylomonas, and Methylotenera) and enrichment of nitrifiers (especially Nitrosomonas and Nitrospira) and anammox bacteria (especially Ca. Annamoxoglobus and Ca. Brocadia) in high-O2 and low-O2 reactors, respectively. Supplementation of the influent with nitrite supported evidence that anammox bacteria in the low-O2 condition were nitrite-limited. This work highlights coupling of aerobic methanotrophy and nitrogen removal in AD digestate-fed reactors, demonstrating the potential application of ME-SND in MBfRs for the treatment of AD's residual liquids and wastewater. Sensor-based tuning of membrane O2 pressure holds promise for the optimization of bubble-less treatment of excess CH4 and NH4+ in wastewater.

Decoupling the simultaneous effects of NO2−, pH and free nitrous acid on N2O and NO production from enriched nitrifying activated sludge

In the pursuit of energy and carbon neutrality, nitrogen removal technologies have been developed featuring nitrite (NO2−) accumulation. However, high NO2− accumulations are often associated with stimulated greenhouse gas (i.e., nitrous oxide, N2O) emissions. Furthermore, the coexistence of free nitrous acid (FNA) formed by NO2− and proton (pH) makes the consequence of NO2− accumulation on N2O emissions complicated. The concurrent three factors, NO2−, pH and FNA may play different roles on N2O and nitric oxide (NO) emissions simultaneously, which has not been systematically studied. This study aims to decouple the effects of NO2− (0-200 mg N/L), pH (6.5-8) and FNA (0-0.15 mg N/L) on the N2O and NO production rates and the production pathways by ammonia oxidizing bacteria (AOB), with the use of a series of precisely executed batch tests and isotope site-preference analysis. Results suggested the dominant factors affecting the N2O production rate were NO2− and FNA concentrations, while pH alone played a relatively insignificant role. The most influential factor shifted from NO2− to FNA as FNA concentrations increased from 0 to 0.15 mg N/L. At concentrations below 0.0045 mg HNO2-N/L, nitrite rather than FNA played a significant role stimulating N2O production at elevated nitrite concentrations. The inhibition effect of FNA emerged with further increase of FNA between 0.0045-0.015 mg HNO2-N/L, weakening the promoting effect of increased nitrite. While at concentrations above 0.015 mg HNO2-N/L, FNA inhibited N2O production especially from nitrifier denitrification pathway with the level of inhibition linearly correlated with the FNA concentration. pH and the nitrite concentration regulated the production pathways, with elevated pH promoting the nitrifier nitrification pathway, while elevated NO2− concentrations promoting the nitrifier denitrification pathway. In contrast to N2O, NO emission was less susceptible to FNA at concentrations up to 0.015 mg N/L but was stimulated by increasing NO2− concentrations. This study, for the first time, distinguished the effects of pH, NO2− and FNA on N2O and NO production, thereby providing support to the design and operation of novel nitrogen removal systems with NO2− accumulation.

Quantifying the Relative Roles of Different Microorganisms on Trimethoprim Degradation by Nitrifying Activated Sludge

AbstractThe cometabolism of ammonia‐oxidizing bacteria (AOB) in nitrifying activated sludge can effectively degrade antibiotics. However, the roles of other microorganisms in antibiotic removal processes remain uncharacterized. In this study, the effects of AOB, nitrite oxidizing bacteria (NOB), and heterotrophs on the removal of the antibiotic trimethoprim (TMP) at environmentally relevant concentrations in an enriched nitrifying activated sludge are quantified. AOB is found to be the most efficient for TMP removal, which is related with ammonium concentration. TMP removal is increased from 48.0% to 64.0% as the ammonium concentration increased from 50 to 150 mg L−1. However, with a further increase in ammonium concentration to 250 mg L−1, TMP removal is decreased to 45.6%. The percentages of TMP removed by AOB, NOB, and heterotrophs are quantified by batch experiments, and are 78.7%, 18.6%, and 2.7%, respectively. The results demonstrate that in nitrifying activated sludge system, the contribution of NOB and heterotrophs on TMP removal should also be considered.

Performance enhancement, membrane fouling mitigation and eco-friendly strategy by electric field coupled membrane bioreactor for treating mariculture wastewater

An eco-friendly strategy for mariculture wastewater treatment using an electric field attached membrane bioreactor (E-MBR) was evaluated and compared with a conventional membrane bioreactor (C-MBR). The removal efficiencies of total nitrogen (TN) and chemical oxygen demand (COD) increased significantly and the membrane fouling rate reduced by 44.8% in the E-MBR. The underlying mechanisms included the enriched nitrifiers and denitrifiers, the enhanced salinity-resistance, the increased activities and upregulated genes of key enzymes involved in nitrification and denitrification for improving the performance of mariculture wastewater treatment, and the enriched extracellular polymeric substance (EPS)-degrading genera, the downregulated EPS biosynthesis genes, the repressed biofilm-forming bacteria, the enhanced zeta potential absolute value and the generated H2O2 for membrane fouling mitigation by electrical stimulation. Compared with the C-MBR, the energy consumption, carbon emissions, and nitrogen footprint were reduced. These findings provide novel insights into mariculture wastewater treatment using an applied electric field.

Enhanced Nitrogen Removal in a Pilot-Scale Anoxic/Aerobic (A/O) Process Coupling PE Carrier and Nitrifying Bacteria PE Carrier: Performance and Microbial Shift

Integrated fixed-film activated sludge technology (IFAS) has a great advantage in improving nitrogen removal performance and increasing treatment capacity of municipal wastewater treatment plants with limited land for upgrading and reconstruction. This research aims at investigating the enhancing effects of polyethylene (PE) carrier and nitrifying bacteria PE (NBPE) carrier on nitrogen removal efficiency of an anoxic/aerobic (A/O) system from municipal wastewater and revealing temporal changes in microbial community evolution. A pilot-scale A/O system and a pilot-scale IFAS system were operated for nearly 200 days, respectively. Traditional PE and NBPE carriers were added to the IFAS system at different operating phases. Results showed that the treatment capacity of the IFAS system was enhanced by almost 50% and 100% by coupling the PE carrier and NBPE carrier, respectively. For the PE carrier, nitrifying bacteria abundance was maintained at 7.05%. In contrast, the nitrifying bacteria on the NBPE carrier was enriched from 6.66% to 23.17%, which could improve the nitrogen removal and treating capacity of the IFAS system. Finally, the ammonia efficiency of the IFAS system with NBPE carrier reached 73.0 ± 7.9% under 400% influent shock load and hydraulic retention time of 1.8 h. The study supplies a suitable nitrifying bacteria enrichment method that can be used to help enhance the nitrogen removal performance of municipal wastewater treatment plants. The study’s results advance the understanding of this enrichment method that effectively improves nitrogen removal and anti-resistance shock-load capacity.

Elucidating nitrifying performance, nitrite accumulation and microbial community in a three-stage plug flow moving bed biofilm reactor (PF - MBBR)

A three-stage plug flow moving bed biofilm reactor (PF - MBBR, consisting of three identical chambers of N1, N2 and N3) was proposed for nitrifier enrichment using synthetic wastewater. During the stable operation, the average NH4+-N effluent was 0.67 mg/L and NH4+-N removal was as high as 97.19% with the nitrite accumulation ratio (NAR) of 54.23%, although the biofilm thickness and biomass both presented downward trends from N1 (296 μm, 2280 mg/L), N2 (248 μm, 1850 mg/L) to N3 (198 μm, 1545 mg/L). Particularly, the comparative results of three stages revealed that N2 showed the optimum NH4+-N removal (77.27%) and NAR (75.21%) in the continuous-flow, while NAR of N3 unexpectedly maintained a high level of 65.83% in the batch test, suggesting that ammonia oxidizing bacteria (AOB) accounted for absolute advantage over nitrite oxidizing bacteria (NOB). High-throughput sequencing initially verified different distribution of bacterial community structure, where N2 was far away from N1 and N3 with the lowest community richness and community diversity (operational taxonomic units (OTUs): 454(N2)<527(N3)<621(N1)). Proteobacteria (77.60%–83.09%), Bacteroidetes (1.66%–3.66%), Acidobacteria (2.28%–4.67%), and Planctomycetes (1.19%–6.63%) were the major phyla. At the genus level, AOB (mainly Nitrosomonas) accounted for 5.08% (N1), 20.74% (N2) and 14.24% (N3) while NOB (mainly Nitrospira) increased from 0.14% (N1), 7.06% (N2) to 4.91% (N3) with the total percentages of 5.22%, 27.80% and 19.15%. Finally, the application feasibility of MBBR optimization linked with nitrite (NO2−-N) accumulation for deep-level nutrient removal was discussed.

Unravelling the contribution of nitrifying and methanotrophic bacteria to micropollutant co-metabolism in rapid sand filters

The presence of organic micropollutant (OMP) in groundwater threatens drinking water quality and public health. Rapid sand filter (RSF) rely on biofilms with nitrifying and methanotrophic bacteria to remove ammonia and methane during drinking water production. Previous research observed the partial removal of OMPs with active nitrification and methane oxidation due to co-metabolic conversion of OMPs. However, the contribution of indigenous nitrifying and methanotrophic communities from RSF has yet to be fully explored. Accordingly, experiments were carried out with biofilm-covered sand collected from field-scale RSF, to assess the removal of nine OMPs by nitrifying and methanotrophic bacteria. Results indicated that stimulating nitrification resulted in significantly more removal of caffeine, 2,4-dichlorophenoxyacetic acid and bentazone. Stimulating methanotrophic conditions enhanced the removal of caffeine, benzotriazole, 2,4-dichlorophenoxyacetic acid and bentazone. Microbial community analysis based on 16 S rRNA gene sequencing revealed Nitrosomonas and Nitrospira are the dominant genus in the community under nitrifying conditions. The three genera Methylobacter, Methylomonas and Methylotenera were enriched under methanotrophic conditions. This study highlights that nitrifying and methanotrophic bacteria play important roles during OMP removal in field RSF. Furthermore, results suggest that bioaugmentation with an enriched nitrifying and methanotrophic culture is a promising approach to improve OMP removal in RSF.

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.

Effect of carrier particle size on enrichment and shift in nitrifier community behaviors for treating increased strength wastewater.

In activated sludge systems, adding carriers can improve nitrifier enrichment. Different attachment area induced by different particle sizes of carriers may result in different nitrifier community. This research investigated the effect of different particle sizes of coal ash on nitrifier enrichment treating increased strength wastewater. Results indicated efficient nitrifying coal ash was obtained with smaller coal ash. The ammonia removal rates reached over 98%, which outclassed that in negative control (63.28%), and no nitrite accumulated in these systems under high nitrogen concentration of 1123.35mgN/L. The high-throughput sequencing assays indicated carriers changed the microbial community structure significantly, thus facilitated the nitrification capacity. Increase abundance of nitrifier has a negative correlation with particle size of carriers. Nitrosomonas became the biggest beneficiary, which maximum composed 50.29% in fillers system and only 13.69% in negative control, whereas the number of Nitrobacter (less than 3.04%) became much lower than ammonia-oxidizing bacteria (AOB). However, the shift of microbial structures, large number of Dokdonella for instance, may guarantee the complete nitrification in systems with smaller carriers. Batch experiments showed a high dissolved oxygen (DO) concentration (4mg/L) and slightly alkaline condition (pH 8.0) had a positive effect on nitrifying coal ash. PRACTITIONER POINTS: The increase size of nitrifier has a negative correlation with particle size of coal ash. The smaller coal ash reduces the adverse effect of high nitrogen on nitrification. The ammonia removal rate reached 99.82% with influent of 1123.35mg /L in the smallest carriers system.

Biodegradation kinetics of organic micropollutants and microbial community dynamics in a moving bed biofilm reactor

Feast-famine moving bed biofilm reactors (MBBRs) have shown high potential for removing organic micropollutants from wastewater. However, the relationship between biofilm community during feast-famine adaptation and micropollutant removal is yet unclear. In this study, we determined the biotransformation kinetics of 36 micropollutants and characterized the microbial communities in an MBBR during a 71-day adaptation period of feast-famine regime (raw/effluent wastewater). The feast-famine regime significantly changed the biodegradation rate constants (k) of 24 micropollutants in different ways: 66 times enhanced degradation for propranolol, while more than 10 times for atenolol, metoprolol, tramadol and venlafaxine, less than 2.8 times for losartan, iomeprol and iohexol were detected. 25–60 days of adaptation time was needed to reach the maximum k. Biofilm accumulated during the adaptation, but the kDNA (k relative to the biofilm with DNA concentration as a proxy for kbiomass) of most micropollutants (except propranolol, metoprolol and venlafaxine) declined. This might indicate that the proliferation of potential degraders for micropollutants was slower than other microorganisms under the feast-famine regime. The microbial community changed significantly during the first 8 days of operation, followed by a relatively steady evolution towards the enrichment of nitrifiers until day 71. A multivariate statistical correlation analysis revealed that the development of occurrence of 88 individual taxonomic groups were found to exhibit a significant positive correlation to the kDNA of micropollutants (p < 0.05, r > 0.5), which represent potential biomarkers linking to biotransformation of micropollutants. These results fill the knowledge gaps between dynamics of biofilm communities and micropollutant removal in the feast-famine regime, which is essential for designing highly efficient MBBR.

Performance and bacterial community dynamics of aerobic granular sludge working at low temperature enhanced by melamine framework embedding

Aerobic granular sludge (AGS) is becoming a well-established technology for wastewater treatment, which has shown outstanding performance in practical applications. However, the cultivation of AGS at low temperature is still a challenge. In this study, melamine framework (MF) was applied to promote sludge granulation at 10 ℃. Stable AGS with compact structure and excellent settle-ability was successfully cultured within 70 d. MF embedded inoculated sludge in the early stage to avoid sludge loss, and provided skeleton support for the granules in the later stage, which effectively accelerated the granulation process and improved the stability of AGS. Extracellular polysaccharides content increased significantly during granulation, with the decreased of protein to polysaccharides ratio. The system achieved high ammonium nitrogen, total nitrogen and total phosphorus removal efficiencies, reaching 96.6%, 70% and 99%, respectively, which were attributed to the enrichment of nitrifiers and denitrifying phosphorus accumulating organisms (DPAOs). The dominant DPAO changed from Dechlormonas to Flavobacterium during granulation, accompanied by the decrease of extracellular polymers (EPS) secreting genus Zoogloea and the increase of EPS degrading genus norank_f__Saprospiraceae. Even though, the AGS maintained structural stability, suggesting that MF pre-embedding and supporting might be an effective method for cultivation and stable operation of AGS at low temperature.

Lumen air pressure (LAP) affecting greywater treatment in an oxygen-based membrane biofilm reactor (O2-MBfR)

Several technologies have been employed to treat greywater (GW) for domestic use. Aerobic biological treatment has achieved high efficiency, the main cost being the necessary source of oxygen (O2). This study explores the effects of lumen air pressure (LAP) on reactor performance and microbial community succession in an O2-based membrane biofilm reactor (O2-MBfR) treating GW. At high LAP (≥0.8 psi), the dissolved oxygen (DO) concentration inside the reactor was higher than 0.38 ± 0.02 mg/L, leading to removal efficiencies of 90%, 98%, and 80%, of total chemical oxygen demand, total linear alkylbenzene sulfonate (LAS), and total nitrogen, respectively. Lower LAP (<0.8 psi) led to a decrease in DO inside the system, and a less effective GW treatment. Low O2 pressure decreased organic biodegradation and ammoniation, and caused LAS accumulation in the biofilm, leading to the solubilization of extracellular polymeric substances and cell lysis. Comprehensive consideration of reactor performance and energy input, DO inside the MBfR at 0.38 ± 0.02 mg/L could be selected as the optimized condition for GW treatment. Microbial community analyses results also revealed that improved LAP was favorable for the enrichment of LAS-biodegradation related genus (Pseudomonas, Parvibaculum, Magnetospirillum, Clostridium, Zoogloea, Dechloromonas and Mycobacterium), nitrifiers (Nitrosomonas and Sphingomonas) and facultative microorganisms (Dechloromonas, Flavobacterium, Pseudomonas, Aeromonas and Zoogloea) that can carry out denitrification under relatively high DO conditions (>0.38 mg/L), but led to the reduction of the relative abundance of heterotrophs (Acidovorax, Thermomonas, Brevundimonas and Enterobacter) that are more sensitive towards high DO conditions.

Analysis of the Effect of Temperature on the Microbial Flora Structure During the Nitrite Oxidation Process Using 16S rRNA High-throughput Sequencing

In order to further understand the influence of high temperature shock on the microbial community structure of activated sludge during the process of nitrite oxidation, the enriched nitrifying activated sludge under different NO2--N concentration was taken as the research object in this study. 16S rRNA high-throughput sequencing technology was used to analyze the changes in the microbial community abundance and structural characteristics of activated sludge by changing the environmental temperature. The results of high-throughput sequencing showed that microorganisms were more likely to grow at 25℃, and the diversity of the microbial community in the activated sludge was the most abundant. With increased temperature, the richness, evenness, and diversity of the flora in the system decreased. In addition, it was found that the main nitrifying bacterium in the system was Nitrospira of Nitrospirae, whereby 35℃ was more suitable for its growth. Meanwhile, a higher temperature also caused differences in the structure of non-nitrifying functional microorganisms (e.g., Bacteroidetes, Chlorofulexi, Halomonas, and Pseudomonas) in the activated sludge. The results of this study provide some theoretical reference for the investigation of the distribution characteristics of microbial flora during the process of nitrite oxidation under high temperature shock, and can also be used as reference for relevant high temperature shock tests.

Grain size tunes microbial community assembly and nitrogen transformation activity under frequent hyporheic exchange: A column experiment

Hyporheic zones (HZ) are hotspots for biogeochemical reactions where groundwater and surface water mix. River dam buildings and other hydrologic controls alter the sediment grain size distribution and modify the downstream hyporheic exchange, with cascading effects on geochemical and microbial processes in river corridors. In this lab-scale column experiment, the N transformations in HZ filled with sediments in different grain sizes were investigated with a focus on understanding the interplay among variational hydraulic connectivity, microbial community structure, functional potential under frequent groundwater−surface water exchange. Porosity was identified as the main driver determining bacterial community assembly in HZ sediments. Significant microbial zonation was observed along the columns and the degree of co-occurrence of bacterial communities in the Fine column was lower than that in the Coarse and Mix columns. The Coarse column allowed for almost 2.47 times the exchange flux relative to the Fine column, and generates the fastest DO consumption rate (−6.52 μg O2/L·s). The enrichment of nitrifiers, i.e., Cytophagaceae and Bacillaceae and nitrification functional genes, i.e., amoA_AOA and amoA_AOB revealed the higher nitrification potential in column filled with coarse sediments. In comparison, the highest NH4+ production rates (2.4 × 10−3 μg N/L·s) took place in Fine column. The higher abundancies of denitrifiers such as Comamonadaceae and Lysobacter and enrichment of functional genes of nirK and nirS interestingly suggested the elevated denitrification potential in Fine column in a more anaerobic environment. The results implied that variations in microbial functional potential and associated nitrogen transformation may occur in size-fractioned HZ to dynamic hyporheic exchange, which added new knowledge to the underlying biogeochemical and ecological processes in regulated river corridors.

Effects of light and mass ratio of microalgae and nitrifiers on the rates of ammonia oxidation and nitrate production

This study investigated the effects of microalgae and light on nitrifying bacteria. The oxygen production rates of microalgae were measured at various cell concentrations, and it was observed that 500–2000 mg/L of microalgal concentration supplied sufficient oxygen for bacterial nitrification without forced aeration. The effects of light on nitrification were investigated by determining the ammonia oxidation rate (AOR) and nitrate production rate (NPR) of enriched nitrifiers in the absence of microalgae with forced aeration, and it was observed that those rates were significantly lowered in the presence of light. However, the inhibitory effects of light on AOR and NPR were partially reduced in the co-culture owing to the self-shading effect. AOR and NPR of co-cultures with Chlorella vulgaris and Scenedesmus quadricauda were high at a low mass ratio of microalgae to enriched nitrifiers, while those with Desmedesmus communis increased as the mass ratio increased. These species-specific phenomena could be attributed to the different production of reactive oxygen species. Despite the negative effects of light and reactive oxygen species, it was estimated that the energy consumption of the co-culture was lower than that of the mechanically-aerated nitrifiers.

Co-culture of microalgae and enriched nitrifying bacteria for energy-efficient nitrification

The aim of this study was to investigate the possibility of using microalgae as an oxygen supplier for nitrification. In order to measure oxygen demands of nitrification, the activated sludge were cultured in a selective medium for 150 days, and enriched nitrifying biomass was successfully obtained. The enriched nitrifiers were used to estimate oxygen demands for nitrification and used for co-culturing with microalgae. Oxygen transfer rates (OTR) were also measured for various mixing conditions to determine if mixing without forced aeration could satisfy oxygen demands of nitrification. For all mixing conditions, OTR were lower than the oxygen uptake rates (OUR) of nitrifiers. As a result, nitrification without forced aeration was incomplete due to oxygen deficiency. Meanwhile, oxygen production rates (OPR) of 6 microalgae species were measured, and it was demonstrated that C. vulgaris, S quadricauda, D. communism and C. emersonii could supply sufficient oxygen for nitrification at all mixing modes. Based on these results, 4 species of microalgae with sufficient oxygen supplying capabilities were selected for co-culturing with enriched nitrifiers, and full nitrification was accomplished without forced aeration. Finally, the electric energy of co-culture was estimated and showed that energy consumption by co-culture was lower than that of aerated nitrifiers.

Comparative assessment of endocrine disrupting compounds removal in heterotrophic and enriched nitrifying biomass

Despite the number of studies that have investigated the fate of endocrine disrupting compounds (EDCs), to date results are still contradictory and more research is required to evaluate the contribution of the microbial communities present in different engineered treatment systems. Thus, autotrophic and heterotrophic types of biomass were here compared in terms of efficiency in the removal of estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethynilestradiol (EE2) and bisphenol A (BPA). Experiments were performed with enriched nitrifying activated sludge (NAS) and enriched ammonia oxidizing bacteria (AOB) sludge cultivated at lab-scale, as well as with conventional activated sludge (CAS) from a full-scale wastewater treatment plant. Both enriched NAS and AOB demonstrated a negligible degrading capacity. In both cases, the studied EDCs exhibited low removals (<14%) and showed no correlation with the increasing nitrification rates contradicting some of the hypothesis present in literature. Contrariwise, the biodegradation capabilities of the heterotrophic fraction of CAS were highlighted. E2 and E3 were removed by up to 100% and 78%, respectively. E1 was found to be the main transformation product of E2 (almost quantitative oxidation) and it was also highly eliminated. Finally, EE2 and BPA were more persistent biologically with removals ranging from 10% to 39%. For these two compounds similar removals were obtained during experiments with heat-inactivated biomass suggesting that sorption could be a relevant route of elimination.

Multi-Parameter Laser Imaging Reveals Complex Microscale Biofilm Matrix in a Thick (4,000 μm) Aerobic Methanol Oxidizing Community

Although methanol has frequently been used as an inexpensive supplementary carbon source to support treatment processes, knowledge of the resultant microbial biofilms, their 3D architecture, microenvironments, exopolymer chemistry and populations remains limited. We supplied methanol as a supplementary carbon source to biofilms developing in rotating annular reactors. Analysis of circulation waters (1.0 l d−1) indicated that dissolved organic carbon was reduced by 25%, NO3-nitrogen by 95%, and total phosphorus by 70%. Analyses of populations using culture based techniques and fluorescence in situ hybridization indicated enrichment of nitrifiers, denitrifiers, and methylotrophic bacteria relative to reference biofilms not receiving methanol. The biofilms that developed were up to 4,000 μm thick. Staining with fluor conjugated lectins in combination with nucleic acid stains, revealed the presence of discrete bacterial cells inside complex globular polymeric structures. These structures were in turn surrounded by an interstitial polymer containing a variety of bacterial cell types. The globular structures bound FITC-conjugated lectins, from Canavalia ensiformis and Ulex europeaus. The FITC-lectin of Phaseolus vulgaris bound the surface of the globular structures and more generally within the matrix. Chemical analyses of the polymer paralleled the results of lectin analyses indicating that the dominant neutral sugars were glucose, galactose, mannose, rhamnose, with fucose and ribose as minor constituents. Amino sugars were not detected. Dual channel imaging with pH sensitive probes indicated that pH gradients from pH 4 to 7 occurred across the globular microcolonies. Critically for the maintenance of aerobic conditions throughout the thick biofilm it was extensively penetrated by a fine fissure network revealed by the location of fluorescent latex microbeads as detected by confocal laser scanning microscopy. Microelectrode studies confirmed the absence of any detectable Eh gradients within the biofilm. However, mobility of various size-fractionated fluorescent probes indicated that the basal region was only penetrated by the lowest molecular weight probes with a hydrated radius of 2.2 nm or less. These observations indicate the selection of a unique, thick (>4,000 μm) microbial community in which a self-organized architecture promotes the maintenance of optimal conditions and metabolism throughout the biofilm community.

Impact of aerobic acclimation on the nitrification performance and microbial community of landfill leachate sludge

Nitrogenous pollution of water is regarded as a global environmental problem, and nitrogen removal has become an important issue in wastewater treatment processes. Landfill leachate is a typical large source of nitrogenous wastewater. Although the characteristics of leachate vary according to the age of the landfill, leachates of mature landfill have high concentrations of nitrogenous compounds. Most nitrogen in these leachates is in the form of ammonium nitrogen. In this study, we investigated the bacterial community of sludge from a landfill leachate lagoon by pyrosequencing of the bacterial 16S rRNA gene. The sludge was acclimated in a laboratory-scale reactor with aeration using a mechanical stirrer to promote nitrification. On 149 days, nitrification was achieved and then the bacterial community was also analyzed. The bacterial community was also analyzed after nitrification was achieved. Pyrosequencing analyses revealed that the abundances of ammonia-oxidizing and nitrite-oxidizing bacteria were increased by acclimation and their total proportions increased to >15% of total biomass. Changes in the sulfate-reducing and sulfur-oxidizing bacteria were also observed during the acclimation process. The aerobic acclimation process enriched a nitrifying microbial community from the landfill leachate sludge. These results suggested that the aerobic acclimation is a processing method for the nitrification ammonium oxidizing throw the enrichment of nitrifiers. Improvement of this acclimation method would allow nitrogen removal from leachate by nitrification and sulfur denitrification.