Achieving nitrite accumulation in a continuous system treating low-strength domestic wastewater: switchover from batch start-up to continuous operation with process control

Although biological nitrogen removal via nitrite is recognized as one of the cost-effective and sustainable biological nitrogen removal processes, nitrite accumulation has proven difficult to achieve in continuous processes treating low-strength nitrogenous wastewater. Partial nitrification to nitrite was achieved and maintained in a lab-scale completely stirred tank reactor (CSTR) treating real domestic wastewater. During the start-up period, sludge with ammonia-oxidizing bacteria (AOB) but no nitrite-oxidizing bacteria (NOB) was obtained by batch operation with aeration time control. The nitrifying sludge with the dominance of AOB was then directly switched into continuous operation. It was demonstrated that partial nitrification to nitrite in the continuous system could be repeatedly and reliably achieved using this start-up strategy. The ratio of dissolved oxygen to ammonium loading rate (DO/ALR) was critical to maintain high ammonium removal efficiency and nitrite accumulation ratio. Over 85% of nitrite accumulation ratio and more than 95% of ammonium removal efficiency were achieved at DO/ALR ratios in an optimal range of 4.0–6.0 mg O₂/g N d, even under the disturbances of ammonium loading rate. Microbial population shift was investigated, and fluorescence in situ hybridization analysis indicated that AOB were the dominant nitrifying bacteria over NOB when stable partial nitrification was established.     

从典型硝化细菌到全程氨氧化微生物：发现及研究进展

生物硝化过程在全球氮循环中起关键性作用,被认为由氨氮氧化成亚硝酸盐和亚硝酸盐氧化成硝酸盐两个步骤组成,分别由氨氧化微生物(Ammonia oxidizing microorganisms,AOM)和硝化细菌(Nitrite oxidizing bacteria,NOB)催化完成。AOM包括氨氧化细菌(Ammonia oxidizing bacteria,AOB)和氨氧化古菌(Ammonia oxidizing archaea,AOA),AOB与AOA分布广泛,两者的相对丰度和氨氮浓度密切相关。2015年底,3个硝化螺菌属(Nitrospira)谱系Ⅱ的NOB被证实含有AOM的特征功能酶,包括氨单加氧酶(AMO)和羟胺脱氢酶(HAO),并证明NOB同时具有氨氧化和亚硝酸盐氧化的能力,命名为全程氨氧化微生物(Complete ammonia oxidizer,Comammox)。根据AMO的α亚基基因amoA的相似性将Comammox分为两大分支clade A和clade B。它们广泛分布于自然环境和人工系统,包括土壤(稻田、森林)、淡水(湿地、河流、湖泊沉积物、蓄水层)、污水处理厂和自来水厂等。本文综述了Comammox的发现及其最新的研究进展,并展望了Comammox作为氮循环关键功能菌群的研究方向和应用前景。     

Partial nitrification in a sequencing batch reactor treating acrylic fiber wastewater

A sequencing batch reactor was employed to treat the acrylic fiber wastewater. The dissolved oxygen and mixed liquor suspended solids were 2–3 and 3,500–4,000 mg/L, respectively. The results showed ammonium oxidizing bacteria (AOB) had superior growth rate at high temperature than nitrite oxidizing bacteria (NOB). Partial nitrification could be obtained with the temperature of 28 °C. When the pH value was 8.5, the nitrite-N accumulation efficiency was 82 %. The combined inhibitions of high pH and free ammonium to NOB devoted to the nitrite-N buildup. Hydraulic retention time (HRT) was a key factor in partial nitrification control, and the optimal HRT was 20 h for nitrite-N buildup in acrylic fiber wastewater treatment. The ammonium oxidation was almost complete and the transformation from nitrite to nitrate could be avoided. AOB and NOB accounted for 2.9 and 4.7 %, respectively, corresponding to the pH of 7.0. When the pH was 8.5, they were 6.7 and 0.9 %, respectively. AOB dominated nitrifying bacteria, and NOB was actually washed out from the system.     

好氧和缺氧条件下游离亚硝酸对氨氧化菌和亚硝酸盐氧化菌的选择性抑制

【背景】稳定短程硝化是实现城市污水厌氧氨氧化技术的瓶颈,目前国内外关于游离亚硝酸(Free nitrous acid,FNA)对硝化菌活性的影响大多是在曝气条件下进行研究,鲜有关于缺氧条件下FNA对硝化菌活性影响的报道。【目的】探究好氧和缺氧下FNA对氨氧化菌(Ammonia oxidizing bacteria,AOB)和亚硝酸盐氧化菌(Nitrite oxidizing bacteria,NOB:Nitrospira和Nitrobacter)活性的抑制影响。【方法】采用序批式反应器(Sequencing batch reactor,SBR),基于混合液悬浮固体浓度(Mixed liquid suspended solids,MLSS)为8 300 mg/L的全程硝化污泥条件,通过批次试验分别考察好氧和缺氧下FNA(初始浓度为1.16 mg/L)处理48 h后,AOB和NOB活性的变化情况。【结果】好氧FNA处理活性污泥48 h后,FNA浓度维持在1.16-1.17 mg/L,游离氨(Free ammonia,FA)浓度小于0.017 mg/L,AOB、Nitrospira、Nitrobacter丰度均未发生明显变化;过曝气至99 h时,与空白组相比,比氨氮氧化速率(r~+_(NH4-N))、比亚硝酸盐氮氧化速率(r_(NO2-N))均出现小幅下降,分别由3.5、4.828 mg N/(g VSS·h)降至3.3、4.668 mg N/(g VSS·h),且亚硝酸盐氮累积率(Nitrite accumulation rate,NAR)始终低于33.2%。缺氧FNA处理活性污泥48 h后,FNA浓度维持在0.64-1.16 mg/L,FA浓度低于0.039 mg/L,AOB丰度变化较小,而Nitrospira、Nitrobacter丰度均明显下降,分别由3.002 9×10~9、4.245×10~8 copies/g VSS降至1.666 5×10~8、5.163 8×10~7 copies/g VSS;过曝气至99 h时,与空白组相比,r~+_(NH4-N)值下降幅度较小,而r_(NO2-N)值明显降低,由4.828 mg N/(g VSS·h)降至0.007 mg N/(g VSS·h),且在过曝气0-292 h内,NAR均大于94%。【结论】好氧FNA处理活性污泥48 h后对AOB和NOB无明显抑制作用,但缺氧FNA处理活性污泥48 h后对AOB具有轻微抑制作用,而对NOB具有强烈的抑制作用,可以实现稳定的短程硝化。     

Effects of loading rate and aeration on nitrogen removal and N<Subscript>2</Subscript>O emissions in intermittently aerated sequencing batch reactors treating slaughterhouse wastewater at 11 °C

This study aimed to find optimal operation conditions for nitrogen removal from high strength slaughterhouse wastewater at 11 °C using the intermittently aerated sequencing batch reactors (IASBRs) so as to provide an engineering control strategy for the IASBR technology. Two operational parameters were examined: (1) loading rates and (2) aeration rates. Both the two parameters affected variation of DO concentrations in the IASBR operation cycles. It was found that to achieve efficient nitrogen removal via partial nitrification–denitrification (PND), “DO elbow” point must appear at the end of the last aeration period. There was a correlation between the ammonium oxidizing bacteria (AOB)/nitrite oxidizing bacteria (NOB) ratio and the average DO concentrations in the last aeration periods; when the average DO concentrations in the last aeration periods were lower than 4.86 mg/L, AOB became the dominant nitrifier population, which benefited nitrogen removal via PND. Both the nitrogen loading rate and the aeration rate influenced the population sizes of AOB and NOB. To accomplish efficient nitrogen removal via PND, the optimum aeration rate (A, L air/min) applied can be predicted according to the average organic loading rates based on mathematical equations developed in this study. The research shows that the amount of N₂O generation in the aeration period was reduced with increasing the aeration rate; however, the highest N₂O generation in the non-aeration period was observed at the optimum aeration rates.     

硝化菌保藏特性及降解动力学研究

投加硝化菌是有效降解水体中亚硝酸盐的方法之一,而作为商品化的硝化菌产品却非常少见。究其原因,保藏过程中的菌体衰亡速率过快是重要影响因素之一,而有关其保藏衰亡的文献几乎没有。本文通过对影响硝化菌衰亡因素及其动力学的研究,发现温度、pH和离子强度是影响其衰亡的重要因素,通过研究使得硝化菌液的衰减指数（Km）由原有的0.25降至0.013,半衰期延长至55d,为硝化菌的产业化提供了理论依据和实现方法。     

Effects of aeration cycles on nitrifying bacterial populations and nitrogen removal in intermittently aerated reactors

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using amoA gene denaturant gradient gel electrophoresis. Nitrosomonas and Nitrosococcus mobilis were the dominant AOB and Nitrospira spp. were the dominant NOB in all reactors, although Nitrosospira and Nitrobacter were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest Nitrosospira rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of Nitrobacter, compared to the other reactors. Nitrosomonas sp. strain Nm107 was detected in all reactors, regardless of the reactor's performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. strain ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.     

Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure

In this study, the performance of partial nitrification via nitrite and microbial community structure were investigated and compared in two sequencing batch reactors (SBR) with different dissolved oxygen (DO) levels. Both reactors achieved stable partial nitrification with nitrite accumulation ratio of above 95% by using real-time aeration duration control. Compared with high DO (above 3 mg/l on average) SBR, simultaneous nitrification and denitrification (SND) via nitrite was carried out in low DO (0.4–0.8 mg/l) SBR. The average efficiencies of SND in high DO and low DO reactor were 7.7% and 44.9%, and the specific SND rates were 0.20 and 0.83 mg N/(mg MLSS h), respectively. Low DO did not produce sludge with poorer settling properties but attained lower turbidities of the effluent than high DO. Fluorescence in situ hybridization (FISH) analysis in both the reactors showed that ammonia-oxidizing bacteria (AOB) were the dominant nitrifying bacteria and nitrite-oxidizing bacteria (NOB) did not be recovered in spite of exposing nitrifying sludge to high DO. The morphology of the sludge from both two reactors according to scanning electron microscope indicated that small rod-shaped and spherical clusters were dominant, although filamentous bacteria and few long rod-shaped coexisted in the low DO reactor. By selecting properly DO level and adopting process control method is not only of benefit to the achievement of novel biological nitrogen removal technology, but also favorable to sludge population optimization.     

Operation of partial nitrification to nitrite of landfill leachate and its performance with respect to different oxygen conditions

The coupled system of partial nitrification and anaerobic ammonium oxidation (Anammox) is efficient in nitrogen removal from wastewater. In this study, the effect of different oxygen concentrations on partial nitrification performance with a sequencing batch reactor (SBR) was investigated. Results indicate that, partial nitrification of landfill leachate could be successfully achieved under the 1.0–2.0 mg L⁻¹ dissolved oxygen (DO) condition after 118 d long-term operation, and that the effluent is suitable for an Anammox reactor. Further decreasing or increasing the DO concentration, however, would lead to a decay of nitrification performance. Additionally, the MLSS concentration in the reactor increased with increasing DO concentration. Respirometric assays suggest that low DO conditions (<2 mg L⁻¹) favor the ammonia-oxidizing bacteria (AOB) and significantly inhibit nitrite oxidizing bacteria (NOB) and aerobic heterotrophic bacteria (AHB); whereas high DO conditions (>3 mg L⁻¹) allow AHB to dominate and significantly inhibit AOB. Therefore, the optimal condition for partial nitrification of landfill leachate is 1.0–2.0 mg L⁻¹ DO concentration.     

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.     

Control and optimization of nitrifying communities for nitritation from domestic wastewater at room temperatures

To achieve nitritation from complete-nitrification seed sludge at room temperature of 19 ± 1 °C, a lab-scale sequencing batch reactor (SBR) treating domestic wastewater with low C/N ratios was operated to investigate the control and optimization of nitrifying communities. Ammonia oxidizing bacteria (AOB) dominance was enhanced through the combination of low DO concentrations (<1.0 mg/L) and preset short-cycle control of aeration time. Nitritation was successfully established with NO₂^?-N/NO_x^?-N over 95%. To avoid the adverse impact of low DO concentrations on AOB activities, DO concentrations were increased to 1–2 mg/L. At the normal DO levels and temperatures, on-line control strategy of aerobic durations maintained the stability of nitritation with nitrite accumulation rate over 95% and ammonia removal above 97%. Fluorescence in-situ hybridization (FISH) analysis presented that the maximal percentage of AOB in biomass reached 10.9% and nitrite oxidizing bacteria (NOB) were washed out.     

Estimation of nitrifier abundances in a partial nitrification reactor treating ammonium-rich saline wastewater using DGGE, T-RFLP and mathematical modeling

The bacterial community in a partial nitrification reactor was analyzed on the basis of 16S rRNA gene by cloning–sequencing method, and the percentages of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the activated sludge were quantified by three independent methods, namely, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and Double Monod modeling. The clone library results suggested that there were only a dominant AOB and a dominant NOB species in the reactor, belonging to Nitrosomonas genus and Nitrospira genus, respectively. The percentages of NOB in total bacterial community increased from almost 0% to 30% when dissolved oxygen (DO) levels were changed from 0.15 mg/L to 0.5 mg/L, coinciding with the accumulation and conversion of nitrite, while the percentages of AOB changed little in the two phases. The results confirmed the importance of low DO level for inhibiting NOB to achieve partial nitrification. Furthermore, the percentages of AOB and NOB in the total bacteria community were estimated based on the results of batch experiments using Double Monod model, and the results were comparable with those determined according to profiles of DGGE and T-RFLP.     

Partial nitrification to nitrite using low dissolved oxygen concentration as the main selection factor

Partial nitrification to nitrite (nitritation) can be achieved in a continuous process without sludge retention by wash out of nitrite oxidising bacteria (NOB) while retaining ammonia oxidising bacteria (AOB), at elevated temperatures (the SHARON process) and, as demonstrated in this paper, also at low dissolved oxygen (DO) concentrations. Enriched AOB was attained at a low DO concentration (0.4 mg l⁻¹) and a dilution rate of 0.42 day⁻¹ in a continuous process. A higher oxygen affinity of AOB compared to NOB seemed critical to achieving this. This was verified by determining the oxygen half saturation constant, K _o, with similar oxygen mass transfer resistances for enriched AOB and NOB as 0.033 ± 0.003 mg l⁻¹ and 0.43 ± 0.08 mg l⁻¹, respectively. However, the extent of nitritation attained was found to be highly sensitive to process upsets.     

Activated sludge under free ammonia treatment using gel immobilization technology for long-term partial nitrification with different initial biomass

To achieve stable partial nitrification, activated sludge from a wastewater treatment plant using free ammonia (FA) inhibition was immobilized in a polyvinyl alcohol carrier. After FA treatment at 16.44 mg L⁻¹ for 1 day, due to the increased growth rate gap between ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), AOB enrichment and NOB inhibition were achieved within 12 days, with AOB and NOB accounting for 65.61 and 0.05%, respectively. Subsequently, with dissolved oxygen concentrations of 4−5 mg L⁻¹, pH of 7.6–7.8 and temperature of 25 ± 1 °C, the immobilized carrier made of activated sludge achieved more than 90% and more than 86% of nitrite accumulation rate at the influent ammonia concentration of 90−110 mg L⁻¹ and 35−50 mg L⁻¹, respectively. After 50 days operation, the NOB content was 0.10%, indicating the immobilized carrier provided favorable conditions for maintaining the low NOB content. Furthermore, due to the low NOB content in the inoculum and the oxygen-limited environment formed by the increase in the AOB numbers in the carrier, immobilized carrier with different initial biomass (1, 2.5 and 5%) can achieve stable partial nitrification.     

Distinctive microbial ecology and biokinetics of autotrophic ammonia and nitrite oxidation in a partial nitrification bioreactor

Biological nitrogen removal (BNR) based on partial nitrification and denitrification via nitrite is a cost-effective alternate to conventional nitrification and denitrification (via nitrate). The goal of this study was to investigate the microbial ecology, biokinetics, and stability of partial nitrification. Stable long-term partial nitrification resulting in 82.1 +/- 17.2% ammonia oxidation, primarily to nitrite (77.3 +/- 19.5% of the ammonia oxidized) was achieved in a lab-scale bioreactor by operation at a pH, dissolved oxygen and solids retention time of 7.5 +/- 0.1, 1.54 +/- 0.87 mg O(2)/L, and 3.0 days, respectively. Bioreactor ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) populations were most closely related to Nitrosomonas europaea and Nitrobacter spp., respectively. The AOB population fraction varied in the range 61 +/- 45% and was much higher than the NOB fraction, 0.71 +/- 1.1%. Using direct measures of bacterial concentrations in conjunction with independent activity measures and mass balances, the maximum specific growth rate (micro(max)), specific decay (b) and observed biomass yield coefficients (Y(obs)) for AOB were 1.08 +/- 1.03 day(-1), 0.32 +/- 0.34 day(-1), and 0.15 +/- 0.06 mg biomass COD/mg N oxidized, respectively. Corresponding micro(max), b, and Y(obs) values for NOB were 2.6 +/- 2.05 day(-1), 1.7 +/- 1.9 day(-1), and 0.04 +/- 0.02 mg biomass COD/mg N oxidized, respectively. The results of this study demonstrate that the highly selective partial nitrification operating conditions enriched for a narrow diversity of rapidly growing AOB and NOB populations unlike conventional BNR reactors, which host a broader diversity of nitrifying bacteria. Further, direct measures of microbial abundance enabled not only elucidation of mixed community microbial ecology but also estimation of key engineering parameters describing bioreactor systems supporting these communities.     

泉州西湖沉积物中硝化细菌的分布及其作用

比较研究泉州西湖沉积物中氨氧化细菌(AOB)和亚硝酸盐氧化细菌(NOB)的分布及氨氧化潜力和亚硝酸盐(NO2?)氧化潜力。结果表明: 西湖沉积物中存在高浓度的有机质(OM)、总氮(TN)和氨氮。AOB生物量为1.1×106?6.4×106 个/g干土, 显著高于NOB生物量4.2×105?7.4×105 个/g 干土(配对t-检验, P<0.05)。对于NOB, 硝化杆菌属(Nitrobacter)和硝化螺菌属(Nitrospira)同时存在于西湖沉积物中, 以Nitrobacter为优势种群。AOB和NOB生物量的差异一定程度上导致西湖沉积物中氨氧化潜力显著高于NO2?氧化潜力(配对t-检验, P<0.05), NO2?氧化过程成为硝化作用的限制步骤。另外, 西湖沉积物中存在的较高浓度氨氮, 一方面促进了AOB的生长和活性, 导致较高速率的氨氧化过程, 另一方面却对亚硝酸盐氧化过程产生选择性抑制, 这也是导致NO2?氧化潜力较低的主要原因之一。     

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3)d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L(-1). Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosomonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662.     

Kinetic analysis on the two-step processes of AOB and NOB in aerobic nitrifying granules

Complete granulation of nitrifying sludge was achieved in a sequencing batch reactor. For the granular sludge, batch experiments were conducted to characterize the kinetic features of ammonia oxidizers (AOB) and nitrite oxidizers (NOB) in the granules using the respirometric method. A two-step nitrification model was established to determine the kinetic parameters of both AOB and NOB. In addition to nitrification reactions, the new model also took into account biomass maintenance and mass transfer through the granules. The yield coefficient, maximum specific growth rate, and affinity constant for ammonium for AOB were 0.21 g chemical oxygen demand (COD) g⁻¹ N, 0.09 h⁻¹, and 9.1 mg N L⁻¹, respectively, whereas the corresponding values for NOB were 0.05 g COD g⁻¹ N, 0.11 h⁻¹, and 4.85 mg N L⁻¹, respectively. The model developed in this study performed well in simulating the oxygen uptake rate and nitrogen conversion kinetics and in predicting the oxygen consumption of the AOB and NOB in aerobic granules.     

Relationship between respirometric activity and community of entrapped nitrifying bacteria: Implications for partial nitrification

Activated sludge obtained from two municipal wastewater treatment facilities (WWTF) was used as seed sludge for enriched nitrifiers, which were later entrapped in polyvinyl alcohol. Seed sludge from one WWTF was acclimated to high ammonia level (1813 mg NH₃-N l^?1) through the return of sludge digester supernatant back to primary clarifier while seed sludge from the other WWTF was un-acclimated. To elucidate on how to control partial nitrification by entrapped cells, which could be different from suspended cells, kinetics of entrapped enriched nitrifiers were studied using a respirometric assay. The community of nitrifiers within the entrapment matrix, which was observed by fluorescence in situ hybridization (FISH) technique, was related to the nitritation and nitratation kinetics based on oxygen uptake rate. Maximum oxygen uptake rate, and substrate and oxygen affinities of both ammonia oxidizing bacteria (AOB) for nitritation and nitrite oxidizing bacteria (NOB) for nitratation in entrapped cells were lower than those of corresponding suspended cells. Under dissolved oxygen (DO) limiting conditions, nitratation was more suppressed than nitritation for suspended cells, while for the entrapped cells, the results were the contrary. A free ammonia (FA) inhibition affected only the un-acclimated sludge. Either FA inhibition or DO limitation might not be a sole effective control parameter to achieve partial nitrification by entrapped cells. FISH results revealed that Nitrosomonas europaea was the dominant AOB while Nitrobacter species was the dominant NOB in all cases. Heterotrophs were also present in the entrapment at 22.8 ± 18.6% and 41.5 ± 4.3% of total bacteria for acclimated and un-acclimated originated sludge. The availability of substrate and oxygen governed the distributions of AOB, NOB and heterotrophs within the entrapment and nitritation kinetics of entrapped nitrifiers.     

Fabrication of Bacteria Environment Cubes with Dry Lift-Off Fabrication Process for Enhanced Nitrification

We have developed a 3D dry lift-off process to localize multiple types of nitrifying bacteria in polyethylene glycol diacrylate (PEGDA) cubes for enhanced nitrification, a two-step biological process that converts ammonium to nitrite and then to nitrate. Ammonia-oxidizing bacteria (AOB) is responsible for converting ammonia into nitrite, and nitrite-oxidizing bacteria (NOB) is responsible for converting nitrite to nitrate. Successful nitrification is often challenging to accomplish, in part because AOB and NOB are slow growers and highly susceptible to many organic and inorganic chemicals in wastewater. Most importantly, the transportation of chemicals among scattered bacteria is extremely inefficient and can be problematic. For example, nitrite, produced from ammonia oxidation, is toxic to AOB and can lead to the failure of nitrification. To address these challenges, we closely localize AOB and NOB in PEGDA cubes as microenvironment modules to promote synergetic interactions. The AOB is first localized in the vicinity of the surface of the PEGDA cubes that enable AOB to efficiently uptake ammonia from a liquid medium and convert it into nitrite. The produced nitrite is then efficiently transported to the NOB localized at the center of the PEGDA particle and converted into non-toxic nitrate. Additionally, the nanoscale PEGDA fibrous structures offer a protective environment for these strains, defending them from sudden toxic chemical shocks and immobilize in cubes. This engineered microenvironment cube significantly enhances nitrification and improves the overall ammonia removal rate per single AOB cell. This approach—encapsulation of multiple strains at close range in cube in order to control their interactions—not only offers a new strategy for enhancing nitrification, but also can be adapted to improve the production of fermentation products and biofuel, because microbial processes require synergetic reactions among multiple species.