Akihiko Terada

Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox

ABSTRACT Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.

Simultaneous nitrification and denitrification by controlling vertical and horizontal microenvironment in a membrane-aerated biofilm reactor

Nitrogen and carbon components in domestic modified wastewater were completely removed by simultaneous nitrification and denitrification using a membrane-aerated biofilm reactor where biofilm was fixed on a hollow-fiber membrane. To measure the spatial distribution of pH, ammonium and nitrate ions and to observe microbes inside the biofilm fixed on the membrane, microelectrodes and the fluorescence in situ hybridization (FISH) method were applied. Due to plug flow in the vertical direction (from the bottom to the top of the reactor), ammonium nitrogen was gradually removed and negligible nitrate nitrogen was detected throughout the reactor. FISH revealed that ammonia-oxidizing bacteria were mainly distributed inside the biofilm and other bacteria, which included denitrifying bacteria, were mainly distributed outside the biofilm and over the suspended sludge. In order to characterize bacterial activity in the vertical direction of the reactor, nitrification rates at lower, central and upper points were calculated using microelectrode data. The nitrification rate at the lower point was 7 and 125 times higher than those at the central and upper points, respectively. These results show that the removal of carbon and nitrogen compounds was accomplished efficiently by using various kinds of bacteria distributed vertically and horizontally in a single reactor.

Nitrogen removal characteristics and biofilm analysis of a membrane-aerated biofilm reactor applicable to high-strength nitrogenous wastewater treatment

A membrane-aerated biofilm reactor (MABR) capable of simultaneous nitrification and denitrification in a single reactor vessel was developed to investigate the characteristics of nitrogen removal from high-strength nitrogenous wastewater, and biofilm analysis using microelectrodes and the fluorescence in situ hybridization (FISH) technique was performed. Mean removal percentages of total organic carbon (TOC) and nitrogen were 96% and 83% at removal rates of 5.76 g-C m(-2) d(-1) and 4.48 g-N m(-2) d(-1), respectively. For stable removal efficiency, constant washing of the biofilm was needed. Dissolved oxygen microelectrode measurement revealed that the biofilm thickness was about 1600 microm, and that oxygen penetrated about 300 to 700 microm, from the outer surface of the membrane. Furthermore, FISH analysis revealed that ammonia-oxidizing bacteria (AOB) were located near the outer surface of the membrane, whereas other bacteria were located from the inner to the outer part of the biofilm. Combining these results demonstrated that simultaneous nitrification and denitrification occurred in the biofilm of the MABR system. In addition, stoichiometric analysis revealed that after 130 d(-1), the free ammonia (FA) concentration ranged within the concentration causing inhibition of the growth of nitrite oxidizing bacteria (NOB) and that AOB consumed 86% of the oxygen supplied through the intra-membrane. These results indicate that nitrogen removal not via nitrate but via nitrite was mainly achieved in the MABR system.

Shifts between Nitrospira- and Nitrobacter-like nitrite oxidizers underlie the response of soil potential nitrite oxidation to changes in tillage practices

Despite their role in soil functioning, the ecology of nitrite-oxidizing bacteria, NOB, and their response to disturbances such as those generated by agricultural practices are scarcely known. Over the course of 17 months, we surveyed the potential nitrite oxidation, PNO, the abundance of the Nitrobacter- and Nitrospira-like NOB (by quantitative PCR) and the community structure of the Nitrobacter-like NOB (by PCR-DGGE and cloning-sequencing targeting the nxrA gene) in soils for four treatments: after establishment of tillage on a previously no-tillage system, after cessation of tillage on a previously tillage system, and on control tillage and no-tillage systems. Key soil variables (moisture, organic carbon content and gross mineralization--i.e. ammonification--measured by the 15N dilution technique) were also surveyed. PNO was always higher for the no-tillage than tillage treatments. Establishment of tillage led to a strong and rapid decrease in PNO whereas cessation of tillage did not change PNO even after 17 months. PNO was strongly and positively correlated to the abundance of Nitrobacter-like NOB and was also strongly related to gross mineralization, a proxy of N-availability; in contrast, PNO was weakly and negatively correlated to the abundance of Nitrospira-like NOB. Selection of a dominant population was observed under no-tillage, and PNO was loosely correlated to the community structure of Nitrobacter-like NOB. Our results demonstrate that Nitrobacter-like NOB are the key functional players within the NOB community in soils with high N availability and high activity level, and that changes in PNO are due to shifts between Nitrospira-like and Nitrobacter-like NOB and to a weaker extent by shifts of populations within Nitrobacter-like NOB.

Microbial community structure in autotrophic nitrifying granules characterized by experimental and simulation analyses

This study evaluates the community structure in nitrifying granules (average diameter of 1600 mum) produced in an aerobic reactor fed with ammonia as the sole energy source by a multivalent approach combining molecular techniques, microelectrode measurements and mathematical modelling. Fluorescence in situ hybridization revealed that ammonia-oxidizing bacteria dominated within the first 200 mum below the granule surface, nitrite-oxidizing bacteria a deeper layer between 200 and 300 mum, while heterotrophic bacteria were present in the core of the nitrifying granule. Presence of these groups also became evident from a 16S rRNA clone library. Microprofiles of NH(4)(+), NO(2)(-), NO(3)(-) and O(2) concentrations measured with microelectrodes showed good agreement with the spatial organization of nitrifying bacteria. One- and two-dimensional numerical biofilm models were constructed to explain the observed granule development as a result of the multiple bacteria-substrate interactions. The interaction between nitrifying and heterotrophic bacteria was evaluated by assuming three types of heterotrophic bacterial growth on soluble microbial products from nitrifying bacteria. The models described well the bacterial distribution obtained by fluorescence in situ hybridization analysis, as well as the measured oxygen, nitrite, nitrate and ammonium concentration profiles. Results of this study are important because they show that a combination of simulation and experimental techniques can better explain the interaction between nitrifying bacteria and heterotrophic bacteria in the granules than individual approaches alone.

Sequential Aeration of Membrane-Aerated Biofilm Reactors for High-Rate Autotrophic Nitrogen Removal: Experimental Demonstration

One-stage autotrophic nitrogen (N) removal, requiring the simultaneous activity of aerobic and anaerobic ammonium oxidizing bacteria (AOB and AnAOB), can be obtained in spatially redox-stratified biofilms. However, previous experience with Membrane-Aerated Biofilm Reactors (MABRs) has revealed a difficulty in reducing the abundance and activity of nitrite oxidizing bacteria (NOB), which drastically lowers process efficiency. Here we show how sequential aeration is an effective strategy to attain autotrophic N removal in MABRs: Two separate MABRs, which displayed limited or no N removal under continuous aeration, could remove more than 5.5 g N/m(2)/day (at loads up to 8 g N/m(2)/day) by controlled variation of sequential aeration regimes. Daily averaged ratios of the surficial loads of O(2) (oxygen) to NH(4)(+) (ammonium) (L(O(2))/L(NH(4))) were close to 1.73 at this optimum. Real-time quantitative PCR based on 16S rRNA gene confirmed that sequential aeration, even at elevated average O(2) loads, stimulated the abundance of AnAOB and AOB and prevented the increase in NOB. Nitrous oxide (N(2)O) emissions were 100-fold lower compared to other anaerobic ammonium oxidation (Anammox)-nitritation systems. Hence, by applying periodic aeration to MABRs, one-stage autotrophic N removal biofilm reactors can be easily obtained, displaying very competitive removal rates, and negligible N(2)O emissions.

Inoculum effects on community composition and nitritation performance of autotrophic nitrifying biofilm reactors with counter-diffusion geometry.

The link between nitritation success in a membrane-aerated biofilm reactor (MABR) and the composition of the initial ammonia- and nitrite-oxidizing bacterial (AOB and NOB) population was investigated. Four identically operated flat-sheet type MABRs were initiated with two different inocula: from an autotrophic nitrifying bioreactor (Inoculum A) or from a municipal wastewater treatment plant (Inoculum B). Higher nitritation efficiencies (NO(2)(-)-N/NH(4)(+)-N) were obtained in the Inoculum B- (55.2-56.4%) versus the Inoculum A- (20.2-22.1%) initiated reactors. The biofilms had similar oxygen penetration depths (100-150 µm), but the AOB profiles [based on 16S rRNA gene targeted real-time quantitative PCR (qPCR)] revealed different peak densities at or distant from the membrane surface in the Inoculum B- versus A-initiated reactors, respectively. Quantitative fluorescence in situ hybridization (FISH) revealed that the predominant AOB in the Inoculum A- and B-initiated reactors were Nitrosospira spp. (48.9-61.2%) versus halophilic and halotolerant Nitrosomonas spp. (54.8-63.7%), respectively. The latter biofilm displayed a higher specific AOB activity than the former biofilm (1.65 fmol cell(-1) h(-1) versus 0.79 fmol cell(-1) h(-1) ). These observations suggest that the AOB and NOB population compositions of the inoculum may determine dominant AOB in the MABR biofilm, which in turn affects the degree of attainable nitritation in an MABR.

The effect of surface charge property on Escherichia coli initial adhesion and subsequent biofilm formation

Polyethylene (PE) sheets were modified by radiation‐induced graft polymerization (RIGP) of an epoxy‐group containing monomer glycidyl methacrylate (GMA). The epoxy group of GMA was opened by introducing sodium sulfite (SS) and diethylamine (DEA) as representatives of negatively and positively charged functional groups, respectively. These modified surfaces by RIGP, termed GMA, SS, and DEA sheets, were investigated to elucidate their effects on initial adhesion and subsequent biofilm formation of Escherichia coli. Initial adhesion test revealed that E. coli density and viability were governed by sheet surface electrostatic property: E. coli cell density on the DEA sheet was 23 times higher than that on the SS sheet after 8 h incubation. The viability of E. coli cells dramatically decreased after contact with the DEA sheet, but remained high on the SS sheet. E. coli biofilm structure on the DEA sheet was dense, homogeneous, and uniform, with biomass higher than that of the GMA and SS sheets by factors of 14.0 and 37.5, respectively. On the contrary, biofilm structure on the SS sheet was sparse, heterogeneous, and mushroom‐shaped. More than 40% of E. coli biofilm on the DEA sheet was retained under a high liquid shear force condition (5,000 s−1), whereas 97% and 100% of biofilms on the GMA and SS sheets were sloughed, indicating that E. coli biofilm robustness depends on surface charge property of the substratum. This suggests that substratum surface fabrication by RIGP may enhance or suppress biofilm formation, a finding with potentially important practical implications. Biotechnol. Bioeng. 2012; 109:1745–1754.

Behavior of PCDDs/PCDFs in remediation of PCBs-contaminated sediments by thermal desorption.

Thermal desorption is an effective method for removing volatile and semivolatile organic matter in contaminated solid remediation. Formation of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in many polychlorinated biphenyls (PCBs) destruction processes has been reported, but the removal pathways are poorly understood. We therefore investigated the behaviors of PCBs and PCDDs/PCDFs in thermal desorption of PCBs-contaminated sediment and predicted the reaction pathways. Four thermal desorption experiments using PCB-contaminated sediments containing different doses of PCBs were carried out. In all the experiments, decomposition of 48-70% of PCBs was achieved, resulting in formation of PCDFs. Despite the PCBs decomposition levels, toxic equivalencies (TEQs) in the treated and volatilized samples were 2.8-6.3 times and 8.0-10.5 times as high as the TEQs in the initial samples, respectively, indicating increased toxicity after treatment. Further analysis revealed that PCDFs with higher numbers of chlorine atoms are likely to remain in the sediments than those of volatilized PCDFs; this is supported by the positive correlation between the vapor pressures of PCDFs and the ratios of volatilized PCDFs.

Assessing nitrification and denitrification in a paddy soil with different water dynamics and applied liquid cattle waste using the 15N isotopic technique

Using livestock wastewater for rice production in paddy fields can remove nitrogen and supplement the use of chemical fertilizers. However, paddy fields have complicated water dynamics owing to varying characteristics and would influence nitrogen removal through nitrification followed by denitrification. Quantification of nitrification and denitrification is of great importance in assessing the influence of water dynamics on nitrogen removal in paddy fields. In this study, nitrification and nitrate reduction rates with different water dynamics after liquid cattle waste application were evaluated, and the in situ denitrification rate was determined directly using the (15)N isotopic technique in a laboratory experiment. A significant linear regression correlation between nitrification and the nitrate reduction rate was observed and showed different regression coefficients under different water dynamics. The regression coefficient in the continuously flooded paddy soil was higher than in the drained-reflooded paddy soil, suggesting that nitrate would be consumed faster in the flooded paddy soil. However, nitrification was limited and the maximum rate was only 13.3 μg Ng(-1)day(-1) in the flooded paddy soil with rice plants, which limited the supply of nitrate. In contrast, the drained-reflooded paddy soil had an enhanced nitrification rate up to 56.8 μg Ng(-1)day(-1), which was four times higher than the flooded paddy soil and further stimulated nitrate reduction rates. Correspondingly, the in situ denitrification rates determined directly in the drained-reflooded paddy soil ranged from 5 to 1035 mg Nm(-2)day(-1), which was higher than the continuously flooded paddy soil (from 5 to 318 mg Nm(-2)day(-1)) during the vegetation period. The nitrogen removal through denitrification accounted for 38.9% and 9.9% of applied nitrogen in the drained-reflooded paddy soil and continuously flooded paddy soil, respectively.