Application of nitrifying and denitrifying processes to waste management of aquatic life support in space

Since a biological filter with nitrifying bacteria was firstly applied to aquatic animal experiments in IML-2 mission, the reactor system has been further studied to combine both nitrifying and denitrifying reactions under aerobic environment allowing an efficient removal of inorganic nitrogen from animal wastes. The isolated denitrifying bacteria had an activity under aerobic condition with rice straw providing a metabolic carbon source for the reaction. The advantage of the aerobic biological filter having both nitrifying and denitrifying activities may allow to reduce the size of the life support system and also for its manageability. The paper reports characteristics of the biological filter systems used for the IML-2 mission and the improved combined filter system having both nitrifying and denitrifying activities, and discuss its application to space experiments.     

Modeling the partial nitrification in sequencing batch reactor for biomass adapted to high ammonia concentrations

Partial nitrification has proven to be an economic way for treatment of industrial N-rich effluent, reducing oxygen and external COD requirements during nitrification/denitrification process. One of the key issues of this system is the intermediate nitrite accumulation stability. This work presents a control strategy and a modeling tool for maintaining nitrite build-up. Partial nitrification process has been carried out in a sequencing batch reactor at 30 degrees C, maintaining strong changing ammonia concentration in the reactor (sequencing feed). Stable nitrite accumulation has been obtained with the help of an on-line oxygen uptake rate (OUR)-based control system, with removal rate of 2 kg NH4 (+)-N x m(-3)/day and 90%-95% of conversion of ammonium into nitrite. A mathematical model, identified through the occurring biological reactions, is proposed to optimize the process (preventing nitrate production). Most of the kinetic parameters have been estimated from specific respirometric tests on biomass and validated on pilot-scale experiments of one-cycle duration. Comparison of dynamic data at different pH confirms that NH3 and NO2- should be considered as the true substrate of nitritation and nitratation, respectively. The proposed model represents major features: the inhibition of ammonia-oxidizing bacteria by its substrate (NH3) and product (HNO2), the inhibition of nitrite-oxidizing bacteria by free ammonia (NH3), the INFluence of pH. It appears that the model correctly describes the short-term dynamics of nitrogenous compounds in SBR, when both ammonia oxidizers and nitrite oxidizers are present and active in the reactor. The model proposed represents a useful tool for process design and optimization.     

A critical comparison of extant batch respirometric and substrate depletion assays for estimation of nitrification biokinetics

Estimation of nitrification biokinetics has been conducted using different batch techniques via measurement of nitrogen species or surrogates such as oxygen (respirometry). However, there are no reports that specifically compare kinetic parameters estimated from respirometry with those from direct nitrogen species measurements. In this study, we evaluated the ability of parameter estimates from isolated and optimally designed complete extant respirometric assays to describe concurrently obtained ammonia and nitrite depletion profiles. Additionally, we mapped the different parameter sets to steady-state bioreactor performance. Using multivariate analysis of variance, we found that estimates from respirometric and substrate depletion assays were predominantly statistically different at the 95% confidence level. The sensitivity of predicted stead-state nitrifying reactor performance to differences in parameter estimates was highest close to the limiting solids retention time (SRT). However, at characteristic nitrifying SRTs the predicted reactor performance using parameter estimates from respirometry and substrate depletion assays were in very close correspondence. Therefore, parameters estimated from extant respirometric assays can be used to adequately predict nitrifying reactor performance.     

VFEU water quality control in STS-95 mission.

In STS-95 Space Shuttle mission, an aquatic animal research facility, Vestibular Function Experiment Unit (VFEU), was flown to perform neurobiological experiment with marine fish, oyster toadfish. For this purpose, we have developed a sea water purification system using highly active nitrifying bacteria at low temperature. With this system, the water quality in the VFEU was maintained in sufficient condition to keep the toadfish in healthy state for 9 days of the mission. This report summarizes the efficiency of the filter system based on the results from pre-flight bacterial preparation, water analysis of samples taken during flight, and the post-flight analysis of the bacterial filter.     

Analysis of Microbial Population Dynamics in a Partial Nitrifying SBR at Ambient Temperature

In this study, a lab-scale partial nitrifying sequencing batch reactor (SBR) was developed to investigate partial nitrification at ambient temperature (16–22 °C). Techniques of denaturing gradient gel electrophoresis (DGGE), cloning, and fluorescence in situ hybridization (FISH) were utilized simultaneously to study microbial population dynamics. Partial nitrification was effectively achieved in response to shifts of influent ammonium concentrations. DGGE results showed that higher ammonia concentration referred to lower ammonia-oxidizing bacteria (AOB) diversity in the SBR. Phylogenetic analysis revealed that all the predominant AOB was affiliated with Nitrosomonas genus. FISH analysis illustrated AOB was the predominant nitrifying bacteria of microbial compositions when SBR achieved partial nitrification (PN) at ambient temperature.     

Competition and coexistence of aerobic ammonium- and nitrite-oxidizing bacteria at low oxygen concentrations

In natural and man-made ecosystems nitrifying bacteria experience frequent exposure to oxygen-limited conditions and thus have to compete for oxygen. In several reactor systems (retentostat, chemostat and sequencing batch reactors) it was possible to establish co-cultures of aerobic ammonium- and nitrite-oxidizing bacteria at very low oxygen concentrations (2–8 μM) provided that ammonium was the limiting N compound. When ammonia was in excess of oxygen, the nitrite-oxidizing bacteria were washed out of the reactors, and ammonium was converted to mainly nitrite, nitric oxide and nitrous oxide by Nitrosomonas-related bacteria. The situation could be rapidly reversed by adjusting the oxygen to ammonium ratio in the reactor. In batch and continuous tests, no inhibitory effect of ammonium, nitric oxide or nitrous oxide on nitrite-oxidizing bacteria could be detected in our studies. The recently developed oxygen microsensors may be helpful to determine the kinetic parameters of the nitrifying bacteria, which are needed to make predictive kinetic models of their competition.     

Molecular and ecological analyses of microbial community structures in biofilms of a full-scale Aerated Up-Flow Biobead process

Molecular and cultivation techniques were used to characterize the bacterial communities of biobead reactor biofilms in a sewage treatment plant to which an Aerated Up-Flow Biobead process was applied. With this biobead process, the monthly average values of various chemical parameters in the effluent were generally kept under the regulation limits of the effluent quality of the sewage treatment plant during the operation period. Most probable number (MPN) analysis revealed that the population of denitrifying bacteria was abundant in the biobead #1 reactor, denitrifying and nitrifying bacteria coexisted in the biobead #2 reactor, and nitrifying bacteria prevailed over denitrifying bacteria in the biobead #3 reactor. The results of the MPN test suggested that the biobead #2 reactor was a transition zone leading to acclimated nitrifying biofilms in the biobead #3 reactor. Phylogenetic analysis of 16S rDNA sequences cloned from biofilms showed that the biobead #1 reactor, which received a high organic loading rate, had much diverse microorganisms, whereas the biobead #2 and #3 reactors were dominated by the members of Proteobacteria. DGGE analysis with the ammonia monooxygenase (amoA) gene supported the observation from the MPN test that the biofilms of September were fully developed and specialized for nitrification in the biobead reactor #3. All of the DNA sequences of the amoA DGGE bands were very similar to the sequence of the amoA gene of Nitrosomonas species, the presence of which is typical in the biological aerated filters. The results of this study showed that organic and inorganic nutrients were efficiently removed by both denitrifying microbial populations in the anaerobic tank and heterotrophic and nitrifying bacterial biofilms well-formed in the three functional biobead reactors in the Aerated Up-Flow Biobead process.     

Dynamic mathematical modeling of sulfate reducing gas-lift reactors

This paper presents a mathematical model able to simulate under dynamic conditions the physical, chemical and biological processes prevailing in a biological sulfate reducing gas-lift reactor. The proposed model is based on differential mass balance equations for substrates, products and bacterial groups involved in a sulfate reduction process. Heterotrophic sulfate reducing bacteria (HSRB), autotrophic sulfate reducing bacteria (ASRB), homoacetogenic bacteria (HB), methanogenic archaea (MA) and acetate degraders (AD) are the microbial groups taken into account in the model. The model is also used to validate a steady-state design model previously proposed by Esposito et al. [1].The proposed model is able to simulate the competition between the biological bacteria growing in the reactor, and predict the performance of a gas-lift reactor. The model includes two main parts: (1) a kinetic part including growth, metabolism and competition of SRB, HB, MA and AD in the system and (2) a mass-transfer part describing the thermodynamic concentration equilibria of gaseous components in the liquid and gas phase. The model has been validated using experimental data obtained by operating a laboratory-scale gas-lift reactor as described in Esposito et al. [2].The model can be applied to simulate the sulfate reduction process in a gas-lift reactor for several purposes, such as the evaluation of the optimal process conditions in terms of COD:SO₄^2? ratio, hydraulic retention time and gas input flow. In particular, model simulations reported in this paper show the model capability to predict the prevailing bacterial species and concentrations in the reactor as a function of the hydraulic retention time.     

Novel application of nitrifying bacterial consortia to ease ammonia toxicity in ornamental fish transport units: trials with zebrafish

Aims: To evaluate whether two commercial nitrifying bacterial consortia can function as biocontrol agents in ornamental fish transporting systems. Methods and Results: The consortia were applied in a simulated set‐up using zebrafish as the model organism in three trials. The efficacy of the bacterial consortia in controlling the ammonia level was validated by measuring water quality parameters such as total ammonia, nitrate and pH of the transport water. The bacterial community structure in the transport unit was studied using denaturing gradient gel electrophoresis. The consortia tested improved the nitrifying activity that in turn facilitated the reduction of ammonia that had accumulated during the transport. Bacterial profiles revealed the presence of both ammonia‐oxidizing and nitrite‐oxidizing bacteria in the transport bags. Conclusions: The application of the consortia during the transportation of zebrafish could profoundly improve the water quality by curbing ammonia accumulation. Significance and Impact of the Study: The potential of applying nitrifying bacteria as a bioremediation practice during the transport of ornamental fish has been demonstrated and this innovative approach contributes to the amelioration of current fish welfare in ornamental fish trade.     

STS-95 Space Experiments (plants and cell biology).

We report the outline of Space Experiments conducted on Space Shuttle (STS-95) launched in autumn of 1998. In this STS-95 mission, Japanese astronaut Dr. Chiaki Mukai achieved her 2nd space flight and conducted a part of 82 space experiments including Japanese experiments. US astronaut Senator John Glenn also achieved his second space flight, 36 years after his first space flight. Senator Glenn was a leader of the original (the first) 7 US astronauts and very famous in US because he succeeded US first orbital space flight around the earth. NASDA had started the project of space experiment using STS-95 at the summer of 1997, therefore we had only one year for the all preparation Yamashita, et al. Biological Sciences in Space, Vol.12 No.3(1998). Scientific results will be reported by investigators, therefore we report here how we had been developing the space experiment plan, on board operation procedure and ground operations including ground control experiments about four plant experiments and one cell biology experiment.     

Wastewater nitrification kinetics using reciprocating jet bioreactor

Kinetic investigations on growth parameters of nitrifying and COD oxidizing bacteria were carried out with recourse to a three stage reciprocating jet bioreactor system using real life wastewater. The system employed in this investigation essentially consisted of separate aerobic oxidation stage along with nitrification stage and anaerobic denitrification stage with facility for biomass recirculation whenever necessary. Steady-state COD oxidation reactor performance was assessed for various values of residence time. Yield coefficient and decay coefficient of COD oxidizing biomass were obtained as 0.3329 kg BM/kg COD and 0.0032 (1/h) respectively.It was observed that COD oxidizing bacteria co-existed with nitrifying bacteria during nitrification process due to the nature of wastewater used. Steady-state nitrification reactor performance was also assessed for various residence time values. Exact concentration of nitrifying and COD oxidizing biomass in the nitrification reactor was then estimated with the help of kinetic growth parameters of COD oxidizing biomass and extent of COD oxidation achieved in nitrification reactor. This further enabled evaluation of corrected kinetic growth parameters estimated as 0.4272 kg BM/kg NH ₄ ⁺ -N and 0.00626 (1/h) for nitrifier biomass yield coefficient and decay coefficient respectively.     

Ecological study of a bioaugmentation failure

A nitrifying sequencing batch reactor was inoculated twice with the aerobic denitrifying bacterium Microvirgula aerodenitrificans and fed with acetate. No improvement was obtained on nitrogen removal. The second more massive inoculation was even followed by a nitrification breakdown, while at the same time, nitrification remained stable in a second reactor operated under the same conditions without bioaugmentation. Fluorescent in situ hybridization with rRNA-targeted probes revealed that the added bacteria almost disappeared from the reactor within 2 days, and that digestive vacuoles of protozoa gave strong hybridization signals with the M. aerodenitrificans -specific probe. An overgrowth of protozoa, coincident with the disappearance of free-living bacteria, was monitored by radioactive dot-blot hybridization only in the bioaugmented reactor. Population dynamics were analysed with a newly developed in situ quantification procedure of the probe-targeted bacteria. The nitrifying groups of bacteria decreased in a similar way in the bioaugmented and non-bioaugmented reactors. Other bacterial groups evolved differently. The involvement of different ecological parameters are discussed separately for each reactor. These results underline the importance of predator–prey interaction and illustrate the undesirable effects of massive bioaugmentation.     

Microbial community analysis by FISH for mathematical modelling of selective enrichment of gel-entrapped nitrifiers obtained from domestic wastewater

Nitrifying activated sludge from natural domestic sewage was entrapped in hydrogel beads, which were subsequently enriched for nitrifiers in a continuous stirred tank reactor (CSTR). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidisers were used in combination with DAPI staining to monitor the selectivity of the enrichment process. The growth of both nitrifying and heterotrophic bacteria was more pronounced in the periphery of the beads, leading to a biofilm-like stratification of the biomass during the enrichment. Quantitatively, the relative number of nitrifiers increased from 20% immediately after immobilisation up to 64% after 30 days, but decreased again due to extensive heterotrophic growth. These changes were accompanied by an increase in nitrifying activity for about 30 days, whereupon it reached a stable level. This selective enrichment was mathematically modelled by applying finite difference techniques to the diffusion-reaction mass balances of all soluble substrates relevant in the nitrification process. To model biomass growth and spreading, balanced by both decay and detachment at the surface of the beads, the differential methods were combined with a descrete cellular automaton approach. The spatially two-dimensional model was used to calculate radial concentration profiles within a gel bead, as well as to estimate the corresponding total activity of the reactor. Qualitatively, this model could simulate all essential aspects observed experimentally. However, more and better population data as well as independent estimates of decay and hydrolysis rates are needed to refine and verify the quantitative model. In conclusion, even in the absence of an external carbon source and with excess ammonium, it was only possible to obtain a moderate enrichment of nitrifying cells compared to heterotrophs. Under long-term cultivation, the biofilm-like structure developed in the outer gel layers led to a vigorous competition between auto- and heterotrophs for space, and thereby, access to oxygen. FISH analysis in combination with mathematical modelling constitute a suitable toolbox for analysing the population dynamics and biocatalytic performance of such an ecosystem based on lithoautotrophic primary production.     

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.     

Nitrifying bacterial growth inhibition in the presence of algae and cyanobacteria

Nitrifying bacteria, cyanobacteria, and algae are important microorganisms in open pond wastewater treatment systems. Nitrification involving the sequential oxidation of ammonia to nitrite and nitrate, mainly due to autotrophic nitrifying bacteria, is essential to biological nitrogen removal in wastewater and global nitrogen cycling. A continuous flow autotrophic bioreactor was initially designed for nitrifying bacterial growth only. In the presence of cyanobacteria and algae, we monitored both the microbial activity by measuring specific oxygen production rate (SOPR) for microalgae and cyanobacteria and specific oxygen uptake rate (SOUR) for nitrifying bacteria. The growth of cyanobacteria and algae inhibited the maximum nitrification rate by a factor of 4 although the ammonium nitrogen fed to the reactor was almost completely removed. Terminal restriction fragment length polymorphism (T‐RFLP) analysis indicated that the community structures of nitrifying bacteria remained unchanged, containing the dominant Nitrosospira, Nitrospira, and Nitrobacter species. PCR amplification coupled with cloning and sequencing analysis resulted in identifying Chlorella emersonii and an uncultured cyanobacterium as the dominant species in the autotrophic bioreactor. Notwithstanding their fast growth rate and their toxicity to nitrifiers, microalgae and cyanobacteria were more easily lost in effluent than nitrifying bacteria because of their poor settling characteristics. The microorganisms were able to grow together in the bioreactor with constant individual biomass fractions because of the uncoupled solids retention times for algae/cyanobacteria and nitrifiers. The results indicate that compared to conventional wastewater treatment systems, longer solids retention times (e.g., by a factor of 4) should be considered in phototrophic bioreactors for complete nitrification and nitrogen removal. Biotechnol. Bioeng. 2010;107: 1004–1011. © 2010 Wiley Periodicals, Inc.     

Novel mathematical modeling on pilot-scale of plug-flow aerated submerged biofilm reactor for dissolved organic matter and nitrogen removal

The investigation aimed to present mathematical models for describing the dynamic behavior of the dissolved organic matter removal and nitrification in the Aerated Submerged Bio-Film (ASBF) for a plug-flow reactor. Based on the experimental data from the batch system of the ASBF pilot plant, mathematical models for the plug-flow reactor were developed to predict dissolved organic matter and ammonia nitrogen removal rates as a function of heterotrophic and autotrophic bacteria populations, dissolved organic matter concentrations, ammonia nitrogen concentrations, dissolved oxygen concentrations, and temperature. The mathematical models for dissolved organic matter and ammonia nitrogen removal in ASBF include two differential equations reflecting heterotrophic and autotrophic bacteria populations, and a number of kinetic parameters. Consequently, the results present a better insight into the dynamics behavior of heterotrophic and autotrophic biofilm growth and their practical application to wastewater for dissolved organic matter and ammonia nitrogen removal process. The mathematical model for ammonia nitrogen and dissolved organic matter removals present good results for the plug-flow reactor.     

In situ PCR for visualizing distribution of a functional gene "amoA" in a biofilm regardless of activity

In this study, ammonia-oxidizing bacteria present in biofilms resulting from a nitrifying reactor were detected by both a conventional FISH technique and an original in situ PCR technique. Both techniques showed that ammonia-oxidizing bacteria were found near the surface of the biofilms. However, after the biofilm had been exposed to 2 weeks of ammonia starvation, ammonia-oxidizing bacteria present in the biofilm could not be detected by fluorescence in situ hybridization (FISH) because they did not have sufficient copies of rRNA. In contrast, ammonia-oxidizing bacteria could be detected by in situ PCR with strong signal. It was thus demonstrated that a cell possessing a specific functional gene is detectable by in situ PCR regardless of its activity.     

羧酶体的组成、结构、功能和检测及其在脱氮细菌中的意义

羧酶体(Carboxysome)是一种具有CO2浓缩功能的"类细胞器",它存在于自养型脱氮细菌中,可增强细菌的自养生长能力。硝化细菌、厌氧氨氧化细菌和部分反硝化细菌都是重要的自养型脱氮细菌,探明其羧酶体的组成、结构和功能,将有助于揭示自养型脱氮菌的生长规律,进而强化生物脱氮过程。基于文献阅读和相关研究,本文对自养型细菌中羧酶体在组成、结构、功能和检测等方面的研究进展进行综述,以期为自养生物脱氮过程的深入理解和优化改进提供参考。     

Water quality management for low temperature marine fishes in space.

Vestibular Function Experiment Unit (VFEU), one of the Spacelab facility flown in Neurolab mission (STS-90) in April, 1998, was to support neurophysiological research using a marine fish, Opsanus tau (oyster toadfish). The functions of the VFEU were primarily a quality management of environmental water during the mission at 14 degrees C and for acquiring physiological signals from implanted micro-electrodes in the otolith nerves as well as the spatial acceleration of the fish. A key element of the life support system was a balanced biological filter containing two types of nitrifying bacteria, Nitrosomonas for ammonia oxidization and Nitrobacter for nitrite oxidization. Although the 16 days mission was successful, two toadfishes died in late phase of the mission. Ammonium concentration in those two life support systems elevated to remarkably high level at the end of the mission whereas the other two indicated very low. This report summarizes the results of the water quality management of the VFEU during the Neurolab mission based on analysis of water samples taken during the flight and those taken prior and just after the flight.