Enhanced nitrogen removal by bioelectrochemical coupling anammox and characteristics of microbial communities / 生物工程学报

To investigate the bioelectrochemical enhanced anaerobic ammonia oxidation (anammox) nitrogen removal process, a bioelectrochemical system with coupled anammox cathode was constructed using a dual-chamber microbial electrolysis cell (MEC). Specifically, a dark incubation batch experiment was conducted at 30 ℃ with different influent total nitrogen concentrations under an applied voltage of 0.2 V, and the enhanced denitrification mechanism was investigated by combining various characterization methods such as cyclic voltammetry, electrochemical impedance spectroscopy and high-throughput sequencing methods. The results showed that the total nitrogen removal rates of 96.9%±0.3%, 97.3%±0.4% and 99.0%±0.3% were obtained when the initial total nitrogen concentration was 200, 300 and 400 mg/L, respectively. In addition, the cathode electrode biofilm showed good electrochemical activity. High-throughput sequencing results showed that the applied voltage enriched other denitrifying functional groups, including Denitratisoma, Limnobacter, and ammonia oxidizing bacteria SM1A02 and Anaerolineaceae, Nitrosomonas europaea and Nitrospira, besides the anammox bacteria. These electrochemically active microorganisms comprised of ammonium oxidizing exoelectrogens (AOE) and denitrifying electrotrophs (DNE). Together with anammox bacteria Candidatus Brocadia, they constituted the microbial community structure of denitrification system. Enhanced direct interspecies electron transfer between AOE and DNE was the fundamental reason for the further improvement of the total nitrogen removal rate of the system.

Denitrifying phosphate accumulating organisms and its mechanism of nitrogen and phosphorus removal / 生物工程学报

Water eutrophication poses great threats to protection of water environment. Microbial remediation of water eutrophication has shown high efficiency, low consumption and no secondary pollution, thus becoming an important approach for ecological remediation. In recent years, researches on denitrifying phosphate accumulating organisms and their application in wastewater treatment processes have received increasing attention. Different from the traditional nitrogen and phosphorus removal process conducted by denitrifying bacteria and phosphate accumulating organisms, the denitrifying phosphate accumulating organisms can simultaneously remove nitrogen and phosphorus under alternated anaerobic and anoxic/aerobic conditions. It is worth noting that microorganisms capable of simultaneously removing nitrogen and phosphorus absolutely under aerobic conditions have been reported in recent years, but the mechanisms remain unclear. This review summarizes the species and characteristics of denitrifying phosphate accumulating organisms and the microorganisms capable of performing simultaneous nitrification-denitrification and phosphorous removal. Moreover, this review analyzes the relationship between nitrogen removal and phosphorus removal and the underlying mechanisms, discusses the challenges of denitrifying phosphorus removal, and prospects future research directions, with the aim to facilitate process improvement of denitrifying phosphate accumulating organisms.

Mechanism of trehalose-enhanced metabolism of heterotrophic nitrification-aerobic denitrification community under high-salt stress / 生物工程学报

Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria are aerobic microorganisms that can remove nitrogen under high-salt conditions, but their performance in practical applications are not satisfactory. As a compatible solute, trehalose helps microorganisms to cope with high salt stress by participating in the regulation of cellular osmotic pressure, and plays an important role in promoting the nitrogen removal efficiency of microbial populations in the high-salt environment. We investigated the mechanism of exogenous-trehalose-enhanced metabolism of HN-AD community under high-salt stress by starting up a membrane aerobic biofilm reactor (MABR) to enrich HN-AD bacteria, and designed a C150 experimental group with 150 μmol/L trehalose addition and a C0 control group without trehalose. The reactor performance and the community structure showed that NH4+-N, total nitrogen (TN) and chemical oxygen demand (COD) removal efficiency were increased by 29.7%, 28.0% and 29.1%, respectively. The total relative abundance of salt-tolerant HN-AD bacteria (with Acinetobacter and Pseudofulvimonas as the dominant genus) in the C150 group reached 66.8%, an 18.2% increase compared with that of the C0 group. This demonstrated that trehalose addition promoted the enrichment of salt-tolerant HN-AD bacteria in the high-salt environment to enhance the nitrogen removal performance of the system. In-depth metabolomics analysis showed that the exogenous trehalose was utilized by microorganisms to improve proline synthesis to increase resistance to high-salt stress. By regulating the activity of cell proliferation signaling pathways (cGMP-PKG, PI3K-Akt), phospholipid metabolism pathway and aminoacyl-tRNA synthesis pathway, the abundances of phosphoethanolamine, which was one of the glycerophospholipid metabolites, and purine and pyrimidine were up-regulated to stimulate bacterial aggregation and cell proliferation to promote the growth of HN-AD bacteria in the high-salt environment. Meanwhile, the addition of trehalose accelerated the tricarboxylic acid (TCA) cycle, which might provide more electron donors and energy to the carbon and nitrogen metabolisms of HN-AD bacteria and promote the nitrogen removal performance of the system. These results may facilitate using HN-AD bacteria in the treatment of high-salt and high-nitrogen wastewater.

Research progress of combined anaerobic ammonium oxidation (ANAMMOX) process for nitrogen removal / 生物工程学报

Anaerobic ammonia oxidation (ANAMMOX) process is an efficient and low-cost biological nitrogen removal process. However, it still faces some challenges in mainstream applications due to the limitation of substrate types and nitrate accumulation. In recent years, the combined process of anammox has been widely studied to solve the above problems. In this paper, the combined processes of anammox developed in recent years are reviewed, and discussed from the process principle, advantages and disadvantages, influencing factors, process extensibility and the key bottlenecks existing in the promotion and application, as well as the relevant work of the subject group. Finally, we take an outlook on the development of the combined anaerobic ammonia oxidation process in municipal domestic wastewater treatment.

Reatores híbridos anaeróbio e aeróbio para remoção de matéria orgânica e nitrogênio em esgoto doméstico diluído / Hybrid anaerobic and aerobic reactors for organic matter and nitrogen removal in diluted domestic sewage

RESUMO A remoção de matéria orgânica e de nitrogênio em esgoto doméstico diluído foi avaliada em dois reatores híbridos, um anaeróbio (RHAN) e outro aeróbio (RHAE). O RHAN era formado por uma câmara tipo upflow anaerobic sludge blanket sobreposta por outra de filtro anaeróbio, enquanto o RHAE tinha uma câmara de lodo ativado sobreposta por outra de biofilme aerado submerso. A operação do sistema foi dividida em duas fases, FI e FII, com razões de recirculação de 50 e 75% e duração de 94 e 110 dias, respectivamente. Para a remoção de nitrogênio, o RHAE foi operado com oxigênio dissolvido de 3,0 mg.L-1. A técnica da reação em cadeia da polimerase foi empregada tanto para o lodo suspenso das câmaras inferiores, como para o biofilme aderido nas câmaras superiores, para identificar a presença de micro-organismos desnitrificantes e nitrificantes. As maiores eficiências de remoção em termos de demanda química de oxigênio e nitrogênio total foram obtidas em FII, sendo 91% e ~50%, respectivamente; as concentrações no efluente foram ~40 mg O2.L-1 e ~15 mg N-NT.L-1. A presença de três grupos de bactérias, as desnitrificantes, as oxidantes de amônia e as oxidantes de nitrito, foi confirmada no biofilme aderido do RHAE, indicando uma biomassa mixotrófica e sugerindo a possibilidade do processo de nitrificação e desnitrificação simultânea.

ABSTRACT The removal of organic matter and nitrogen in diluted domestic sewage was evaluated in two hybrid reactors, one anaerobic (ANHR) and another aerobic (AEHR). ANHR was formed by a upflow anaerobic sludge blanket (UASB)-type chamber overlaid by an anaerobic filter, while AEHR had an activated sludge chamber overlaid by a submerged aerated biofilm chamber. The experimental period was divided into two phases, FI and FII, with recirculation ratios of 50 and 75%, and duration of 94 and 110 days, respectively. For nitrogen removal, AEHR was operated with a 3.0-mg.L-1 dissolved oxygen. Polymerase chain reaction technique was used for both suspended sludge in the down-chambers and attached biofilm in the upper-chambers, to identify the presence of denitrifying and nitrifying microorganisms. The highest removal efficiencies in terms of chemical oxygen demand and total nitrogen were obtained in FII, being 91 and ~50%, respectively; effluent concentrations were ~40 mg O2.L-1 and ~15 mg N-TN.L-1. The presence of three groups of bacteria, the denitrifiers, the ammonia oxidants and the nitrite oxidants, was confirmed in the attached biofilm of the RHAE, indicating a mixotrophic biomass and suggesting the possibility of simultaneous nitrification and denitrification process.

Effects of Fe₃O₄ on the denitrification performance of Pseudomonas stutzeri / 生物工程学报

Biological denitrification is the most widely used technology for nitrate removal in wastewater treatment. Conventional denitrification requires long hydraulic retention time, and the nitrate removal efficiency in winter is low due to the low temperature. Therefore, it is expected to develop new approaches to enhance the denitrification process. In this paper, the effect of adding different concentrations of Fe₃O₄ nanoparticles on the denitrification catalyzed by Pseudomonas stutzeri was investigated. The maximum specific degradation rate of nitrate nitrogen improved from 18.0 h⁻¹ to 23.7 h⁻¹ when the concentration of Fe₃O₄ increased from 0 mg/L to 4 000 mg/L. Total proteins and intracellular iron content also increased along with increasing the concentration of Fe₃O₄. RT-qPCR and label-free proteomics analyses showed that the relative expression level of denitrifying genes napA, narJ, nirB, norR, nosZ of P. stutzeri increased by 55.7%, 24.9%, 24.5%, 36.5%, 120% upon addition of Fe₃O₄, and that of denitrifying reductase Nap, Nar, Nir, Nor, Nos increased by 85.0%, 147%, 16.5%, 47.1%, 95.9%, respectively. No significant difference was observed on the relative expression level of denitrifying genes and denitrifying reductases between the bacteria suspended and the bacteria adhered to Fe₃O₄. Interestingly, the relative expression level of electron transfer proteins of bacteria adhered to Fe₃O₄ was higher than that of the bacteria suspended. The results indicated that Fe₃O₄ promoted cell growth and metabolism through direct contact with bacteria, thereby improving the denitrification. These findings may provide theoretical support for the development of enhanced denitrification.

Characteristics of the rhizosphere and phyllosphere microbial community of three submerged plants in the dragon-shaped water system of Beijing / 生物工程学报

Aquatic plants and the epiphytic microorganisms are important contributors to the purification of constructed wetlands. Taking the dragon-shaped water system of Beijing Olympic Park as a model, this study analyzed the structure and function of the microbial communities reside the sediment, the water body and the rhizosphere and phyllosphere of three submerged plants-Vallisneria natans, Myriophyllum verticillatum, and Potamogeton pectinatus using high-throughput sequencing technology. The results showed that the microbial diversity from the highest to the lowest were samples from sediment, plant rhizosphere, plant phyllosphere and water. The microbial diversity of plant phyllosphere samples were significantly higher than those of the water body. LEfSe analysis showed that different habitats enriched different microbial groups. The sediments mainly enriched anaerobic microbes, while the water body and the phyllosphere of plants mainly enriched aerobic microbes, and the rhizosphere of plants had the both. Functional prediction analysis showed that the abundance of denitrification marker genes in phyllosphere samples was higher than that in samples from rhizosphere, sediment and water body, and the abundance of denitrification marker genes in phyllosphere samples of M. verticillatum and P. pectinatus was higher than that of V. natans. This study could serve as a guidance for the selection of submerged plants and functional microorganisms for constructed wetlands.

Advances in denitrification microorganisms and processes / 生物工程学报

Denitrification is an indispensable part of most sewage treatment systems. The biological denitrification process has attracted much attention in the past decades due to the advantages such as cost-effectiveness, process simplicity, and absence of secondary pollution. This review summarized the advances on biological denitrification processes in recent years according to the different physiological characteristics and denitrification mechanisms of denitrification microorganisms. The pros and cons of different biological denitrification processes developed based on nitrifying bacteria, denitrifying bacteria, and anaerobic ammonia-oxidizing bacteria were compared with the aim to identify the best strategy for denitrification in a complex wastewater environment. The rapid development of synthetic biology provides possibilities to develop highly-efficient denitrifying strains based on mechanistic understandings. Combined with the applications of automatic simulation to obtain the optimal denitrification conditions, cost-effective and highly-efficient denitrification processed can be envisioned in the foreseeable future.

Craft Brewery Wastewater Treatment: a Fixed-Bed Single-Batch Reactor with Intermittent Aeration to Remove COD and TN

Abstract This study evaluated an intermittently aerated, fixed-bed, single-batch reactor, with mini BioBob© as biofilm media support, as an alternative treatment of craft brewery wastewater. In order to remove chemical oxygen demand (COD) and total nitrogen (TN), seven conditions were performed in a central composite experimental design (CCD) with different aeration times (1, 2 and 3 h in a 4 h cycle) and hydraulic retention times (HRT) (12, 16 and 20 h). The results showed that the removal of COD and TN were positively affected by increased aeration time and HRT. The condition that presented the best quality effluent was Condition No. 1 (20 h HRT and 3 h aeration), with 209 ± 28 mg COD L-1; 3.00 ± 0.15 mg TKN L-1 ; and 0.67 ± 0.11 mg NO3-N L-1. Kinetic assays showed that the highest values for the substrate removal rate constant, kCOD = 0.1774 h-1 were obtained with the longest aeration time (3 h). The most probable number (MPN) test showed a higher concentration of denitrifying bacteria (heterotrophic), 3.3 x 106, than for AOB and NOB bacteria (autotrophic), which were 4.9 x 103 and 2.7 x 103, respectively. Moreover, it was possible to verify that correcting the influent alkalinity with 7.14 mg CaCO3 for each 1 mg of TKN resulted in better process efficiency. It was concluded that COD and TN can be removed from craft brewery wastewater using an intermittently aerated, fixed-bed, single-batch reactor with mini Biobob© as biofilm media support.

Characterization of a novel heterotrophic nitrifying and aerobically denitrifying bacterium, Halomonas sp. Z8, as a potential bioagent for wastewater treatment

Aim: The aim of the present study was to identify and characterize the functions of key microbes mediating nitrification.Methodology: After sampling the biofilm from a submerged biofilter in a marine aquaculture system, selective media were used to isolate microbial strains involved in nitrification. Isolates were identified using physiological and biochemical assays and sequencing of 16S rRNA gene. Nitrogen removal under different conditions was characterized. Nitrogen removal pathway was characterized by a 15N tracer experiment. Representation of key microbes in the biolfim was characterized by metagenomics analysis. Results: Single-factor tests showed that Halomonas sp. strain Z8 exhibited good heterotrophic nitrification and aerobic denitrification abilities, with maximum NH4+-N, NO2--N and No3--N removal rates of 2.37, 1.28 and 1.7 mg N l-1 hr-1, respectively. The 15N isotope tracer experiment confirmed the aerobic nitrogen removal pathway of strain Z8. Average NO3-removal efficiencies were all above 80% in an aerated moving bed bioreactor inoculated with strain Z8 and employed to treat synthetic marine aquaculture wastewater. Metagenomic microbial community analysis revealed that Halomonas sp. Z8 was one of the dominant taxa at genus level, suggesting a vital role in removing nitrate from bioreactor

Capacidade metabólica de bactérias heterotróficas de sistemas de lodo ativado em ambientes aeróbios e anóxicos / Metabolic capacity of heterotrophic bacteria of activated sludge systems in aerobic and anoxic environments

RESUMO Para projetos de sistemas de lodo ativado em países com temperaturas elevadas, como a do Brasil, sugere-se a introdução de um ou mais reatores anóxicos, visto que a nitrificação torna-se um fator quase inevitável. A desnitrificação é um processo capaz de reduzir custos com aeração em até 20%, recuperar a alcalinidade perdida com a nitrificação e ainda remover matéria orgânica e nitrogênio. Este estudo teve como objetivo avaliar a capacidade metabólica de bactérias heterotróficas desses sistemas em ambiente aeróbio e anóxico via nitrato e via nitrito. Para os testes foram comparadas duas configurações: Bardenpho e University of Cape Town (UCT). Os resultados mostraram significativa viabilidade da desnitrificação via nitrato, pois as taxas médias obtidas de consumo de oxigênio e oxigênio equivalente foram semelhantes para ambos os sistemas, indicando que, além da remoção do nitrogênio, a matéria orgânica é removida com a mesma eficiência de um reator aeróbio, possibilitando a redução de custos com aeração devido à possibilidade de limitá-lo apenas ao processo de nitrificação. Contudo, na desnitrificação pelo uso do nitrito, percebeu-se uma redução na capacidade de remoção de matéria orgânica em até 60%, que pode inferir em uma vantagem duvidosa na opção de um processo de nitrificação e desnitrificação simultâneo, mesmo que este apresente menor demanda de oxigênio dissolvido (OD). Entretanto, pode ser viável a sua utilização quando houver também a pretensão de remoção de fósforo via nitrito por organismos acumuladores de fósforo.

ABSTRACT For projects of activated sludge systems in countries with high temperatures such as Brazil, the introduction of one or more anoxic reactors is suggested, since nitrification becomes an almost inevitable factor. Denitrification is a process able to reduce aeration costs by up to 20%, recover the alkalinity lost with nitrification and still remove organic matter and nitrogen. This study aimed to evaluate the metabolic capacity of heterotrophic bacteria of these systems in aerobic and anoxic environment via nitrate and nitrite. Two configurations were compared for the tests: Bardenpho and University of Cape Town (UCT). The results showed significant viability of denitrification via nitrate, as the average rates obtained from oxygen consumption and oxygen equivalents were similar for both systems, indicating that beyond the removal of nitrogen, organic matter is removed with the same efficiency as an aerobic reactor enabling cost reduction with aeration due to the possibility of limiting it only to the nitrification process. However, denitrification by the use of nitrite showed a reduction in the ability of removal of organic matter by 60%, which may lead to a doubtful advantage in the option of a simultaneous nitrification and denitrification process, even if it presents minor oxygen demand. However, it may present exceptions, such as when there is also the claim of phosphorus removal via nitrite by phosphorus-accumulating organisms that may overcome the weakness of this rate.

Bioprocess of nitrite accumulation in water - a review / 生物工程学报

Nitrite is a by-product of the nitrogen cycle. The excessive nitrite not only constrains growth and metabolism of bacteria, but also impairs health of humans and aquatic organisms. On the other hand, the continuous maintaining of nitrite accumulation could achieve the shortcut nitrification process, and reduce energy consumption of biological nitrogen removal to save cost. This article reviews the biological processes and causes of nitrite accumulation in the water environment, and summarizes the factors that affect the accumulation of nitrite, to provide reference for wastewater treatments, including improving the nitrogen removal efficiency, reducing operating costs, decreasing discharge of sewage and nitrite nitrogen in natural water.

Lodo ativado com aeração prolongada operado sob baixa concentração de oxigênio dissolvido: comportamento cinético das bactérias heterotróficas e autotróficas nitrificantes / Extended aeration activated sludge process operated under low dissolved oxygen concentration: Kinetic behavior of nitrifying heterotrophic and autotrophic bacteria

RESUMO Este trabalho teve como objetivo a determinação dos parâmetros cinéticos que descrevem o crescimento das bactérias heterotróficas e autotróficas nitrificantes em três sistemas de lodos ativados com aeração prolongada, operados com baixa aeração, a fim de se obter a remoção de nitrogênio de esgoto sanitário pelo processo de nitrificação e desnitrificação simultâneas (NDS). Em relação às remoções de matéria orgânica e nitrogênio, aplicando-se idade do lodo na faixa de 12 a 20 dias e concentrações de oxigênio dissolvido (OD) nos reatores em torno de 0,5 mg O2.L-1, foi possível obter eficiências superiores a 90% e um esgoto tratado com concentrações de nitrato inferiores a 7,0 mg N.L-1. Os experimentos realizados comprovam que, nas condições operacionais adotadas durante os testes respirométricos, as bactérias heterotróficas e autotróficas presentes nos lodos ativados estavam ativos e, ainda que baixa, a concentração de OD não foi limitante para o desenvolvimento da cinética dos processos envolvidos na remoção de materiais orgânico e nitrogenado.

ABSTRACT The objective of this work was to determine the kinetic parameters that describe the growth of heterotrophic and autotrophic nitrifying bacteria in three systems of activated sludge with prolonged aeration, operated with low aeration, with the objective of obtaining nitrogen removal of sanitary sewage by simultaneous nitrification and denitrification (SND). In relation to the removal of organic matter and nitrogen, by using sludge ages ranging from 12 to 20 days and OD concentrations in the reactors around 0.5 mgO2.L-1, it was possible to obtain efficiencies higher than 90% and a sewer treated with nitrate concentrations below 7.0 mgN.L-1. The experiments carried out prove that, under the operating conditions adopted during the respirometric tests, heterotrophic and autotrophic bacteria present in activated sludge were active and, although the low concentration of OD was not limiting for the development of the kinetics of the processes involved in the removal of organic and nitrogenous material.

Remoção simultânea de material orgânico, nitrogênio e fósforo em um reator em bateladas sequenciais com biofilme de leito móvel operado pelo processo anaeróbio-anóxico-óxico / Simultaneous removal of organic material, nitrogen and phosphorus in a reactor in batches of mobile bed biofilm sequencing by the anaerobic-anoxic-oxic process

RESUMO Neste estudo, foi avaliado o desempenho de um reator em bateladas sequenciais com biofilme de leito móvel operado pelo processo anaeróbio-anóxico-óxico (A2O), aplicado à remoção simultânea de material orgânico, nitrogênio e fósforo de esgoto sanitário. Utilizaram-se dois reatores com volume útil de 36 L cada, sendo que um deles recebeu anéis plásticos móveis do tipo Kaldnes®, com área superficial específica estimada em 310 m2/m3 que ocuparam 50% do volume útil do reator (denominado aqui de MBSBBR). O estudo foi conduzido com tempo de retenção de sólidos (TRS) de 12 dias, tempo de retenção hidráulico (TRH) de 6 horas, taxa de alimento/microrganismo (A/M) de 0,4 a 0,5 gDQO/SSV.d, oxigênio dissolvido (OD) na faixa de 0,3-0,8 mgO2/L e taxas de aplicação de carga orgânica, nitrogênio e fósforo de 47,2 gDQO/L.d, 5,4 gNTK/L.d e 0,4 gP/L.d, respectivamente. Os resultados de eficiência de remoção de demanda química de oxigênio (DQO), N-NH3 e fósforo foram de 91,1, 90,5 e 85,4% para o reator MBSBBR e de 89,5, 91,6 e 84,4% para o reator RBS, respectivamente. Os resultados do teste estatístico de ANOVA foram aplicados às principais variáveis estudadas, e os valores convergiram para valores de f observado < f crítico . Dessa forma, considera-se que houve similaridade de desempenho nos reatores MBSBBR e RBS. Conclui-se que a introdução do meio suporte não trouxe benefícios ao tratamento em termos de remoção de nutrientes. No entanto, o estudo mostrou que é possível de se obter elevada remoção de nitrogênio, em baixas concentrações de OD, o que se torna vantajoso para o tratamento de esgoto com elevada concentração de nitrogênio.

ABSTRACT In this study, the performance of a reactor in sequential batch with mobile bed biofilm operated by the anaerobic-anoxic-oxic (A2O) process, applied to the simultaneous removal of organic material, nitrogen and phosphorus from sanitary sewage was evaluated. Two reactors with a useful volume of 36 liters each were used, one of them receiving Kaldnes® type mobile plastic rings, with a specific surface area estimated at 310 m2/m3, which occupied 50% of the reactor's useful volume (MBSBBR). The study was conducted with 12-day solid retention time (SRT), 6 hour hydraulic retention time (HRT), food/microorganism (F/M) ratio of 0.4 to 0.5 gCOD/VSS.d, dissolved oxygen (DO) in the range of 0.3-0.8 mgO2/L and application rates of organic load, nitrogen and phosphorus of 47.2 gCOD/L.d, 5.4 gTNK/L.d and 0.4 gP/L.d, respectively. The efficiency of chemical oxygen demand (COD), N-NH3 and Phosphorus removal was of 91.1%, 90.5% and 85.4% for the MBSBBR reactor and 89.5%, 91.6% and 84.4% for the RBS reactor, respectively. The results of the statistical ANOVA test were applied to the main variables studied and the values converged to values of f observed < f critical . In this way, it is considered that there was a similarity of performance between the MBSBBR and RBS reactors. It is concluded that the introduction of the support medium did not bring benefits to the treatment in terms of nutrient removal. However, the study showed that it is possible to obtain high nitrogen removal at low concentrations of DO, which is advantageous for the sewage treatment with high nitrogen concentration.

Advances in heterotrophic nitrification-aerobic denitrifying bacteria for nitrogen removal under extreme conditions / 生物工程学报

Heterotrophic nitrification-aerobic denitrification (HN-AD) is an enrichment and breakthrough theory of traditional autotrophic nitrification heterotrophic denitrification. Heterotrophic nitrification-aerobic denitrifiers with the feature of wide distribution, strong adaptability and unique metabolic mechanism have many special advantages, including fast-growing, rapid biodegradability and long lasting activity, which can rapidly remove ammonia nitrogen, nitrate nitrogen (NO₃⁻-N) and nitrite nitrogen (NO₂⁻-N) under aerobic conditions simultaneously. Therefore, HN-AD bacteria show the important potential for denitrification under extreme conditions with high-salt, low-temperature or high-ammonia nitrogen environment, and HN-AD bacteria attract extensive attention in the field of biological denitrification of wastewater. In this review, we first introduce the previously reported HN-AD bacterial species which have denitrification performance in the extreme environments and state their typical metabolic mechanism. Then, we systematically analyze the nitrogen removal characteristics and potential under extreme conditions. We also briefly describe the progress in the application of HN-AD bacterial. Finally, we outlook the application prospects and research directions of HN-AD denitrification technology.

Draft genome sequence of Vitellibacter aquimaris D-24T isolated from seawater

ABSTRACT Vitellibacter aquimaris D-24T (=KCTC 42708T = DSM 101732T), a halophilic marine bacterium, was isolated from seawater collected from Desaru beach, Malaysia. Here, we present the draft genome sequence of D-24T with a genome size of approximately 3.1 Mbp and G + C content of 39.93%. The genome of D-24T contains genes involved in reducing a potent greenhouse gas (N2O) in the environment and the degradation of proteinaceous compounds. Genome availability will provide insights into potential biotechnological and environmental applications of this bacterium.

Emisión de óxido nitroso, nitrificación, desnitrificación y mineralización de nitrógeno durante el cultivo del arroz en 2 suelos de Uruguay / Nitrous oxide emission, nitrification, denitrification and nitrogen mineralization during rice growing season in 2 soils from Uruguay

Los procesos microbianos como la mineralización, la nitrificación y la desnitrificación regulan la dinámica del nitrógeno en el suelo. Estos 2 últimos son los principales responsables de la emisión de óxido nitroso (N2O). En este trabajo se determinaron los flujos de N2O en momentos clave del ciclo de cultivo del arroz en 2 sitios que diferían principalmente en el contenido de materia orgánica del suelo (MO), en las localidades de Salto (mayor MO) y de Treinta y Tres. Dichos momentos clave fueron a la siembra, en macollaje, en primordio floral y a la madurez. También se determinó el potencial de mineralización neta de N y las actividades y los números más probables (NMP) de oxidantes de NH4+ y de desnitrificantes. El potencial de mineralización de N, así como la actividad y el NMP de oxidantes de NH4+, no variaron con el tipo de suelo. Sin embargo, la actividad y el NMP de desnitrificantes fueron mayores en el suelo con mayor contenido de MO, independiente de la etapa del cultivo. A su vez, en las etapas finales del ciclo del cultivo, el NMP de desnitrificantes aumentó coincidiendo con el mayor potencial de mineralización y el mayor contenido de N mineral del suelo. Solo se observó un incremento en el flujo de N2O en el suelo de Salto a la madurez del arroz y cuando el suelo ya había sido drenado (44,2g N-N2O/ha d, frente a 20,8g N-N2O/ha d en Treinta y Tres). Esta investigación señala la importancia de estudiar las emisiones en distintos tipos de suelos y de continuar la medición luego del drenaje del cultivo de arroz para la elaboración de los inventarios de gases de efecto invernadero.

Microbial processes such as mineralization, nitrification and denitrification regulate nitrogen dynamics in the soil. The last two processes may produce nitrous oxide (N2O). In this work N2O fluxes were quantified at four moments of the rice cycle, sowing, tillering, panicle initiation and maturity, in two sites that differed mainly in their soil organic matter (OM) content, Salto (higher OM) and Treinta y Tres. Potential net N mineralization, ammonium oxidation and denitrification as well as the most probable numbers (MPN) of ammonia oxidizers and denitrifiers were determined. Potential N mineralization did not vary with the soil type and increased at rice maturity. Neither ammonia oxidation potential nor MPN were different among the soils. However, the soil with higher OM exhibited higher activity and MPN of denitrifiers, irrespective of the rice stage. In turn, at the latest phases of the crop, the MPN of denitrifiers increased coinciding with the highest mineralization potential and mineral N content of the soil. Significant differences in N2O flux were observed in Salto, where the highest emissions were detected at rice maturity, after the soil was drained (44.2 vs 20.8g N-N2O/ha d in Treinta y Tres). This work shows the importance of considering the soil type and end-of-season drainage of the rice field to elaborate GHGs (greenhouse gases) inventories.

Effects of different external carbon sources and electron acceptors on interactions between denitrification and phosphorus removal in biological nutrient removal processes / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The effects of two different external carbon sources (acetate and ethanol) and electron acceptors (dissolved oxygen, nitrate, and nitrite) were investigated under aerobic and anoxic conditions with non-acclimated process biomass from a full-scale biological nutrient removal-activated sludge system. When acetate was added as an external carbon source, phosphate release was observed even in the presence of electron acceptors. The release rates were 1.7, 7.8, and 3.5 mg P/(g MLVSS·h) (MLVSS: mixed liquor volatile suspended solids), respectively, for dissolved oxygen, nitrate, and nitrite. In the case of ethanol, no phosphate release was observed in the presence of electron acceptors. Results of the experiments with nitrite showed that approximately 25 mg NO2-N/L of nitrite inhibited anoxic phosphorus uptake regardless of the concentration of the tested external carbon sources. Furthermore, higher denitrification rates were obtained with acetate (1.4 and 0.8 mg N/(g MLVSS·h)) compared to ethanol (1.1 and 0.7 mg N/ (g MLVSS·h)) for both anoxic electron acceptors (nitrate and nitrite).

Novos reguladores de resposta envolvidos na virulência de Pseudomonas aeruginosa / New response regulators involved in Pseudomonas aeruginosa virulence

Os sistemas de sinalização de dois componentes são sistemas prevalentes em bactérias, permitindo a adaptação a diferentes condições ambientais. O sistema de dois componentes classicamente possui uma proteína histidina quinase, o primeiro componente, capaz de reconhecer o estímulo ambiental e fosforilar o regulador de resposta, o segundo componente. Pseudomonas aeruginosa é uma proteobactéria ubíqua, capaz de infectar hospedeiros filogeneticamente distintos. Esse patógeno oportunista apresenta um dos maiores conjuntos de sistemas de dois componentes em bactérias, que permite que ela sobreviva numa grande gama de ambientes, incluindo humanos. P. aeruginosa UCBPP-PA14 apresenta pelo menos 64 histidina quinases e 76 reguladores de resposta codificados em seu genoma. Diversos sistemas de dois componentes já foram correlacionados com a virulência, sendo o sistema GacSA o exemplo melhor caracterizado. Há poucos estudos sistemáticos sobre o envolvimendo dos reguladores de resposta na virulência de P. aeruginosa e os sinais que induzem a ativação dos reguladores de resposta precisam ser encontrados. Para identificar novos reguladores de resposta envolvidos na patogenicidade, infecções in vitro em macrófagos e in vivo em Drosophila melanogaster foram realizadas neste trabalho. Os macrófagos foram infectados com cada mutante dos reguladores de resposta ou com a linhagem selvagem, e a produção da citocina pró-inflamatória TNF-α e o clearance bacteriano foram determinados. Alternativamente, as moscas foram infectadas utilizando-se a estratégia de feeding e a sobrevivência foi verificada. Utilizando-se essas abordagens, a identificação de diversos reguladores de resposta com papel na virulência foi alcançada, além de se corfirmar o papel de reguladores de resposta já estudados. Um dos novos genes envolvidos em virulência, PA14_26570 (nomeado neste trabalho de atvR), codifica um regulador de resposta atípico com substituição no aspartato fosforilável para glutamato, o que usualmente induz um estado sempre ativo. Um mutante não polar em atvR foi construído e macrófagos infectados com a linhagem ΔatvR confirmaram um maior clearance bacteriano e maior produção de TNF-α em comparação aos macrófagos infectados com a linhagem selvagem. Para comprovar a participação de AtvR durante a patogênese, um modelo de pneumonia aguda em camundongos foi utilizado. Camundongos infectados com a linhagem ΔatvR apresentaram uma maior sobrevivência em comparação aos camundongos infectados com a linhagem selvagem. Além disso, os camundongos infectados com ΔatvR apresentaram menor carga bacteriana, aumento no recrutamento de neutrófilos ativados e aumento na produção de citocinas pró-inflamatórias (TNF-α e IFN-γ). Utilizando-se uma abordagem transcritômica (RNA-Seq), foi determindo diversos genes são regulados positivamente na linhagem superexpressando AtvR em relação à linhagem controle. Dentre esses, os clusters de respiração anaeróbia nar, nir, nor e nos estão incluídos. Esse resultado foi confirmado por qRT-PCR e análises fenotípicas, em que a linhagem ΔatvR apresentou menor crescimento e expressão da nitrato redutase durante condições de hipóxia em comparação à linhagem selvagem. Em suma, neste trabalho foi demonstrado que diversos reguladores de resposta são importantes para a virulência de P. aeruginosa em macrófagos in vitro e in vivo em Drosophila, além de caracterizar o regulador de resposta atípico AtvR, que regula a respiração anaeróbica por desnitrificação, permitindo que P. aeruginosa possa infectar e colonizar o hospedeiro com maior eficiência

Two-component systems are widespread in bacteria, allowing the adaptation to environmental changes. A two-component system is classically composed by a sensor kinase that phosphorylates a cognate response regulator. Pseudomonas aeruginosa is a ubiquitous proteobacterium able to cause disease in several hosts. This opportunistic pathogen presents one of the largest sets of two-component systems known in bacteria, which certainly contributes to its ability to thrive in a wide range of environmental settings, including humans. P. aeruginosa UCBPP-PA14 genome codes for at least 64 sensor kinases and 76 response regulators. Some response regulators are already known to be related to virulence, with the GacSA system as the best characterized. There are no systematic studies about the involvement of P. aeruginosa response regulators in virulence. Moreover, the input signal that triggers the response regulator activation is yet to be uncovered for most systems. To find new response regulators involved in virulence, in vitro infections werecarried out using macrophages. Briefly, the macrophages were infected with each response regulator mutant or the wild-type strain, the pro-inflammatory cytokine production (TNF-α) and the bacterial clearance were evaluated. Using this approach, we identified several response regulators involved in virulence, and we also confirmed the involvement of known response regulators in this process. One of the novel virulence-related response regulators, PA14_26570 (named here as AtvR), is an atypical response regulator with a substitution in the phosphorylable aspartate to glutamate, that usually leads to an always-on state. A non-polar mutant was constructed, and macrophage infection with ΔatvR confirmed an increased bacterial clearance as well as a higher TNF-α production as compared to the wild-type strain. To ascertain the role of AtvR during the pathogenic process, an acute pneumonia model was used. Mice infected with ΔatvR showed an increased survival as compared to mice infected with the wildtype strain. In addition, ΔatvR infected mice showed reduced bacterial burden, increased neutrophil recruitment and activation, as well as increased pro-inflammatory cytokine production (TNF-α and IFN-γ). Also, using a transcriptomic approach (RNASeq), we showed that several genes were upregulated in the strain overexpressing AtvR. These genes include the anaerobic respiration clusters nar, nir, nor and nos. This result was confirmed by qRT-PCR and phenotypic analysis, in which ΔatvR showed reduced growth and nitrate reductase expression during hypoxic conditions as compared to the wild-type strain. In conclusion, we have demonstrated that several response regulators are important for P. aeruginosa virulence in vitro. In addition, we further characterized the atypical response regulator AtvR, which regulates anaerobic respiration via denitrification, allowing this bacterium to infect and colonize the host more efficiently

Complete genome sequence of the aerobically denitrifying thermophilic bacterium Chelatococcus daeguensis TAD1

ABSTRACT Chelatococcus daeguensis TAD1 is a themophilic bacterium isolated from a biotrickling filter used to treat NOx in Ruiming Power Plant, located in Guangzhou, China, which shows an excellent aerobic denitrification activity at high temperature. The complete genome sequence of this strain was reported in the present study. Genes related to the aerobic denitrification were identified through whole genome analysis. This work will facilitate the mechanism of aerobic denitrification and provide evidence for its potential application in the nitrogen removal.