Evolution of bacterial denitrification and denitrifier diversity

Little is known about the role of nitrate in evolution of bacterial energy-generating mechanisms. Denitrifying bacteria are commonly regarded to have evolved from nitrate-respiring bacteria. Some researchers regard denitrification to be the precursor of aerobic respiration; others feel the opposite is true. Currently recognized denitrifying bacteria such as Hyphomicrobium, Paracoccus, Pseudomonas and Thiobacillus form a very diverse group. However, inadequate testing procedures and uncertain taxonomic identification of many isolates may have overstated the number of genera with species capable of denitrification. Nitrate reductases are structurally similar among denitrifying bacteria, but distinct from the enzymes in other nitrate-reducing organisms. Denitryfying bacteria have one of two types of nitrite reductase, either a copper-containing enzyme or an enzyme containing a cytochrome cd moiety. Both types are distinct from other nitrate reductases. Organisms capable of dissimilatory nitrate reduction are widely distributed among eubacterial groups defined by 16S ribosomal RNA phylogeny. Indeed, nitrate reduction is an almost universal property of actinomycetes and enteric organisms. However, denitrification is restricted to genera within the purple photosynthetic group. Denitrification within the genus Pseudomonas is distributed in accordance with DNA and RNA homology complexes. Denitrifiers seem to have evolved from a common ancestor within the purple photosynthetic bacterial group, but not from a nitrate-reducing organism such as those found today. Although denitrification seems to have arisen at the same time as aerobic respiration, the evolutionary relationship between the two cannot be determined at this time.     

Soil and sediment bacteria capable of aerobic nitrate respiration.

Several laboratory strains of gram-negative bacteria are known to be able to respire nitrate in the presence of oxygen, although the physiological advantage gained from this process is not entirely clear. The contribution that aerobic nitrate respiration makes to the environmental nitrogen cycle has not been studied. As a first step in addressing this question, a strategy which allows for the isolation of organisms capable of reducing nitrate to nitrite following aerobic growth has been developed. Twenty-nine such strains have been isolated from three soils and a freshwater sediment and shown to comprise members of three genera (Pseudomonas, Aeromonas, and Moraxella). All of these strains expressed a nitrate reductase with an active site located in the periplasmic compartment. Twenty-two of the strains showed significant rates of nitrate respiration in the presence of oxygen when assayed with physiological electron donors. Also isolated was one member of the gram-positive genus Arthrobacter, which was likewise able to respire nitrate in the presence of oxygen but appeared to express a different type of nitrate reductase. In the four environments studied, culturable bacteria capable of aerobic nitrate respiration were isolated in significant numbers (10(4) to 10(7) per g of soil or sediment) and in three cases were as abundant as, or more abundant than, culturable bacteria capable of denitrification. Thus, it seems likely that the corespiration of nitrate and oxygen may indeed make a significant contribution to the flux of nitrate to nitrite in the environment.     

Perchlorate and nitrate remediation efficiency and microbial diversity in a containerized wetland bioreactor

We have developed a method to remove perchlorate (14-27 microg/L) and nitrate (48 mg/L) from contaminated groundwater using a wetland bioreactor. The bioreactor has operated continuously in a remote field location for more than 2 yr with a stable ecosystem of indigenous organisms. This study assesses the bioreactorfor long-term perchlorate and nitrate remediation by evaluating influent and effluent groundwater for oxidation-reduction conditions and nitrate and perchlorate concentrations. Total community DNA was extracted and purified from 10-g sediment samples retrieved from vertical coring of the bioreactor during winter. Analysis by denaturing gradient gel electrophoresis of short, 16S rDNA, polymerase-chainreaction products was used to identify dominant microorganisms. Bacteria genera identified were closely affiliated with bacteria widely distributed in soils, mud layers, and fresh water. Of the 17 dominant bands sequenced, most were gram negative and capable of aerobic or anaerobic respiration with nitrate as the terminal electron acceptor (Pseudomonas, Acinetobacter, Halomonas, and Nitrospira). Several identified genera (Rhizobium, Acinetobactor, and Xanthomonas) are capable of fixing atmospheric nitrogen into a combined form (ammonia) usable by host plants. Isolates were identified from the Proteobacteria class, known for the ability to reduce perchlorate. Initial bacterial assessments of sediments confirm the prevalence of facultative anaerobic bacteria capable of reducing perchlorate and nitrate in situ.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The s springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Sulfide persistence in oil field waters amended with nitrate and acetate

Nitrate amendment is normally an effective method for sulfide control in oil field-produced waters. However, this approach has occasionally failed to prevent sulfide accumulation, despite the presence of active nitrate-reducing bacterial populations. Here, we report our study of bulk chemical transformations in microcosms of oil field waters containing nitrate-reducing, sulfide-oxidizing bacteria, but lacking denitrifying heterotrophs. Amendment with combinations of nitrate, acetate, and phosphate altered the microbial sulfur and nitrogen transformations. Elemental sulfur produced by chemotrophic nitrate-reducing bacteria was re-reduced heterotrophically to sulfide. Ammonification, rather than denitrification, was the predominant pathway for nitrate reduction. The application of nitrite led to transient sulfide depletion, possibly due to higher rates of nitrite reduction. The addition of molybdate suppressed both the accumulation of sulfide and the heterotrophic reduction of nitrate. Therefore, sulfidogenesis was likely due to elemental sulfur-reducing heterotrophic bacteria, and the nitrate-reducing microbial community consisted mainly of facultatively chemotrophic microbes. This study describes one set of conditions for continued sulfidogenesis during nitrate reduction, with important implications for nitrate control of sulfide production in oil fields.     

The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification

Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase in Pseudomonas sp. strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap- mutant, but that activity could be restored by complementation with the nap region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.     

洱海沉积物中反硝化细菌的分离与反硝化作用研究

自洱海十个点位的沉积物中富集筛选出101株反硝化细菌并从中筛选出1株较强反硝化能力的细菌,命名为EH314。该细菌接触酶(过氧化氢酶)试验、产硫化氢试验和淀粉水解均为阳性,葡萄糖氧化发酵实验结果为氧化菌,产脂酶(Tween 80)试验结果为阴性;初步鉴定该菌为产碱杆菌属细菌;对细菌反硝化能力进行测定发现,菌株EH314能有效地降解水体中的硝酸盐且反硝化可在有氧条件下进行。     

Effect of periplasmic nitrate reductase on diauxic lag of Paracoccus pantotrophus

Paracoccus pantotrophus expresses two nitrate reductases—membrane bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap). In growth experiments with two denitrifying species (Paracoccus pantotrophus and Alcaligenes eutrophus) that have both Nap and Nar and two species (Pseudomonas denitrificans and Pseudomonas fluorescens) with Nar only, it was found that diauxic lag is shorter for bacteria that express Nap. In P. pantotrophus, napEDABC encodes the periplasmic nitrate reductase. To analyze the effect of Nap on diauxic lag, the nap operon was deleted from P. pantotrophus. The growth experiments with nap^? mutant resulted in increased diauxic lag when switched from aerobic to anoxic respiration, suggesting Nap is responsible for shorter lags and helps in adaptation to anoxic metabolism after transition from aerobic conditions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

好氧反硝化细菌碳氮代谢特点及途径的研究进展

好氧反硝化作用的发现打破了反硝化只能在严格厌氧条件下进行的传统认知,为生物脱氮提供了一条新的途径,已成为近些年的研究热点。碳源可为好氧反硝化过程提供能量和电子供体,其代谢难易程度直接影响着好氧反硝化细菌的脱氮效率,因此有必要明确碳源在好氧反硝化脱氮过程中的代谢机理。基于此,本文阐述了好氧反硝化细菌的种类及其对硝态氮与亚硝态氮的代谢途径;系统分析了不同好氧反硝化细菌对碳氮源代谢的差异与代谢机理;综合分析了碳代谢差异对好氧反硝化脱氮过程的影响,并对未来的研究方向进行了展望,旨在深入理解好氧反硝化细菌同时去除碳氮的机理,为提高废水生物脱氮除碳效率提供理论依据。     

喹啉废水反硝化反应器中优势菌的代谢功能分析

采用纯培养技术对一个降解喹啉的反硝化反应器筛选到的26株优势菌株进行反硝化能力以及好氧降解喹啉能力研究,其中的反硝化菌还测定了反硝化条件下的喹啉降解能力。结果发现Bacillus、Staphylococcus、Pseudomonas、Brucella、Delftia等5个属的10株菌具有反硝化能力,Rhodococcus属的9株细菌能够好氧降解喹啉,揭示了反硝化喹啉降解反应器中主要细菌类型的代谢特性,发现在缺氧反硝化反应器中存在多样的代谢类型的细菌。     

基于响应面法对一株好氧反硝化菌脱氮效能优化

【目的】水体富营养化是当今我国水环境面临的重大水域环境问题,氮素超标排放是主要的引发因素之一。好氧反硝化菌构建同步硝化反硝化工艺比传统脱氮工艺优势更大。获得高效的好氧反硝化菌株并通过生长因子优化使脱氮效率达到最高。【方法】经过序批式生物反应器(Sequencing batch reactor,SBR)的定向驯化,筛选获得高效好氧反硝化菌株,采用响应面法优化好氧反硝化过程影响总氮去除效率的关键因子(碳氮、溶解氧、pH、温度)。【结果】从运行稳定的SBR反应器中定向筛选高效好氧反硝化菌株Pseudomonas T13,采用响应面法对碳氮比、pH和溶解氧关键因子综合优化获得在18 h内最高硝酸盐去除率95%,总氮去除率90%。该菌株的高效反硝化效果的适宜温度范围为25?30 °C;最适pH为中性偏碱;适宜的COD/NO3?-N为4:1以上;最佳溶解氧浓度在2.5 mg/L。【结论】从长期稳定运行的SBR反应器中筛选获得一株高效好氧反硝化菌Pseudomonas T13,硝酸盐还原酶比例占脱氮酶基因的30%以上,通过运行条件优化获得硝氮去除率达到90%以上,对强化废水脱氮工艺具有良好应用价值。     

Identification of two new denitrifying strains of Rhodobacter sphaeroides

Abstract Highly specific polyclonal and antibodies against either nitrate, nitrite or nitrous oxide reductases from a photosynthetic denitrifying bacterium Rhodobacter sphaeroides f. sp. denitrificans were used to show the presence of immunologically reactive proteins in strains that Pellerin and Gest had shown to grow in the dark with nitrate as a terminal acceptor [9]. Two strains of this bacterium, namely 81-3 and 2.4.3 synthesized the three denitrifying enzymes and were capable of denitrification. Strains 81-1 and 2.4.1 (neotype) both expressed nitrate reductase activities but nitrite reductase was not detected since these strains did not reduce nitrite. They also did not grow in the dark with nitrate as a terminal acceptor. Each of strains 81-1, 81-3, 2.4.1 and 2.4.3 contain four plasmids. R. sphaeroides f. sp. denitrificans , however, contains only one large 108 kb plasmid, which is distinctly different in size from those detected in the other strains. This indicates that the 108 kb plasmid is not necessarily specific for denitrification.     

Aerobic denitrification: a controversy revived

During studies on the denitrifying mixotroph, Thiosphaera pantotropha, it has been found that this organism is capable of simultaneously utilizing nitrate and oxygen as terminal electron acceptors in respiration. This phenomenon, termed aerobic denitrification, has been found in cultures maintained at dissolved oxygen concentrations up to 90% of air saturation.The evidence for aerobic denitrification was obtained from a number of independant experiments. Denitrifying enzymes were present even in organisms growing aerobically without nitrate. Aerobic yields on acetate were higher (8.1 g protein/mol) without than with (6.0 g protein/mol) nitrate, while the anaerobic yield with nitrate was even lower (4 g protein/mol). The maximum specific growth rate of Tsa. pantotropha was higher (0.34 h^-1) in the presence of both oxygen (>80% air saturation) and nitrate than in similar cultures not supplied with nitrate (0.27 h^-1), indicating that the rate of electron transport to oxygen was limiting. This was confirmed by oxygen uptake experiments which showed that although the rate of respiration on acetate was not affected by nitrate, the total oxygen uptake was reduced in its presence. The original oxygen uptake could be restored by the addition of denitrification inhibitors.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

好氧反硝化生物脱氮技术的研究进展

好氧反硝化生物脱氮技术自提出以来,凭借能实现同步硝化反硝化、节省基建投资及运行费用等诸多优点,受到国内外环境领域学者的广泛关注。本文首先总结了近年来好氧反硝化菌种的筛选分离情况,以及环境因子对好氧反硝化菌脱氮效能的影响,包括溶解氧(dissolved oxygen,DO)、碳氮比(C/N)、温度等。然后深入探讨了好氧反硝化生物脱氮技术的原理,好氧反硝化过程中的关键功能基因及酶,同时介绍了分子生物技术在好氧反硝化研究过程中的应用,以及好氧反硝化生物脱氮技术在实际应用方面的研究现状。最后,基于目前的研究瓶颈问题,对未来好氧反硝化生物脱氮技术的研究方向提出了科学展望。     

Use of Tn5 Mutants To Assess the Role of the Dissimilatory Nitrite Reductase in the Competitive Abilities of Two Pseudomonas Strains in Soil

We examined the influence of soil aeration state and plant root presence on the comparative survival of wild-type bacteria and isogenic Tn5 (Nir(sup-)) mutants lacking the ability to synthesize nitrite reductase. Two denitrifying Pseudomonas strains with different nitrite reductase types were used. Enumeration of bacteria in sterile and nonsterile soils was based on differential antibiotic resistance. The validity of the bacterial models studied (i.e., equal growth of wild-type and mutant bacteria under aerobic conditions and significantly better growth of wild-type bacteria under denitrifying conditions) was verified in pure-culture studies. In sterile soil, both strains survived better under aerobic than under anaerobic conditions. The lower efficiency of denitrification than O(inf2) respiration in supporting bacterial growth explained this result, and the physical heterogeneity of soil did not strongly modify the results obtained in pure-culture studies. In nonsterile soil, one of the Pseudomonas strains survived better under anaerobic conditions while the other competed equally with the indigenous soil microflora under aerobic and anaerobic conditions. However, when the Nir(sup-)-to-total inoculant ratios (wild type plus Nir(sup-) mutant) were analyzed, it appeared that the presence of nitrite reductase conferred on both Pseudomonas strains a competitive advantage for anaerobic environment or rhizosphere colonization. This is the first attempt to demonstrate with isogenic nondenitrifying mutants that denitrification can contribute to the persistence and distribution of bacteria in fluctuating soil environments.     

Fungal denitrification and nitric oxide reductase cytochrome P450nor

We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite to nitrous oxide (N(2)O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene, modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N(2)O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated. Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a hybrid N(2) or N(2)O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen donor (amines and imines). Possible involvement of NirK and P450nor is suggested.     

好氧反硝化微生物多样性及其反硝化功能初步研究

为研究污水厂/养殖池中好氧反硝化微生物的多样性及菌株反硝化能力,本研究采集了位于福建省厦门市和漳州市的污水处理厂、排污口、污水池、对虾养殖池的污水和污泥样品进行好氧反硝化微生物的富集、分离、鉴定和功能筛选。分别以NaNO3、NaNO2作为唯一氮源共分离纯化获得128株单菌。其中以NaNO3为唯一氮源分离得到63株,以NaNO2为唯一氮源分离得到65株。16SrRNA基因序列分析表明,128株单菌分属于γ-变形菌纲(Gammaproteobacteria,58.6%)、芽胞杆菌纲(Bacilli,6.4%)、放线菌纲(Actinobacteria,11.7%)、α-变形菌纲(Alphaproteobacteria,8.6%)、纤维菌纲(Cytophagia,2.3%)、鞘脂杆菌纲(Sphingobacteria,0.8%)和黄杆菌纲(Flavobacteria,1.6%)7个纲中的38个属。其中盐单胞菌属(Halomonas,29.7%)和芽胞杆菌属(Bacillus,12.5%)为优势菌属,并且广泛存在于各个样品中。反硝化功能初筛结果表明,35株菌能在72h内将20mg·L-1 NO-3-N/NO-2-N完全去除;复筛结果表明,21株菌能在72h内将100 mg·L-1 NO-3-N/NO-2-N完全去除,并且盐单胞菌属、卓贝尔氏菌属(Zobellella)、斯塔普氏菌属(Stappia)及节杆菌属(Arthrobactor)反硝化效果较好,其中斯塔普氏属是首次报道具有好氧反硝化功能。本研究结果表明,污水场/养殖池等环境中可培养反硝化细菌多样性丰富,同时高效反硝化菌的获得也为含氮废水的生物处理提供了良好的菌种资源。     

Physiological roles for two periplasmic nitrate reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025)

The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth.     

A dual functional redox enzyme maturation protein for respiratory and assimilatory nitrate reductases in bacteria

Nitrate is available to microbes in many environments due to sustained use of inorganic fertilizers on agricultural soils and many bacterial and archaeal lineages have the capacity to express respiratory (Nar) and assimilatory (Nas) nitrate reductases to utilize this abundant respiratory substrate and nutrient for growth. Here, we show that in the denitrifying bacterium Paracoccus denitrificans, NarJ serves as a chaperone for both the anaerobic respiratory nitrate reductase (NarG) and the assimilatory nitrate reductase (NasC), the latter of which is active during both aerobic and anaerobic nitrate assimilation. Bioinformatic analysis suggests that the potential for this previously unrecognized role for NarJ in functional maturation of other cytoplasmic molybdenum‐dependent nitrate reductases may be phylogenetically widespread as many bacteria contain both Nar and Nas systems.