Denitrification Phenotype Research Articles

Incomplete denitrification phenotypes in diverse Thermus species from diverse geothermal spring sediments and adjacent soils in southwest China

A few members of the bacterial genus Thermus have been shown to be incomplete denitrifiers, terminating with nitrite (NO2−) or nitrous oxide (N2O). However, the denitrification abilities of the genus as a whole remain poorly characterized. Here, we describe diverse denitrification phenotypes and genotypes of a collection of 24 strains representing ten species, all isolated from a variety of geothermal systems in China. Confirmed terminal products of nitrate reduction were nitrite or N2O, while nitric oxide (NO) was inferred as the terminal product in some strains. Most strains produced N2O; complete denitrification was not observed. Denitrification phenotypes were largely consistent with the presence of denitrification genes, and strains of the same species often had the same denitrification phenotypes and largely syntenous denitrification gene clusters. Genes for nirS and nirK coexisted in three Thermus brockianus and three Thermus oshimai genomes, which is a unique hallmark of some denitrifying Thermus strains and may be ecologically important. These results show that incomplete denitrification phenotypes are prominent, but variable, within and between Thermus species. The incomplete denitrification phenotypes described here suggest Thermus species may play important roles in consortial denitrification in high-temperature terrestrial biotopes where sufficient supply of oxidized inorganic nitrogen exists.

Nitrite accumulation and impairment of N2O reduction explains contrasting soil denitrification phenotypes

Soil denitrification produces the potent greenhouse gas and ozone depleter nitrous oxide (N2O). We previously linked soil N2O emission potential (N2O/N2O + N2) to a continuum of denitrification phenotypes in 20 pasture soils but were unable to determine their proximal cause. At one end of the continuum, soils carried out completely concurrent production and reduction of N2O (concurrent phenotype) while at the other end soils delayed N2O reduction until almost all added nitrate (NO3−) was accumulated as N2O (sequential phenotype). In an unsealed environment, the later phenotype is predicted to emit most added N as N2O. Here we tested the role of delayed N2O reductase synthesis and nitrite (NO2−) based impairment of N2O reduction as determinants of delayed N2O reduction in soils with sequential phenotypes. Nitric oxide (NO), N2O and N2 accumulation were measured in response to added NO3−, NO2− and N2O in anoxic batch incubations of fresh or pre-incubated soil using automated gas chromatography. Successive NO3− additions drove 6 of 7 soils towards increasingly concurrent N2O production/reduction, suggesting delayed production of N2O reductase may be the cause of delayed N2O reduction in initially sequentially denitrifying soils. NO2− addition (2 mM NO3− + 1 mM NO2− vs. 3 mM NO3− control) to sequentially and concurrently denitrifying soil demonstrated that NO2− impairs N2O reduction, even when N2O reductase was fully induced (pre-incubated soils, 1 mM NO2− + 42 μmol N2O vs. 42 μmol N2O control). Further, 2 pieces of evidence suggest that differences in NO2− accumulation are a probable cause of the previously observed denitrification phenotypes. 1) 48 fold higher NO2− accumulation in the sequential vs. concurrent soil in response to 3 mM NO3− addition. 2) the concurrent timing of NO2− depletion/N2O reduction in the sequential soil. However, these observations should be confirmed in a greater number of soils. Inhibition of N2O reductase by NO is proposed as a potential proximal cause of impaired early N2O reduction which could link separate effectors (NO2−, carbon, successive NO3− additions) to a common inhibitory mechanism.

Distribution of Denitrification among Haloarchaea: A Comprehensive Study.

Microorganisms from the Halobacteria class, also known as haloarchaea, inhabit a wide range of ecosystems of which the main characteristic is the presence of high salt concentration. These environments together with their microbial communities are not well characterized, but some of the common features that they share are high sun radiation and low availability of oxygen. To overcome these stressful conditions, and more particularly to deal with oxygen limitation, some microorganisms drive alternative respiratory pathways such as denitrification. In this paper, denitrification in haloarchaea has been studied from a phylogenetic point of view. It has been demonstrated that the presence of denitrification enzymes is a quite common characteristic in Halobacteria class, being nitrite reductase and nitric oxide reductase the enzymes with higher co-occurrence, maybe due to their possible role not only in denitrification, but also in detoxification. Moreover, copper-nitrite reductase (NirK) is the only class of respiratory nitrite reductase detected in these microorganisms up to date. The distribution of this alternative respiratory pathway and their enzymes among the families of haloarchaea has also been discussed and related with the environment in which they constitute the major populations. Complete denitrification phenotype is more common in some families like Haloarculaceae and Haloferacaceae, whilst less common in families such as Natrialbaceae and Halorubraceae.

Soil N2O emission potential falls along a denitrification phenotype gradient linked to differences in microbiome, rainfall and carbon availability

Soil denitrification produces the potent greenhouse gas nitrous oxide (N2O) and by further reduction of N2O, the harmless inert gas N2. N2O emission is determined by rate and timing of the N2O producing and reducing steps which are sensitive to a series of proximal and distal regulators such as pH and microbial community composition. Microbial community associations to N2O emission potential (N2O/(N2O + N2)) are commonly entangled with pH leaving the true role of community composition unclear. Here, we leverage a set of soil microbiomes strongly linked to rainfall above pH to test the hypothesis that microbiome vs. N2O emission potential (N2O/(N2O + N2)) correlations will be maintained across alternative distal drivers. N2O emission potential (N2O/(N2O + N2)) and denitrification gas (NO, N2O, N2) kinetics were assessed by automated gas chromatography while community composition was assessed by 16 S rRNA gene sequencing and qPCR of nosZI and II genes. Analyses revealed a sustained correlation between microbiome and N2O emission potential (N2O/(N2O + N2)) in the absence of a pH effect. Further, a continuum of gas accumulation phenotypes linked to NO accumulation and sensitive to carbon addition are identified. Separate phenotypes carried out N2O production and reduction steps more concurrently or sequentially and thus determined N2O accumulation and emission potential (N2O/(N2O + N2)). Concurrent N2O producing/reducing soils typically contained NO accumulation to a low steady state, while carbon addition manipulations which increased NO accumulation also increased sequentiality of N2O production/reduction and thus emission potential (N2O/(N2O + N2)). These features may indicate a conserved NO inhibitory mechanism across multiple effectors (rainfall, community composition, carbon availability).

Involvement of the cbb3-Type Terminal Oxidase in Growth Competition of Bacteria, Biofilm Formation, and in Switching between Denitrification and Aerobic Respiration.

Paracoccus denitrificans has a branched electron transport chain with three terminal oxidases transferring electrons to molecular oxygen, namely aa3-type and cbb3-type cytochrome c oxidases and ba3-type ubiquinol oxidase. In the present study, we focused on strains expressing only one of these enzymes. The competition experiments showed that possession of cbb3-type oxidase confers significant fitness advantage during oxygen-limited growth and supports the biofilm lifestyle. The aa3-type oxidase was shown to allow rapid aerobic growth at a high oxygen supply. Activity of the denitrification pathway that had been expressed in cells grown anaerobically with nitrate was fully inhibitable by oxygen only in wild-type and cbb3 strains, while in strains aa3 and ba3 dinitrogen production from nitrate and oxygen consumption occurred simultaneously. Together, the results highlight the importance of the cbb3-type oxidase for the denitrification phenotype and suggest a way of obtaining novel bacterial strains capable of aerobic denitrification.

Distinct Expression of the Two NO-Forming Nitrite Reductases in Thermus antranikianii DSM 12462T Improved Environmental Adaptability

Hot spring ecosystems are analogous to some thermal environments on the early Earth and represent ideal models to understand life forms and element cycling on the early Earth. Denitrification, an important component of biogeochemical nitrogen cycle, is highly active in hot springs. Nitrite (NO2-) reduction to nitric oxide (NO) is the significant and rate-limiting pathway in denitrification and is catalyzed by two types of nitrite reductases, encoded by nirS and nirK genes. NirS and NirK were originally considered incompatible in most denitrifying organisms, although a few strains have been reported to possess both genes. Herein, we report the functional division of nirS and nirK in Thermus, a thermophilic genus widespread in thermal ecosystems. Transcriptional levels of nirS and nirK coexisting in Thermus antranikianii DSM 12462T were measured to assess the effects of nitrite, oxygen, and stimulation time. Thirty-nine Thermus strains were used to analyze the phylogeny and distribution of nirS and nirK; six representative strains were used to assess the denitrification phenotype. The results showed that both genes were actively transcribed and expressed independently in T. antranikianii DSM 12462T. Strains with both nirS and nirK had a wider range of nitrite adaptation and revealed nir-related physiological adaptations in Thermus: nirK facilitated adaptation to rapid changes and extended the adaptation range of nitrite under oxygen-limited conditions, while nirS expression was higher under oxic and relatively stable conditions.

Denitrifying haloarchaea within the genus Haloferax display divergent respiratory phenotypes, with implications for their release of nitrogenous gases.

Haloarchaea are extremophiles, generally thriving at high temperatures and salt concentrations, thus, with limited access to oxygen. As a strategy to maintain a respiratory metabolism, many halophilic archaea are capable of denitrification. Among them are members of the genus Haloferax, which are abundant in saline/hypersaline environments. Three reported haloarchaeal denitrifiers, Haloferax mediterranei, Haloferax denitrificans and Haloferax volcanii, were characterized with respect to their denitrification phenotype. A semi-automatic incubation system was used to monitor the depletion of electron acceptors and accumulation of gaseous intermediates in batch cultures under a range of conditions. Out of the species tested, only H. mediterranei was able to consistently reduce all available N-oxyanions to N2 , while the other two released significant amounts of NO and N2 O, which affect tropospheric and stratospheric chemistries respectively. The prevalence and magnitude of hypersaline ecosystems are on the rise due to climate change and anthropogenic activity. Thus, the biology of halophilic denitrifiers is inherently interesting, due to their contribution to the global nitrogen cycle, and potential application in bioremediation. This work is the first detailed physiological study of denitrification in haloarchaea, and as such a seed for our understanding of the drivers of nitrogen turnover in hypersaline systems.

Expression of nirK and nirS genes in two strains of Pseudomonas stutzeri harbouring both types of NO-forming nitrite reductases

Reduction of nitrite to nitric oxide in denitrification is catalysed by two different nitrite reductases, encoded by nirS or nirK. Long considered mutually exclusive and functionally redundant in denitrifying bacteria, we show expression of both genes co-occurring in Pseudomonas stutzeri. The differential expression patterns between strain AN10 and JM300 in relation to oxygen and nitrate and their different denitrification phenotypes, with AN10 reducing nitrate more rapidly and accumulating nitrite, suggest that nirS and nirK can have different roles. Dissimilar gene arrangements and transcription factors in the nir gene neighbourhoods could explain the observed differences in gene expression and denitrification activity.

The regional variation of denitrification phenotypes under anoxic incubation with soils from eight forested catchments in different climate zones of China

Denitrification characteristics of forest soils from eight headwater catchments in China were investigated in this study, along a climatic gradient from the tropics in the South to the temperate zones. Within each catchment, different landscape positions along hydrological flow paths were also considered, including well-drained soils on hill slopes and poorly drained soils in groundwater discharge zones. The results showed that instantaneous denitrification rates were much greater in soils from the northern sites than those from the southern sites (with the average of 110.0 and 25.4nmolNg−1drysoilh.−1, respectively). Large potentials for nitrous oxide (N2O) loss (evaluated as maximum N2O accumulation before it was reduced to dinitrogen (N2)) were observed in the six tropical and subtropical catchments, particularly in soils with high carbon (C) and nitrogen (N). Meanwhile high N2O/(N2O+N2) stoichiometries were displayed in soils from these southern sites. Within catchments, soils from the groundwater discharge zones showed greater potential denitrification rates but smaller N2O/(N2O+N2) ratios in comparison with those on the hill slopes, implying large N removal potentials of soils from the groundwater discharge zones. Furthermore, our findings suggest soil pH is the key controller for the potential denitrification rates and the N2O/(N2O+N2) stoichiometries. Soil pH, C and N availability affect the potential for N2O loss synergistically. Our findings not only pinpoint the denitrification phenotypes of soils along the climatic gradient, but also confirm the small-scale variations within catchments which reflect the in situ habitat of the denitrifiers. These indicate the importance of discrimination related to different landscape positions when modeling N2O emissions and N removals from regional N loading.

Nitrogen assimilation in denitrifier Bacillus azotoformans LMG 9581T.

Until recently, it has not been generally known that some bacteria can contain the gene inventory for both denitrification and dissimilatory nitrate (NO3-)/nitrite (NO2-) reduction to ammonium (NH4+) (DNRA). Detailed studies of these microorganisms could shed light on the differentiating environmental drivers of both processes without interference of organism-specific variation. Genome analysis of Bacillus azotoformans LMG 9581T shows a remarkable redundancy of dissimilatory nitrogen reduction, with multiple copies of each denitrification gene as well as DNRA genes nrfAH, but a reduced capacity for nitrogen assimilation, with no nas operon nor amtB gene. Here, we explored nitrogen assimilation in detail using growth experiments in media with different organic and inorganic nitrogen sources at different concentrations. Monitoring of growth, NO3- NO2-, NH4+ concentration and N2O production revealed that B. azotoformans LMG 9581T could not grow with NH4+ as sole nitrogen source and confirmed the hypothesis of reduced nitrogen assimilation pathways. However, NH4+ could be assimilated and contributed up to 50% of biomass if yeast extract was also provided. NH4+ also had a significant but concentration-dependent influence on growth rate. The mechanisms behind these observations remain to be resolved but hypotheses for this deficiency in nitrogen assimilation are discussed. In addition, in all growth conditions tested a denitrification phenotype was observed, with all supplied NO3- converted to nitrous oxide (N2O).

Primers for overlooked nirK, qnorB, and nosZ genes of thermophilic Gram-positive denitrifiers.

Although efforts have been made the past few years, knowledge on genomic and phenotypic diversity and occurrence of the denitrification ability in Gram-positive bacteria are still fragmentary. Many environmental monitoring approaches have used nir, nor, and nos genes as marker genes for detection of denitrification or denitrifying bacteria. However, primers used in these methods often fail to detect the genes in specific bacterial taxa, such as Gram-positive denitrifiers. In this study, novel primer sets specifically targeting nirK, qnorB, and nosZ genes of the Firmicute genus Geobacillus were developed by genomic mining and tested in parallel with commonly used primers on a set of phylogenetically closely related denitrifying geobacilli. Novel nirK and qnorB sequences were recovered from all strains tested, whereas nosZ was detected in part of the strain set, which was in agreement with observed phenotypes. Interspecies and modest intraspecies variations in amplified fragment length polymorphism (AFLP) patterns were observed, verifying presence of genomic variation within the strain set. Our study shows that closely related Gram-positive denitrifiers may differ in denitrification phenotype and genotype. But foremost, novel primers targeting very divergent nirK, qnorB, and nosZ gene sequences of Gram-positive denitrifiers, are now available for cultivation-independent environmental surveys.

Role of norEF in Denitrification, Elucidated by Physiological Experiments with Rhodobacter sphaeroides

Many denitrifying organisms contain the norEF gene cluster, which codes for two proteins that are thought to be involved in denitrification because they are expressed during the reduction of nitrite and nitric oxide. The products of both genes are predicted to be membrane associated, and the norE product is a member of the cytochrome c oxidase subunit III family. However, the specific role of norEF is unknown. The denitrification phenotypes of Rhodobacter sphaeroides strains with and without norEF genes were studied, and it was found that loss of norEF lowered the rate of denitrification from nitrate and resulted in accumulation of micromolar concentrations of nitric oxide during denitrification from nitrite. norEF appears to have no direct role in the reduction of nitric oxide; however, since deletion of norEF in the wild-type 2.4.3 strain had essentially no influence on the kinetics of potential nitric oxide reduction (Vmax and Ks), as measured by monitoring the depletion of a bolus of nitric oxide injected into anoxic cultures without any other electron acceptors. However, norEF-deficient cells that had undergone a more chronic exposure to micromolar concentrations of nitric oxide showed an ∼50% reduction in Vmax but no change in apparent Ks. These results can explain the occurrence of norEF in the 2.4.3 strain of R. sphaeroides, which can reduce nitrate to nitrous oxide, and their absence from strains such as 2.4.1, which likely use nitric oxide reductase to mitigate stress due to episodic exposure to nitric oxide from exogenous sources.

Sequential extraction of denitrifying organisms from soils; strongly attached cells produce less N2O than loosely attached cells

Soil denitrifier communities (DC) have been studied intensively because their phenotypic characteristics may influence the emission of NO and N2O. In the present study, we challenge the concept of soil DC as one assemblage of organisms, and propose that DCs are functionally stratified based on their position within the soil matrix. We hypothesize that the soils contain “inner” and “outer” habitats; the inner meaning sites within crevices and cavities of the soil mineral material, structured organic materials and strong biofilms, the outer meaning exposed surfaces and macropores of the soil. We used sequential dispersion/density gradient centrifugation (DGC), to obtain two contrasting fractions of cells differing in the strength of attachment to soil particles. We define loosely attached cells (LAC) as those which are liberated from soil particles by moderate dispersion of the soils. LAC were harvested by DGC, and the pellets at the bottom of the gradients were then re-dispersed with higher shear force to release more strongly attached cells (SAC) which were again harvested by DGC. We hypothesized that SAC are cells situated deeper into biofilms and structures than LAC, hence SAC would experience more frequent and/or longer anoxic spells than LAC. For this reason, we hypothesized 1) that the relative frequency of denitrifying organisms is higher in SAC than LAC and 2) that N2O-reductase is expressed earlier and stronger in SAC than in LAC. The results suggest a rejection of the first hypothesis but lend strong support for the second: In response to oxygen depletion, the two fractions (LAC and SAC) expressed similar rates of denitrification, both produced a mixture of NO, N2O and N2, but the relative amount of N2O produced was much higher for LAC than SAC. The soils' matrix appears to contain strata which select for specific denitrification phenotypes, with implications for N2O-emission.

Thermus oshimai JL-2 and T. thermophilus JL-18 genome analysis illuminates pathways for carbon, nitrogen, and sulfur cycling.

The complete genomes of Thermus oshimai JL-2 and T. thermophilus JL-18 each consist of a circular chromosome, 2.07 Mb and 1.9 Mb, respectively, and two plasmids ranging from 0.27 Mb to 57.2 kb. Comparison of the T. thermophilus JL-18 chromosome with those from other strains of T. thermophilus revealed a high degree of synteny, whereas the megaplasmids from the same strains were highly plastic. The T. oshimai JL-2 chromosome and megaplasmids shared little or no synteny with other sequenced Thermus strains. Phylogenomic analyses using a concatenated set of conserved proteins confirmed the phylogenetic and taxonomic assignments based on 16S rRNA phylogenetics. Both chromosomes encode a complete glycolysis, tricarboxylic acid (TCA) cycle, and pentose phosphate pathway plus glucosidases, glycosidases, proteases, and peptidases, highlighting highly versatile heterotrophic capabilities. Megaplasmids of both strains contained a gene cluster encoding enzymes predicted to catalyze the sequential reduction of nitrate to nitrous oxide; however, the nitrous oxide reductase required for the terminal step in denitrification was absent, consistent with their incomplete denitrification phenotypes. A sox gene cluster was identified in both chromosomes, suggesting a mode of chemolithotrophy. In addition, nrf and psr gene clusters in T. oshmai JL-2 suggest respiratory nitrite ammonification and polysulfide reduction as possible modes of anaerobic respiration.

Whole Genome Sequencing of Thermus oshimai JL-2 and Thermus thermophilus JL-18, Incomplete Denitrifiers from the United States Great Basin

The strains Thermus oshimai JL-2 and Thermus thermophilus JL-18 each have a circular chromosome, 2.07 Mb and 1.9 Mb in size, respectively, and each has two plasmids ranging from 0.27 Mb to 57.2 kb. The megaplasmid of each strain contains a gene cluster for the reduction of nitrate to nitrous oxide, consistent with their incomplete denitrification phenotypes.

The impact of copper, nitrate and carbon status on the emission of nitrous oxide by two species of bacteria with biochemically distinct denitrification pathways

Denitrifying bacteria convert nitrate (NO(3) (-) ) to dinitrogen (N(2) ) gas through an anaerobic respiratory process in which the potent greenhouse gas nitrous oxide (N(2) O) is a free intermediate. These bacteria can be grouped into classes that synthesize a nitrite (NO(2) (-) ) reductase (Nir) that is solely dependent on haem-iron as a cofactor (e.g. Paracoccus denitrificans) or a Nir that is solely dependent on copper (Cu) as a cofactor (e.g. Achromobacter xylosoxidans). Regardless of which form of Nir these groups synthesize, they are both dependent on a Cu-containing nitrous oxide reductase (NosZ) for the conversion of N(2) O to N(2) . Agriculture makes a major contribution to N(2) O release and it is recognized that a number of agricultural lands are becoming Cu-limited but are N-rich because of fertilizer addition. Here we utilize continuous cultures to explore the denitrification phenotypes of P. denitrificans and A. xylosoxidans at a range of extracellular NO(3) (-) , organic carbon and Cu concentrations. Quite distinct phenotypes are observed between the two species. Notably, P. denitrificans emits approximately 40% of NO(3) (-) consumed as N(2) O under NO(3) (-) -rich Cu-deficient conditions, while under the same conditions A. xylosoxidans releases approximately 40% of the NO(3) (-) consumed as NO(2) (-) . However, the denitrification phenotypes are very similar under NO(3) (-) -limited conditions where denitrification intermediates do not accumulate significantly. The results have potential implications for understanding denitrification flux in a range of agricultural environments.

Expression of nitrous oxide reductase in Paracoccus denitrificans is regulated by oxygen and nitric oxide through FnrP and NNR

The reductases performing the four steps of denitrification are controlled by a network of transcriptional regulators and ancillary factors responding to intra- and extracellular signals, amongst which are oxygen and N oxides (NO and ). Although many components of the regulatory network have been identified, there are gaps in our understanding of their role(s) in controlling the expression of the various reductases, in particular the environmentally important N2O reductase (N2OR). We investigated denitrification phenotypes of Paracoccus denitrificans mutants deficient in: (i) regulatory proteins (three FNR-type transcriptional regulators, NarR, NNR and FnrP, and NirI, which is involved in transcription activation of the structural nir cluster); (ii) functional enzymes (NO reductase and N2OR); or (iii) ancillary factors involved in N2O reduction (NirX and NosX). A robotized incubation system allowed us to closely monitor changes in concentrations of oxygen and all gaseous products during the transition from oxic to anoxic respiration. Strains deficient in NO reductase were able to grow during denitrification, despite reaching micromolar concentrations of NO, but were unable to return to oxic respiration. The FnrP mutant showed linear anoxic growth in a medium with nitrate as the sole NOx, but exponential growth was restored by replacing nitrate with nitrite. We interpret this as nitrite limitation, suggesting dual transcriptional control of respiratory nitrate reductase (NAR) by FnrP and NarR. Mutations in either NirX or NosX did not affect the phenotype, but the double mutant lacked the potential to reduce N2O. Finally, we found that FnrP and NNR are alternative and equally effective inducers of N2OR.

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

Abstract Samples of water, sediment and bacterial mat from hot springs in Graendalur 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.

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

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 N 2O 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 → N 2) 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 N 2O 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 N 2 with N 2O as an intermediate but a virtual inability to reduce N 2O when N 2O was the sole oxidant.

Thermophilic Bacillus sp. that shows the denitrification phenotype of Pseudomonas aeruginosa

A thermophilic Bacillus sp. of marine origin was observed to grow anaerobically on nitrite, nitrous oxide (N2O) in the presence of nitrite, and N2O alone for a few hours after exhaustion of nitrite. This represents the second example of a denitrification phenotype originally observed to occur with Pseudomonas aeruginosa.