Nitrification and denitrification by Thiosphaera pantotropha in aerobic chemostat cultures

Abstract: Thiosphaera pantotropha has been reported to denitrify aerobically and nitrify heterotrophically. However, recent evidence has indicated that these properties (particularly aerobic denitrification) have been lost. The occurrence and levels of aerobic denitrification and heterotrophic nitrification by T. pantotropha in chemostat cultures have therefore been re-evaluated. Only low nitrate reduction rates were observed: the apparent nitrogen loss was of the same order of magnitude as the combined error in the calculated nitrogen consumption. However, ¹⁵N mass spectrometry revealed low aerobic denitrification rates (about 10% of the rates originally published by this group). Heterotrophic nitrification rates were about a third of previous observations. N₂ and N₂O were both produced from NH₄⁺, NO₃⁻ and NO₂⁻. Periplasmic nitrate reductase was present in aerobically grown cells.     

The effect of electron acceptor variations on the behaviour of Thiosphaera pantotropha and Paracoccus denitrificans in pure and mixed cultures

Abstract The competitive advantages provided by a capacity for aerobic denitrification have been tested by comparing Thiosphaera pantotropha (which denitrifies aerobically and anaerobically), with a strain of Paracoccus denitrificans (which only denitrifies under anaerobic conditions) in acetate-limited chemostats. A comparison of μ -C_s curves based on K _s and μ _max measurements indicated that Pa. denitrificans could be expected to dominate mixtures of the two species at high growth rates when the dissolved oxygen was above 80% of air saturation and NH₃ was the sole source of nitrogen. The comparison also suggested t that at lower growth rates, lower dissolved oxygen tensions, and/or in the presence of nitrate, Tsa. pantotropha should have the competitive advantage. Chemostat experiments with mixtures of the two species showed that Tsa. pantotropha did, indeed, dominate the population when expected. However, when Pa. denitrificans was expected to dominate, only a small increase in the Pa. denitrificans numbers was possible before Tsa. pantotropha formed a biofilm on the walls of the chemostat instead of washing out, and was again able to out-compete Pa. denitrificans for acetate. Experiments with axenic chemostat cultures subjected to aerobic/anaerobic switches showed that Tsa. pantotropha , with its constitutive denitrifying system, was able to adjust smoothly to the changing environmental conditions and thus continued to grow. Pa. denitrificans does not have constitutive denitrifying enzymes, and could consequently not adjust its metabolism to the lack of oxygen rapidly enough. It therefore washed out at a rate equivalent to the dilution rate.     

Competition between hydrogen peroxide and nitrate for electrons from the respiratory chains of Thiosphaera pantotropha and Rhodobacter capsulatus

Abstract Thiosphaera pantotropha and some strains of Rhodobacter capsulatus express both a periplasmic nitrate reductase and cytochrome c peroxidase when grown under aerobic conditions. Harvested cell suspensions of either species can respire nitrate in the presence of 200 μM O₂ (∼ 80% air saturation), at 70–80% of the anaerobic rate. Addition of hydrogen peroxide to such cells causes a 90% inhibition of nitrate reduction under anaerobic or aerobic conditions. The duration of the inhibition is proportional to the concentration of hydrogen peroxide added and can be ascribed to the expression of periplasmic peroxidases that compete with the nitrate reductase for electrons from the respiratory chain. The results reveal a hitherto unrecognised interaction between reactions of denitrification and the reduction of hydrogen peroxide by a periplasmic peroxidase that may have implications for the denitrification in microaerobic environments. The creation of aerobic conditions in bacterial cultures by addition of hydrogen peroxide, and relying on the generation of oxygen by endogenous catalase activity, is a commonly used technique for studying respiratory processes. The observations presented here demonstrate that results derived from such experiments should be interpreted with caution.     

The use of a metabolically structured model in the study of growth, nitrification, and denitrification by Thiosphaera pantotropha

Thiosphaera pantotropha is capable of aerobic heterotrophic nitrification and both aerobic and anaerobic denitrification. These phenomena have been studied in acetate-limited aerobic and anaerobic continuous cultures supplied with ammonia and nitrate. The internal reaction rates were defined, based on biochemical knowledge. The observable external conversion rates are related through a linear equation on the basis of the specified internal reaction rates. The linear equation is a Pirt relation extended for microbial systems with multiple electron donors (acetate and ammonia) and electron acceptors (oxygen and nitrate). The coefficients in this equation were estimated from the continuous culture measurements, and are composed of parameters involved in ATP production and consumption by the microorganism. It is shown that with realistic values for these parameters, the metabolically structured model describes the aerobic as well as the anaerobic experiments.     

Cytochrome cb-type nitric oxide reductase with cytochrome c oxidase activity from Paracoccus denitrificans ATCC 35512.

A highly active nitric oxide reductase was purified from Paracoccus denitrificans ATCC 35512, formerly named Thiosphaera pantotropha, which was anaerobically cultivated in the presence of nitrate. The enzyme was composed of two subunits with molecular masses of 34 and 15 kDa and contained two hemes b and one heme c per molecule. Copper was not found in the enzyme. The spectral properties suggested that one of the two hemes b and heme c were in six-coordinated low-spin states and another heme b was in a five-coordinated high-spin state and reacted with carbon monoxide. The enzyme showed high cytochrome c-nitric oxide oxidoreductase activity and formed nitrous oxide from nitric oxide with the expected stoichiometry when P. denitrificans ATCC 35512 ferrocytochrome c-550 was used as the electron donor. The V max and Km values for nitric oxide were 84 micromol of nitric oxide per min/mg of protein and 0.25 microM, respectively. Furthermore, the enzyme showed ferrocytochrome c-550-O2 oxidoreductase activity with a V max of 8.4 micromol of O2 per min/mg of protein and a Km value of 0.9 mM. Both activities were 50% inhibited by about 0.3 mM KCN.     

Confirmation of 'aerobic denitrification' in batch cultures, using gas chromatography and 15N mass spectrometry

Abstract: Because of a revival in the controversy surrounding 'aerobic denitrification', especially in relation to Thiosphaera pantotropha , activity in aerobic batch cultures was evaluated using gas chromatography and mass spectrometry after the addition of ¹⁵N-labelled NH₄⁺ and NO₂⁻. Aerobic denitrifying activity in T. pantotropha was present, but only at about 10% of the originally-reported levels. The activity of ' Pseudomonas denitrificans ' was similar to previously-reported values. Alcaligenes faecalis showed significant aerobic denitrifying activity, producing almost equivalent amounts of N₂ and N₂O. An unidentified pseudomonad, isolate G4, presumably requires anoxia for enzyme activity as it did not denitrify aerobically, even though it has a constitutive denitrifying pathway.     

A mathematical description of the behaviour of mixed chemostat cultures of an autotrophic nitrifier and a heterotrophic nitrifier/aerobic denitrifier; a comparison with experimental data

Abstract A general, unstructed mathematical model has been used to describe the behaviour of nutrient-limited growth of two bacteria in a continuous co-culture. The experimental system consisted of a two-membered mixed culture of the heterotrophic nitrifier/aerobic denitrifier, Thiosphaera pantotropha , and the autotrophic nitrifier, Nitrosomonas europaea , competing for ammonia in chemostat culture. The outcome of competition was only dependent on the Monod constants and the growth yields of the two bacteria. The model shows that both bacteria will oxidize equal amounts of ammonia when the cell ratio of T. pantotropha/N. europaea is 260.     

Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system

AIMS: This study compares the ability of Thiosphaera pantotropha ATCC 35512 and the newly isolated Pseudomonas stutzeri SU2 to perform aerobic denitrification. METHODS AND RESULTS: Nitrate-supplemented basal medium in airtight crimp-sealed serum bottles containing an atmosphere of 92% oxygen was inoculated with Ps. stutzeri SU2 or T. pantotropha and incubated at 30 degrees C. During the 92-h incubation period, aerobic denitrification by Ps. stutzeri SU2 (NO3(-) - N removal 99.24%) was more efficient than that by T. pantotropha (NO3(-) - N removal 27.29%). CONCLUSION: Pseudomonas stutzeri SU2, which was isolated from the activated sludge of a sequencing batch reactor treating piggery wastewater, rapidly reduced the nitrate to nitrogen gas without nitrite accumulation. The nitrate removal rate of SU2 was 0.032 mmol NO3(-) - N g cell-1 h-1 after 44 h incubation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas stutzeri SU2 can be used in a full-scale sequencing batch system for efficient in situ aerobic nitrate removal from piggery wastewater.     

The cytochromes c-550 of Paracoccus denitrificans and Thiosphaera pantotropha: a need for re-evaluation of the history of Paracoccus cultures

Abstract The c -type cytochrome and protein profiles were compared for a number of cultures of Paracoccus denitrificans obtained from a range of culture collections. The cultures fell into two groups corresponding to the two original isolates of this bacterial species. One group, which included NCIMB 8944, ATCC 13543, ATCC 17741, ATCC 19367, Pd 1222 and DSM 413, were similar or identical to LMD 22.21. The second group, including DSM 65 and LMG 4218, were similar or identical to LMD 52.44. These groupings were not compatible with the recorded history of culture deposition. Mass spectrometry and amino acid sequence comparisons showed that the cytochrome c -550 of the LMD 52.44 culture group differed by 16% from that of the LMD 22.21 group, and yet was only 1% different from the cytochrome c -550 of Thiosphaera pantotropha . These results suggest that consideration should be given to creation of a new species of Paracoccus pantotropha , which would include Thiosphaera pantotropha and Paracoccus denitrificans LMD 52.44.     

Inhibition of denitrification activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH

The influence of pH on the denitrification activity of a continuous culture of Paracoccus denitrificans was studied in relation to the presence of nitrite. After a transition from aerobic to anaerobic conditions at the suboptimal pH of 6.8, P. denitrificans was not able to build up a functional denitrification pathway. Nitrite accumulated in the medium as the predominant denitrification product. Although the nitrite reductase gene was induced properly, the enzyme could not be detected at sufficient amounts in the culture. These observations indicate that either translation was somehow inhibited, or once synthesized nitrite reductase was inactivated, possibly by the high concentrations of nitrous acid (HNO₂. Interestingly, when a P. denitrificans culture which was grown to steady-state under anaerobic conditions was then exposed to suboptimal pHs, cells exhibited a reduced overall denitrification activity, but neither nitrite nor any other denitrification intermediate accumulated.     

Bioenergetic examination of the heterotrophic nitrifier-denitrifier Thiosphaera pantotropha

The heterotrophic nitrifying-denitrifying bacterium Thiosphaera pantotropha is remarkable as it nitrifies and denitrifies simultaneously. With respect to nitrogenous compounds, whether nitrification or denitrification results in energy conservation is of interest. Proton translocation studies were performed to determine if energy was conserved by the bacterium during heterotrophic nitrification and denitrification. Hydrazine (N₂H _inf5 ^sup+ ) was employed as the heterotrophic nitrification substrate while nitrate, nitrite and nitrous oxide were used as denitrification substrates. Analysis of the data indicate that the bacterium does not conserve energy when hydrazine was the substrate. Conversely, energy was conserved when either nitrate, nitrite or nitrous oxide functioned as the oxidants during denitrification-dependent proton translocation experiments. Thiosphaera pantotropha thus is similar to other heterotrophic nitrifiers-denitrifiers in that it conserves energy while denitrifying but has not been observed to do so when heterotrophically nitrifying.     

The expression of redox proteins of denitrification inThiosphaera pantotropha grown with oxygen,nitrate, and nitrous oxide as electron acceptors

The redox proteins and enzymes involved in denitrification inThiosphaera pantotropha exhibited a differential expression in response to oxygen. Pseudoazurin was completely repressed during batch or continuous culture under oxic conditions. Cytochromecd ₁ nitrite reductase was also heavily repressed after aerobic growth. Nitrite, nitric oxide, and nitrous oxide reductase activities were detected in intact cells under some conditions of aerobic growth, indicating that aerobic denitrification might occur in some circumstances. However, the rates of denitrification were much lower after aerobic growth than after anaerobic growth. Growth with nitrous oxide as sole electron acceptor mimicked aerobic growth in some respects, implying that expression of parts of the denitrification apparatus might be controlled by the redox state of a component of the electron transport chain rather than by oxygen itself. Nevertheless, the regulation of expression of nitrous oxide reductase was linked to the oxygen concentration.     

Metabolic pathways inParacoccus denitrificans and closely related bacteria in relation to the phylogeny of prokaryotes

Denitrification and methylotrophy inParacoccus denitrificans are discussed. The properties of the enzymes of denitrification: the nitrate-nitrite antiporter, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase are described. The genes for none of these proteins have yet been cloned and sequenced fromP. denitrificans. A number of sequences are available for enzymes fromEscherichia coli, Pseudomonas stutzeri andPseudomonas aeruginosa. It is concluded that pathway specificc-type cytochromes are involved in denitrification. At least 40 genes are involved in denitrification.In methanol oxidation at least 20 genes are involved. In this case too pathway specificc-type cytochromes are involved. The sequence homology between the quinoproteins methanol dehydrogenase, alcoholdehydrogenase and glucose dehydrogenase is discussed. This superfamily of proteins is believed to be derived from a common ancestor. ThemoxFJGI operon determines the structural components of methanol dehydrogenase and the associatedc-type cytochrome. Upstream of this operon 3 regulatory proteins were found. The mox Y protein shows the general features of a sensor protein and the moxX protein those of a regulatory protein. Thus a two component regulatory system is involved in both denitrification and methylotrophy.The phylogeny of prokaryotes based on 16S rRNA sequence is discussed. It is remarkable that the 16S rRNA ofThiosphaera pantotropha is identical to that ofP. denitrificans. Still these bacteria show a number of differences.T. pantotropha is able to denitrify under aerobic circumstances and it shows heterotrophic nitrification. Nitrification and heterotrophic nitrification are found in species belonging to the -and -subdivisions of purple non-sulfur bacteria. Thus the occurrence of heterotrophic nitrification inT. pantotropha which belongs to the -subdivision of purple non-sulfur bacteria is a remarkable property. FurthermoreT. pantotropha contains two nitrate reductases of which the periplasmic one is supposed to be involved in aerobic denitrification. The nitrite reductase is of the Cu-type and not of the cytochromecd ₁ type as inP. denitrificans. Also the cytochromeb of theQbc complex ofT. pantotropha is highly similar to its counterpart inP. denitrificans. It is hypothesized that the differences between these two organisms which both contain large megaplasmids is due to a combination of loss of genetic information and plasmid-coded properties. The distribution of a number of complex metabolic systems in eubacteria and in a number of species belonging to the -group of purple non sulphur bacteria is reviewed. Two possibilities to explain this haphazard distribution are considered: 1. Lateral gene transfer between distantly related micro organisms occurs frequently. 2. The eubacterial ancestors must have possessed already these properties. The distribution of these properties is due to sporadic loss during evolutionary divergence.With respect to the occurrence and frequency of lateral gene transfer two opposing views exist. According to molecular biologists lateral gene transfer occurs frequently and is very easy. Bacteria are supposed to form one large gene pool. On the other hand population geneticists have provided evidence that strong systems operate that establish reproductive isolation between diverged species and even between closely related cell lines.Data on amino acid sequences of nitrogenase proteins, cytochromesc, cytochrome oxidases, -subunits of ATP synthase and tryptophan biosynthetic enzymes of various micro organisms were reviewed. In all these cases phylogenetic trees could be constructed based on the amino acid sequence data. In all cases this phylogenetic tree was similar to the one based on 16S rRNA homology. Only in one case evidence for the occurrence of lateral gene transfer was obtained. Therefore it is concluded that lateral gene transfer played a minor role in the distribution of complex metabolic systems among prokaryotes. It must be stressed that this does not exclude the possibility that lateral gene transfer occurred frequently in the initial stage of bacterial evolution. It is hypothesized that the appearance of nitrogen fixation, denitrification and cytochrome oxidase formation were early events in the evolution of micro organisms. Both systems are supposed to have evolved only once. Subsequently the capacity to fix nitrogen or to denitrifymust have been lost many times, just as photosynthetic capacity is supposed to have been lost many times. During evolution many systems have been lost leading to a haphazard distribution of metabolic characters among bacteria. As an example it is suggested that organisms with a respiratory chain similar to that ofEscherichia coli arose by loss of the capacity to form the Qbc complex andc-type cytochromes. The remaining systems could be controlled much better however than in the ancestral organisms.     

Combined heterotrophic nitrification and aerobic denitrification in Thiosphaera pantotropha and other bacteria

Reports of the simultaneous use of oxygen and denitrification by different species of bacteria have become more common over the past few years. Research with some strains (e.g. Thiosphaera pantotropha) has indicated that there might be a link between this aerobic denitrification and a form of nitrification which requires rather than generates energy and is therefore known as heterotrophic nitrification. This paper reviews recent research into heterotrophic nitrification and aerobic denitrification, and presents a preliminary model which, if verified, will provide at least a partial explanation for the simultaneous occurrence of nitrification and denitrification in some bacteria.     

Integration of heterologous DNA into the genome of Paracoccus denitrificans is mediated by a family of IS1248-related elements and a second type of integrative recombination event.

All members of the IS1248 family residing in the genome of Paracoccus denitrificans have been isolated by using a set of insertion sequence entrapment vectors. The family consists of five closely related members that integrate the entrapment vectors at distinct sites. One of these, IS1248b, was sequenced and, except for a single base change, shown to be identical to the previously isolated IS1248a. Southern analysis of genomic DNA with labeled IS1248 revealed different hybridization patterns for different isolates of P. denitrificans and Thiosphaera pantotropha. No hybridization was observed with DNA from Thiobacillus versutus and more distantly related species. From a comparison of the fingerprints it was shown that one of the members of the IS1248 family found in P. denitrificans DSM413 is absent in strain NCIB8944, although they are catalogued in international strain catalogues as identical strains. Furthermore, strains Pd1222 and Pd1235, both derivatives of P. denitrificans DSM413, were shown to have different patterns of IS1248 hybridizing restriction fragments. In 14 of 18 strains, the entrapment vectors used in this study were incorporated into the genome via IS1248-mediated cointegrate formation. In the other four strains, the entrapment vectors were shown to be integrated through a different mechanism not involving IS1248.     

Interactions between respiration and denitrification during growth of Thiosphaera pantotropha in continuous culture

Abstract The effects of oxygen on the use of nitrate as an electron acceptor by the denitrifying bacterium Thiosphaera pantotropha were investigated during growth on acetate. In batch cultures under aerobic conditions nitrate was not utilised and the growth rate constant was 0.55 h⁻¹. The corresponding value for growth on nitrate under anoxic conditions was 0.37 h⁻¹. In acetate-limited continuous cultures with feedback control of the dissolved oxygen concentration, nitrate utilisation was totally inhibited by the lowest concentration of oxygen tested (22 μM). Carbon conversion efficiencies with acetate increased from 0.28 with nitrate to 0.44 with oxygen. The rates of nitrification calculated from nitrogen balance studies were not greater than 1.5% of the rate of anoxic denitrification.     

反硝化细菌在污水处理作用中的研究

反硝化细菌在污水处理过程中起到十分重要的作用。传统理论认为反硝化细菌是异养厌氧的,20世纪80年代发现了好氧反硝化细菌。最近,自养反硝化细菌的发现,特别是脱氮硫杆菌的发现引起了人们的极大兴趣。     

Urea production by Thiosphaera pantotropha and by anaerobic enrichment cultures from marine sediments

Abstract The production of urea by Thiosphaera pantotropha was studied. Batch cultures were grown on acetate as energy source and with NO₃⁻ or O₂ as terminal electron acceptor. Urea accumulated in the media during exponential growth in aerobic and anaerobic cultures of T. pantotropha . Urea production continued after the cells had entered the stationary growth phase. Bacterial ability to produce urea was supported by studies of cultures enriched for denitrifying, sulphate-reducing and fermenting bacteria. The results implied that urea production was common among bacteria normally considered to be important in marine sediments.