Nitrate-reducing Microorganisms Research Articles

Salivary nitrate – an ecological factor in reducing oral acidity

Human oral cavities represent a novel environment with a constant supply of concentrated nitrate. For humans, over 80% of dietary nitrate originates from fruit and vegetables. With a healthy, balanced diet, rich in fruit and vegetables, the concentration of nitrate in saliva can reach up to more than three times the European drinking water standard. The physiological function of the active excretion of salivary nitrate is unknown. Furthermore, little is known of the ecological function of oral nitrate and the effect on the oral environment during its subsequent oral microbial conversions. The objectives of the research were to investigate the effect on salivary pH coupled with oral microbial nitrate and/or nitrite reduction. Human saliva samples were incubated anaerobically in the presence of 111.0 mmol glucose (2%), with and without 1.5 mmol nitrate/nitrite, and pH and nitrate/nitrite consumption were measured during the time-course of the incubations. We found that anaerobic incubation of saliva containing a mixture of oral bacteria in the presence of nitrate/nitrite substrates and glucose resulted in a higher pH than was found in controls in the absence of nitrate/nitrite. These results suggest that the presence of these electron acceptors repressed acid fermentation, or increased alkali production, or consumed acid produced, thus reducing salivary acidity. This finding identifies salivary nitrate as a possible ecological factor in reducing oral acidity. The possibility that a symbiotic relationship between host nitrate excretion and nitrate-reducing microorganisms might help to protect against tooth decay should be explored further.

Functional stability of the nitrate-reducing community in grassland soils towards high nitrate supply

To study the effects of short-term fluctuation of nitrate concentrations on the nitrate-reducing community, repacked soil cores were amended with 0, 100 and 300 μ g NO 3 - ‐ N g - 1 soil and incubated for 3, 7 and 14 days, respectively. The nitrate reductase activity was determined in a laboratory-based enzyme assay. In parallel, the community structure of nitrate-reducing microorganisms was characterised by RFLP-PCR using the functional gene narG, which encodes the catalytic site of the membrane-bound nitrate reductase. The community structure remained constant over the experimental period indicating that this functional community is characterised by a high resistance towards fluctuating nitrate concentrations. Decreases in nitrate concentration as well as increase in pH values indicated a very active nitrate-reducing community under nitrate addition. Surprisingly, inhibition of nitrite reductase by 2,4-dinitrophenol, which is a precondition for the measurement of nitrate reductase activity, could not be achieved in the 100 μ g NO 3 - ‐ N treatment despite increased concentrations of the inhibitor. However, comparison of the nitrate reductase in the control and the 300 μ g NO 3 - ‐ N treatment showed a significant increase in the latter at day 3. No further differences were observed at days 7 and 14, which suggests a high resilience of the nitrate reductase activity.

Microbial succession of nitrate‐reducing bacteria in the rhizosphere of Poa alpina across a glacier foreland in the Central Alps

Changes in community structure and activity of the dissimilatory nitrate-reducing community were investigated across a glacier foreland in the Central Alps to gain insight into the successional pattern of this functional group and the driving environmental factors. Bulk soil and rhizosphere soil of Poa alpina was sampled in five replicates in August during the flowering stage and in September after the first snowfalls along a gradient from 25 to 129 years after deglaciation and at a reference site outside the glacier foreland (>2000 years deglaciated). In a laboratory-based assay, nitrate reductase activity was determined colorimetrically after 24 h of anaerobic incubation. In selected rhizosphere soil samples, the community structure of nitrate-reducing microorganisms was analysed by restriction fragment length polymorphism (RFLP) analysis using degenerate primers for the narG gene encoding the active site of the membrane-bound nitrate reductase. Clone libraries of the early (25 years) and late (129 years) succession were constructed and representative clones sequenced. The activity of the nitrate-reducing community increased significantly with age mainly due to higher carbon and nitrate availability in the late succession. The community structure, however, only showed a small shift over the 100 years of soil formation with pH explaining a major part (19%) of the observed variance. Clone library analysis of the early and late succession pointed to a trend of declining diversity with progressing age. Presumably, the pressure of competition on the nitrate reducers was relatively low in the early successional stage due to minor densities of microorganisms compared with the late stage; hence, a higher diversity could persist in this sparse environment. These results suggest that the nitrate reductase activity is regulated by environmental factors other than those shaping the genetic structure of the nitrate-reducing community.

Characterization of microbial activities and U reduction in a shallow aquifer contaminated by uranium mill tailings.

A characterization of the Shiprock, NM, uranium mill tailing site focused on the geochemical and microbiological factors governing in-situ uranium-redox reactions. Groundwater and aqueous extracts of sediment samples contained a wide concentration range of sulfate, nitrate, and U(VI) with median values of 21.2 mM, 16.1 micro M, and 2.7 micro M, respectively. Iron(III) was not detected in groundwater, but a median value of 0.3 mM in sediment extracts was measured. Bacterial diversity down gradient from the disposal pile reflected the predominant geochemistry with relatively high numbers of sulfate- and nitrate-reducing microorganisms, and smaller numbers of acetogenic, methanogenic, nitrate-dependent Fe(II)-oxidizing, Fe(III)-reducing, and sulfide-oxidizing bacteria. In aquifer slurry incubations, nitrate reduction was always preferred and had a negative impact on sulfate-, Fe(III)-, and U-reduction rates. We also found that sulfate-reduction rates decreased sharply in the presence of clay, while Fe(III)-reduction increased with no clear impact on U reduction. In the absence of clay, iron and sulfate reduction correlated with concentrations of Fe(III) and sulfate, respectively. Rates of U(VI) loss did not correlate with the concentration of any electron acceptor. With the exception of Fe(III), electron donor amendment was largely unsuccessful in stimulating electron acceptor loss over a 2-week incubation period, suggesting that endogenous forms of organic matter were sufficient to support microbial activity. Our findings suggest that efforts to accelerate biological U reduction should initially focus on stimulating nitrate removal.

Anaerobic utilization of essential oils by denitrifying bacteria.

Plant volatile organic compounds are a major carbon source in nature. We studied the degradability of these substances by anaerobic microorganisms in enrichment cultures with representative essential oils as organic substrates and nitrate as electron acceptor. Lemon and pine needle oil supported microbial growth in the presence of pure oil, whereas parsley seed, camphor, sage, fennel, and mint oil supported growth only when the essential oils were dissolved in an overlying phase of 2,2,4,4,6,8,8-heptamethylnonane. Thyme oil did not support denitrification. Analyses of the microbially degraded oils revealed the disappearance of monoterpenes, of several monoterpenoids, and of methoxy-propenyl-benzenes, including apiole and myristicin. Most-probable-number determinations for denitrifying communities in sewage sludge and forest soil yielded 10(6) to 10(7) monoterpene-utilizing cells ml(-1), representing 0.7 to 100% of the total cultivable nitrate-reducing microorganisms. The utilization of essential oils together with the common occurrence of this metabolic trait are indications for an environmentally important, but currently unexplored anaerobic turnover of plant volatile organic compounds in soil.

Isolation and characterization of phenol-degrading denitrifying bacteria.

Phenol is a man-made as well as a naturally occurring aromatic compound and an important intermediate in the biodegradation of natural and industrial aromatic compounds. Whereas many microorganisms that are capable of aerobic phenol degradation have been isolated, only a few phenol-degrading anaerobic organisms have been described to date. In this study, three novel nitrate-reducing microorganisms that are capable of using phenol as a sole source of carbon were isolated and characterized. Phenol-degrading denitrifying pure cultures were obtained by enrichment culture from anaerobic sediments obtained from three different geographic locations, the East River in New York, N.Y., a Florida orange grove, and a rain forest in Costa Rica. The three strains were shown to be different from each other based on physiologic and metabolic properties. Even though analysis of membrane fatty acids did not result in identification of the organisms, the fatty acid profiles were found to be similar to those of Azoarcus species. Sequence analysis of 16S ribosomal DNA also indicated that the phenol-degrading isolates were closely related to members of the genus Azoarcus. The results of this study add three new members to the genus Azoarcus, which previously comprised only nitrogen-fixing species associated with plant roots and denitrifying toluene degraders.

Nitrite adulteration of workplace urine drug-testing specimens. I. Sources and associated concentrations of nitrite in urine and distinction between natural sources and adulteration.

The active ingredient in the commercial workplace urine drug-testing adulterant, Klear, was previously determined to be nitrite ion. Nitrite adulteration compromises the confirmation of some drugs, notably the marijuana metabolite. A previously reported bisulfite step overcomes some nitrite adulteration, but it cannot do so in every case, which leaves the laboratory to report the specimen as not suitable for testing. Unlike many other adulterants, nitrite is found in normal urine at low concentrations. In order to defend a report of nitrite adulteration, it is necessary to provide evidence that the amount of nitrite in a workplace urine specimen could not arise by normal means. The objectives of this study were to identify all sources of nitrite in urine and the range of concentrations associated with these sources and to determine if nitrite adulteration can be supported based upon a quantitative result. The scientific literature was reviewed for internal and external sources of nitrite and their concentration ranges and are reported. The following specimens were obtained and nitrite concentrations measured by a spectrophotometric method: clinical specimens nitrite positive by test strip (< 15 micrograms/mL); specimens culture positive for nitrate-reducing microorganisms (< 36 micrograms/mL); specimens from patients on medications that may metabolize to nitrite (< 6 micrograms/mL); and drug-test specimens, both negative (< 130 micrograms/mL) and others that appeared to be adulterated with nitrite (range 1910-12,200 micrograms/mL, mean 5910). The literature and the nitrite measurements of this study indicate a substantial difference between concentrations from natural sources compared with adulteration. A quantitative measurement of nitrite by a well-structured assay can provide scientifically valid and forensically defensible proof of adulteration with a nitrite-containing substance.

N-nitrosodimethylamine, nitrate and nitrate-reducing microorganisms in human milk.

Of 54 milk samples from 54 healthy nursing women analysed for volatile N-nitrosamines, 42 appeared negative. Trace amounts (below the detection limit 0.5 microgram l-1) of N-nitrosodimethylamine were detected in the milk of 10 mothers and two samples contained this compound at 1.1 and 1.2 micrograms 1-1 respectively. Almost all samples investigated contained nitrate (mean 2.9 +/- 2.3 mg1-1 and nitrate reducing microorganisms (mean 4.2 +/- 1.0 log ml-1). The recent finding of N-nitrosodimethylamine in human milk gives evidence of the continuous endogenous formation of N-nitrosamines.

Nitrous Oxide Emission From Microorganisms.

N2O has been caused more and more attention because of its green-house effect. Microbial activity in biosphere is regarded as an important N2O source. Therefore, to make clear and modify the activity of microorganisms is of fundamental importance to control N2O emission from microorganisms, especially, in Wasetwater treatment situations. In the paper, based on investigation, heterotrophic, nitrate respiring and nitrate reducing microorganisms were emphasized as those being able to produce N2O, besides traditional nitrifiers and denitrifiers. There are different pathways of N2O emission for different organisms under different conditions. The possible mechanisms of N2O yielding were detailed in the paper accompanying with the description of various factors affecting N2O emission. Then, current situation of research on control of N2O emission from wastewater treatment was described and analyzed for making clear the trend of research on N2O emission control in waste water treatment field.

Quantitative relationship between oral nitrate-reducing activity and the endogenous formation of N-nitrosoamino acids in humans

Salivary nitrate and nitrite concentrations, in vitro kinetics of nitrate reduction in saliva, and numbers of salivary nitrate-reducing micro-organisms were each compared with N-nitrosoamino acid excretion in 16 humans eating controlled diets. N-Nitrosoproline (NPRO) and N-nitrosothiazolidine-4-carboxylic acid (NTCA) excretion were measured after intake of nitrate (5.24 mmol) and l-proline (4.35 mmol) before and after treatment with an oral antiseptic (Peridex, 0.12% chlorhexidine gluconate). Peridex treatment resulted in a 94% reduction in the numbers of salivary nitrate-reducing bacteria, a decline from 17 to 4% in the amount of salivary nitrate that was reduced to nitrite in vivo, and an 85% reduction in the rate of in vitro nitrate reduction by saliva. Concurrently, there were 62 and 74% inhibitions of endogenous NPRO and NTCA formation, respectively. Correlations of the numbers of nitrate reductase micro-organisms, in vivo oral nitrate reduction and salivary nitrite concentrations, with individual NPRO excretion, indicated that individuals with higher oral nitrate-reducing capacities formed more N-nitrosoamino acid endogenously. These data suggest that individual differences in oral nitrate reduction are a significant factor in gastric nitrosation and account for a large proportion of the interindividual variability in nitrosoamino acid excretion.

Nitrate-reducing microorganisms in the gastric juice of patients with stomach cancer--experimental data and considerations upon the role of these microorganisms in gastrocarcinogenesis.

During recent years evidence has been accumulated supporting the hypothesis according to which nitroso compounds are involved in the generation of stomach cancer in humans. The present report deals with the occurrence and content of nitrite, nitrate and nitrosamines in the gastric juice of stomach cancer patients with special reference to nitrate-reducing microorganisms. Microorganisms were found to be present in gastric juice in all cases of cancer, mean total count being (5.6 +/- 2.4) log/ml (mean +/- standard deviation) and with limit values 1.4-8.0 log/ml. Nitrate-reducing microorganisms were found in 9 cases from 10. Their mean counts were (5.2 +/- 2.5) log/ml and limit values less than 0.5-8.0 log/ml. The role of nitrate-reducing microorganisms in the formation of nitrite and carcinogenic nitroso compounds is discussed. Some practical measures to control the count of nitrate-reducing microorganisms in stomach are presented.