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Concept: Nitrate

168

A major challenge for the bioremediation of toxic metals is the co-occurrence of nitrate, as it can inhibit metal transformation. Geobacter metallireducens, Desulfovibrio desulfuricans, and Sulfurospirillum barnesii are three soil bacteria that can reduce chromate [Cr(VI)] and nitrate, and may be beneficial for developing bioremediation strategies. All three organisms respire through dissimilatory nitrate reduction to ammonia (DNRA), employing different nitrate reductases but similar nitrite reductase (Nrf). G. metallireducens reduces nitrate to nitrite via the membrane bound nitrate reductase (Nar), while S. barnesii and D. desulfuricans strain 27774 have slightly different forms of periplasmic nitrate reductase (Nap). We investigated the effect of DNRA growth in the presence of Cr(VI) in these three organisms and the ability of each to reduce Cr(VI) to Cr(III), and found that each organisms responded differently. Growth of G. metallireducens on nitrate was completely inhibited by Cr(VI). Cultures of D. desulfuricans on nitrate media was initially delayed (48 h) in the presence of Cr(VI), but ultimately reached comparable cell yields to the non-treated control. This prolonged lag phase accompanied the transformation of Cr(VI) to Cr(III). Viable G. metallireducens cells could reduce Cr(VI), whereas Cr(VI) reduction by D. desulfuricans during growth, was mediated by a filterable and heat stable extracellular metabolite. S. barnesii growth on nitrate was not affected by Cr(VI), and Cr(VI) was reduced to Cr(III). However, Cr(VI) reduction activity in S. barnesii, was detected in both the cell free spent medium and cells, indicating both extracellular and cell associated mechanisms. Taken together, these results have demonstrated that Cr(VI) affects DNRA in the three organisms differently, and that each have a unique mechanism for Cr(VI) reduction.

Concepts: Bacteria, Metabolism, Redox, Nitrogen, Bioremediation, Denitrification, Nitrate, Nitrate reductase

38

Recent studies have suggested that dietary inorganic nitrate (NO(3) (-)) supplementation may improve muscle efficiency and endurance exercise tolerance but possible effects during team sport-specific intense intermittent exercise have not been examined. We hypothesized that NO(3) (-) supplementation would enhance high-intensity intermittent exercise performance. Fourteen male recreational team-sport players were assigned in a double-blind, randomized, crossover design to consume 490 mL of concentrated, nitrate-rich beetroot juice (BR) and nitrate-depleted placebo juice (PL) over ~30 h preceding the completion of a Yo-Yo intermittent recovery level 1 test (Yo-Yo IR1). Resting plasma nitrite concentration ([NO(2) (-)]) was ~400 % greater in BR compared to PL. Plasma [NO(2) (-)] declined by 20 % in PL (P < 0.05) and by 54 % in BR (P < 0.05) from pre-exercise to end-exercise. Performance in the Yo-Yo IR1 was 4.2 % greater (P < 0.05) with BR (1,704 ± 304 m) compared to PL (1,636 ± 288 m). Blood [lactate] was not different between BR and PL, but the mean blood [glucose] was lower (3.8 ± 0.8 vs. 4.2 ± 1.1 mM, P < 0.05) and the rise in plasma [K(+)] tended to be reduced in BR compared to PL (P = 0.08). These findings suggest that NO(3) (-) supplementation may promote NO production via the nitrate-nitrite-NO pathway and enhance Yo-Yo IR1 test performance, perhaps by facilitating greater muscle glucose uptake or by better maintaining muscle excitability. Dietary NO(3) (-) supplementation improves performance during intense intermittent exercise and may be a useful ergogenic aid for team sports players.

Concepts: Better, Protein, Crossover study, Muscle, Lactic acid, Nitrate, Nitrite, Yo-yo

28

Onsite wastewater systems, or septic tanks, serve approximately 25% of the United States population; they are therefore a critical component of the total carbon balance for natural water bodies. Septic tanks operate under strictly anaerobic conditions, and fermentation is the dominant process driving carbon transformation. Nitrate, Fe(III), and sulfate reduction may be operating to a limited extent in any given septic tank. Electron acceptor amendments will increase carbon oxidation, but nitrate is toxic and sulfate generates corrosive sulfides, which may damage septic system infrastructure. Fe(III) reducing microorganisms transform all major classes of organic carbon that are dominant in septic wastewater: low molecular weight organic acids, carbohydrate monomers and polymers, and lipids. Fe(III) is not toxic, and the reduction product Fe(II) is minimally disruptive if the starting Fe(III) is added at 50-150mgL(-1). We used (14)C radiolabeled acetate, lactate, propionate, butyrate, glucose, starch, and oleic acid to demonstrate that short and long-term carbon oxidation is increased when different forms of Fe(III) are amended to septic wastewater. The rates of carbon mineralization to (14)CO(2) increased 2-5times (relative to unamended systems) in the presence of Fe(III). The extent of mineralization reached 90% for some carbon compounds when Fe(III) was present, compared to levels of 50-60% in the absence of Fe(III). (14)CH(4) was not generated when Fe(III) was added, demonstrating that this strategy can limit methane emissions from septic systems. Amplified 16S rDNA restriction analysis indicated that unique Fe(III)-reducing microbial communities increased significantly in Fe(III)-amended incubations, with Fe(III)-reducers becoming the dominant microbial community in several incubations. The form of Fe(III) added had a significant impact on the rate and extent of mineralization; ferrihydrite and lepidocrocite were favored as solid phase Fe(III) and chelated Fe(III) (with nitrilotriacetic acid or EDTA) as soluble Fe(III) forms.

Concepts: Redox, Soil, Sewage treatment, Wastewater, Nitrate, Septic tank, Imhoff tank, Septic drain field

28

The salivary glands of adults concentrate nitrate from plasma into saliva where it is converted to nitrite by bacterial nitrate reductases. Nitrite can play a beneficial role in adult gastrointestinal and cardiovascular physiology. When nitrite is swallowed, some of it is converted to nitric oxide (NO) in the stomach and may then exert protective effects in the gastrointestinal tract and throughout the body. It has yet to be determined either when newborn infants acquire oral nitrate reducing bacteria or what the effects of antimicrobial therapy or premature birth may be on the bacterial processing of nitrate to nitrite. We measured nitrate and nitrite levels in the saliva of adults and both preterm and term human infants in the early weeks of life. We also measured oral bacterial reductase activity in the saliva of both infants and adults, and characterized the species of nitrate reducing bacteria present. Oral bacterial conversion of nitrate to nitrite in infants was either undetectable or markedly lower than the conversion rates of adults. No measurable reductase activity was found in infants within the first two weeks of life, despite the presence of oral nitrate reducing bacteria such as Actinomyces odontolyticus, Veillonella atypica, and Rothia mucilaginosa. We conclude that relatively little nitrite reaches the infant gastrointestinal tract due to the lack of oral bacterial nitrate reductase activity. Given the importance of the nitrate-nitrite-NO axis in adults, the lack of oral nitrate-reducing bacteria in infants may be relevant to the vulnerability of newborns to hypoxic stress and gastrointestinal tract pathologies.

Concepts: Childbirth, Infant, Digestive system, Nitrogen, Infant mortality, Newborn, Nitrate, Nitrate reductase

27

To characterize the denitrifying phosphorus (P) uptake properties of Accumulibacter, a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of Accumulibacter and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized Accumulibacter subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria [Dechloromonas (from 1.2% to 19.2%) and Competibacter (from 16.4% to 20.0%)], while overall Accumulibacter decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses were performed to characterize the denitrifying P-uptake properties of the Accumulibacter clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/L), all Accumulibacter clades successfully took up phosphorus in the presence of nitrate. However, the Accumulibacter clades showed no P-uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/L); under these conditions, reduction of nitrate to nitrite did not occur, whereas P-uptake by Accumulibacter clades occurred when nitrite was added. These results suggest that the Accumulibacter cells lack nitrate reduction capabilities and that P-uptake by Accumulibacter is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and Competibacter.

Concepts: Gene expression, Molecular biology, Ribosomal RNA, Biotechnology, 16S ribosomal RNA, In situ hybridization, Enhanced biological phosphorus removal, Nitrate

27

The pathology of a gastric ulcer is complex and multifactorial. Gastric ulcers affect many people around the world and its development is a result of the imbalance between aggressive and protective factors in the gastric mucosa. In this study, we evaluated the ethanolic extract of Rosmarinus officinalis L. (eeRo); this plant, more commonly known as rosemary, has attracted the interest of the scientific community due to its numerous pharmacological properties and their potential therapeutic applications. Here, we tested the preventive effects of eeRo against gastric ulcer induced by 70% ethanol in male Wistar rats. In addition, we aimed to clarify the mechanism involved in the preventive action of the eeRo in gastric ulcers. Based on the analysis of markers of oxidative damage and enzymatic antioxidant defense systems, the measurement of nitrite and nitrate levels and the assessment of the inflammatory response, the eeRo exhibited significant antioxidant, vasodilator and antiinflammatory properties.

Concepts: Inflammation, Stomach, Anti-inflammatory, Helicobacter pylori, Peptic ulcer, Lamiaceae, Nitrate, Rosemary

26

It has been shown that nitrate supplementation can enhance endurance exercise performance. Recent work suggests that nitrate ingestion can also increase intermittent type exercise performance in recreational athletes. We hypothesized that six days of nitrate supplementation can improve high-intensity intermittent type exercise performance in trained soccer players. Thirty-two male soccer players (age: 23 ± 1 years, height: 181 ± 1 m, weight: 77 ± 1 kg, playing experience: 15.2 ± 0.5 years, playing in the first team of a 2nd or 3rd Dutch amateur league club) participated in this randomized, double-blind cross-over study. All subjects participated in two test days in which high-intensity intermittent running performance was assessed using the Yo-Yo IR1 test. Subjects ingested nitrate-rich (140 mL; ~800 mg nitrate/day; BR) or a nitrate-depleted beetroot juice (PLA) for six subsequent days, with at least eight days of wash-out between trials. The distance covered during the Yo-Yo IR1 was the primary outcome measure, while heart rate (HR) was measured continuously throughout the test, and a single blood and saliva sample were collected just prior to the test. Six days of BR ingestion increased plasma and salivary nitrate and nitrite concentrations in comparison to PLA (p < 0.001), and enhanced Yo-Yo IR1 test performance by 3.4 ± 1.3% (from 1574 ± 47 to 1623 ± 48 m; p = 0.027). Mean HR was lower in the BR (172 ± 2) vs. PLA trial (175 ± 2; p = 0.014). Six days of BR ingestion effectively improves high-intensity intermittent type exercise performance in trained soccer players.

Concepts: Saliva, Measurement, Test method, Kilogram, Nitrate, Endurance, Nitrite, Yo-yo

17

It has been reported that nitrate supplementation can improve exercise performance. Most of the studies have used either beetroot juice or sodium nitrate as a supplement; there is lack of data on the potential ergogenic benefits of an increased dietary nitrate intake from a diet based on fruits and vegetables. Our aim was to assess whether a high-nitrate diet increases nitric oxide bioavailability and to evaluate the effects of this nutritional intervention on exercise performance. Seven healthy male subjects participated in a randomized cross-over study. They were tested before and after 6 days of a high (HND) or control (CD) nitrate diet (~8.2 mmol∙day(-1) or ~2.9 mmol∙day(-1), respectively). Plasma nitrate and nitrite concentrations were significantly higher in HND (127 ± 64 µM and 350 ± 120 nM, respectively) compared to CD (23 ± 10 µM and 240 ± 100 nM, respectively). In HND (vs. CD) were observed: (a) a significant reduction of oxygen consumption during moderate-intensity constant work-rate cycling exercise (1.178 ± 0.141 vs. 1.269 ± 0.136 L·min(-1)); (b) a significantly higher total muscle work during fatiguing, intermittent sub-maximal isometric knee extension (357.3 ± 176.1 vs. 253.6 ± 149.0 Nm·s·kg(-1)); © an improved performance in Repeated Sprint Ability test. These findings suggest that a high-nitrate diet could be a feasible and effective strategy to improve exercise performance.

Concepts: Ammonia, Nutrition, Obesity, Oxide, Diet, Nitric acid, Nitrate, Sodium nitrite

16

Dietary supplementation with beetroot juice (BR) containing ~5-8 mmol of inorganic nitrate (NO3(-)) increases plasma nitrite concentration ([NO2(-)]), reduces blood pressure, and may positively influence the physiological responses to exercise. However, the dose-response relationship between the volume of BR ingested and the physiological effects invoked has not been investigated. In a balanced crossover design, 10 healthy males ingested 70, 140 or 280 ml of concentrated BR (containing 4.2, 8.4 and 16.8 mmol NO3-, respectively) or no supplement to establish the effects of BR on resting plasma [NO3(-)] and [NO2(-)] over 24 h. Subsequently, on six separate occasions, 10 subjects completed moderate-intensity and severe-intensity cycle exercise tests 2.5 h post-ingestion of 70, 140 and 280 ml BR, or NO3(-)-depleted BR as placebo (PL). Following acute BR ingestion, plasma [NO2(-)] increased in a dose-dependent manner, with the peak changes occurring at ~2-3 h. Compared to PL, 70 ml BR did not alter the physiological responses to exercise. However, 140 and 280 ml BR reduced the steady-state VO2 during moderate-intensity exercise by 1.7% (P=0.06) and 3.0% (P<0.05), whilst time to task failure was extended by 14% and 12% (both P<0.05), respectively, compared to PL. The results indicate that, while plasma [NO2(-)] and the O2 cost of moderate-intensity exercise are improved dose-dependently with NO3(-)-rich BR, there is no additional improvement in exercise tolerance after ingesting BR containing 16.8 compared to 8.4 mmol NO3(-). These findings have important implications for the use of BR to enhance cardiovascular health and exercise performance in young adults.

Concepts: Ingestion, Dose-response relationship, Pharmacodynamics, Nitrate

11

Nitrate, the most oxidized form of nitrogen, is regulated to protect people and animals from harmful levels as there is a large over abundance due to anthropogenic factors. Widespread field testing for nitrate could begin to address the nitrate pollution problem, however, the Cadmium Reduction Method, the leading certified method to detect and quantify nitrate, demands the use of a toxic heavy metal. An alternative, the recently proposed Environmental Protection Agency Nitrate Reductase Nitrate-Nitrogen Analysis Method, eliminates this problem but requires an expensive proprietary spectrophotometer. The development of an inexpensive portable, handheld photometer will greatly expedite field nitrate analysis to combat pollution. To accomplish this goal, a methodology for the design, development, and technical validation of an improved open-source water testing platform capable of performing Nitrate Reductase Nitrate-Nitrogen Analysis Method. This approach is evaluated for its potential to i) eliminate the need for toxic chemicals in water testing for nitrate and nitrite, ii) reduce the cost of equipment to perform this method for measurement for water quality, and iii) make the method easier to carryout in the field. The device is able to perform as well as commercial proprietary systems for less than 15% of the cost for materials. This allows for greater access to the technology and the new, safer nitrate testing technique.

Concepts: Hydrogen, Redox, Oxidizing agent, Nitrogen, Heavy metal music, Free software, Nitrate, Proprietary software