Anaerobic degradation of 3-hydroxybenzoate by a newly isolated nitrate-reducing bacterium

A Gram-negative nitrate-reducing bacterium, strain Asl-3, was isolated from activated sludge with nitrate and 3-hydroxybenzoate as sole source of carbon and energy. The new isolate was facultatively anaerobic, catalase- and oxidase-positive and polarly monotrichously flagellated. In addition to nitrate, nitrite, N2O, and O2 served as electron acceptors. Growth with 3-hydroxybenzoate and nitrate was biphasic: nitrate was completely reduced to nitrite before nitrite reduction to N2 started. Benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, protocatechuate or phenyl-acetate served as electron and carbon source under aerobic and anaerobic conditions. During growth with excess carbon source, poly-beta-hydroxybutyrate was formed. These characteristics allow the affiliation of strain Asl-3 with the family Pseudomonadaceae. Analogous to the pathway of 4-hydroxybenzoate degradation in other bacteria, the initial step in anaerobic 3-hydroxybenzoate degradation by this organism was activation to 3-hydroxy-benzoyl-CoA in an ATP-consuming reaction. Cell extracts of 3-hydroxybenzoate-grown cells exhibited 3-hydroxybenzoyl-CoA synthetase activity of 190 nmol min-1 mg protein-1 as well as benzoyl-CoA synthetase activity of 86 nmol min-1 mg protein-1. A reductive dehydroxylation of 3-hydroxybenzoyl-CoA could not be demonstrated due to rapid hydrolysis of chemically synthesized 3-hydroxybenzoyl-CoA by cell extracts.     

Anaerobic Nitrate-Dependent Iron (II) Oxidation by a Novel Autotrophic Bacterium,Citrobacter freundii Strain PXL1

Anaerobic Fe(II) Oxidizing Denitrifiers (AFODN), a type of newly found Fe(II)-oxidizing bacteria, play an important role in iron and nitrogen cycling. In the present study, a novel AFODN strain PXL1 was isolated from anaerobic activated sludge. Phylogenetic analysis of 16S rRNA gene sequence revealed similarity between this strain and Citrobactor freundii. The strain reduced 30% of nitrate and oxidized 85% of Fe(II) over 72 h with an initial Fe(II) concentration of 3.4 mM and nitrate concentration of 9.5 mM. Oxidation of iron was dependent on the reduction of nitrate to nitrite in the absence of other electron donors or acceptors. Nitrate reduction and Fe(II) oxidation followed first-order reaction kinetics. Iron oxides accumulated in the culture were analyzed by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) spectroscopy and scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS). The strain recovered deposited oxidized Fe in the form of amorphous Fe oxides.     

Complete denitrification in coculture of obligately chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria from a hypersaline soda lake

Eight anaerobic enrichment cultures with thiosulfate as electron donor and nitrate as electron acceptor were inoculated with sediment samples from hypersaline alkaline lakes of Wadi Natrun (Egypt) at pH 10; however, only one of the cultures showed stable growth with complete nitrate reduction to dinitrogen gas. The thiosulfate-oxidizing culture subsequently selected after serial dilution developed in two phases. Initially, nitrate was mostly reduced to nitrite, with a coccoid morphotype prevailing in the culture. During the second stage, nitrite was reduced to dinitrogen gas, accompanied by mass development of thin motile rods. Both morphotypes were isolated in pure culture and identified as representatives of the genus Thioalkalivibrio, which includes obligately autotrophic sulfur-oxidizing haloalkaliphilic species. Nitrate-reducing strain ALEN 2 consisted of large nonmotile coccoid cells that accumulated intracellular sulfur. Its anaerobic growth with thiosulfate, sulfide, or polysulfide as electron donor and nitrate as electron acceptor resulted in the formation of nitrite as the major product. The second isolate, strain ALED, was able to grow anaerobically with thiosulfate as electron donor and nitrite or nitrous oxide (but not nitrate) as electron acceptor. Overall, the action of two different sulfur-oxidizing autotrophs resulted in the complete, thiosulfate-dependent denitrification of nitrate under haloalkaliphilic conditions. This process has not yet been demonstrated for any single species of chemolithoautotrophic sulfur-oxidizing haloalkaliphiles.     

ANAMMOX process start up and stabilization with an anaerobic seed in Anaerobic Membrane Bioreactor (AnMBR)

ANaerobic AMMonium OXidation (ANAMMOX) process, an advanced biological nitrogen removal alternative to traditional nitrification--denitrification removes ammonia using nitrite as the electron acceptor without oxygen. The feasibility of enriching anammox bacteria from anaerobic seed culture to start up an Anaerobic Membrane Bioreactor (AnMBR) for N-removal is reported in this paper. The Anammox activity was established in the AnMBR with anaerobic digester seed culture from a Sewage Treatment Plant in batch mode with recirculation followed by semi continuous process and continuous modes of operation. The AnMBR performance under varying Nitrogen Loading Rates (NLR) and HRTs is reported for a year, in terms of nitrogen transformations to ammoniacal nitrogen, nitrite and nitrate along with hydrazine and hydroxylamine. Interestingly ANAMMOX process was evident from simultaneous Amm-N and nitrite reduction, consistent nitrate production, hydrazine and hydroxylamine presence, notable organic load reduction and bicarbonate consumption.     

Denitrifikation bei Hydrogenomonas eutropha Stamm H16

Zusammenfassung Hydrogenomonas eutropha (syn. Alcaligenes eutrophus) Stamm H 16 wächst anaerob mit Fructose und Nitrat bzw. Nitrit. Autotrophanaerobes Wachstum unter einer H₂-CO₂-Atmosphäre (90+10 Vol.-%) mit Nitrat als einzigem Wasserstoff-Acceptor ist minimal.Während des anaeroben Wachstums mit Nitrat sind zwei Phasen zu unterscheiden. In der ersten Phase erfolgt die Zellvermehrung auf Kosten der Reduktion von Nitrat zu Nitrit; dieses wird angehäuft. In der zweiten Phase wird Nitrit unter Bildung von Stickstoff reduziert.Gewaschene, anaerob gewachsene Zellen reduzieren Nitrat und Nitrit unter Bildung von N₂. Stöchiometrische Experimente mit H₂ oder Fructose als H-Donatoren lassen darauf schließen, daß Stickstoff das einzige Produkt der Denitrifikation durch die Zellen ist. Diese Schlußfolgerung wurde durch eine massenspektrometrische Analyse des gebildeten Gases bestätigt. Aerob gewachsene Zellen reduzieren Nitrat nur zu Nitrit. In Gegenwart von Ammonium-Salz gewachsene Zellen reduzieren Nitrat mit sehr geringer Rate.Die Ergebnisse deuten darauf hin, daß Stamm H 16 über nur eine Nitratreductase verfügt. Die Bildung des Enzyms ist durch Ammonium reprimierbar; O₂ ist ohne Einfluß. Die Nitritreductase fand sich sowohl in der löslichen Fraktion als auch in den gereinigten Partikeln lokalisiert. Das Nitritreductase-System wird nur unter anaeroben Bedingungen gebildet.

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Denitrification in Hydrogenomonas eutropha strain H16

Summary The hydrogen bacterium Hydrogenomonas eutropha (syn. Alcaligenes eutrophus) strain H 16 is able to grow anaerobically with fructose and nitrate or nitrite, respectively. Autotrophic anaerobic growth under a gas atmosphere of hydrogen and carbon dioxide (90+10 vol-%) with nitrate as the sole hydrogen acceptor is minimal.During anaerobic growth with nitrate as H-acceptor, two growth phases are distinguishable: During the first phase cell growth occurs with the reduction of nitrate to nitrite, which is accumulated; on the second phase nitrite is reduced with the formation of gaseous nitrogen.Washed, anaerobically grown cells reduce nitrate and nitrite with the formation of N₂. Stoichiometric experiments employing hydrogen or fructose as the hydrogen donors are consistent with the conclusion that nitrogen is the sole product of denitrification by these cells. This was confirmed by mass spectrometric analysis of the gas formed. Aerobically grown cells are able to reduce nitrate only to nitrite; when grown in the presence of ammonia, the reduction rate is very low.The results indicate that strain H 16 contains only one nitrate reductase. The formation of this enzyme system is not influenced by oxygen, however, is repressed by ammonia.When employing a purified soluble fraction and particles, nitrite reductases were found in both fractions. The nitrite reductase system is formed only under anaerobic conditions.

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Abkürzungen MB Methylenblau - PMS Phenazinmethosulfat     

Anaerobic metabolism of cyclohexanol by denitrifying bacteria

Three strains of denitrifying bacteria were anaerobically enriched and isolated from oxic or anoxic habitats with cyclohexanol or cyclohexanone as sole electron donor and carbon source and with nitrate as electron acceptor. The bacteria were facultatively anaerobic, Gramnegative and metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as terminal electron acceptor. Cyclohexanol and cyclohexanone were degraded both anaerobically and aerobically. Aromatic compounds were oxidized in the presence of molecular oxygen only. One of the bacterial strains was further characterized. During anaerobic cyclohexanol degradation approximately 40% of the substrate was oxidized to phenol, which accumulated as dead-endproduct in the growth medium; 60% of cyclohexanol was completely oxidized to CO₂ and assimilated, respectively. In addition to phenol formation, transient accumulation of cyclohexanone, 2-cyclohexenone and 1,3-cyclohexanedione was observed. Based on these findings we propose a pathway for anaerobic cyclohexanol degradation involving these intermediates.     

Mixed culture of nitrifying bacteria and denitrifying bacteria for simultaneous nitrification and denitrification

A mixed culture containing nitrifying bacteria and denitrifying bacteria was investigated for aerobic simultaneous nitrification and denitrification. A mixture of NaHCO₃ and CH₃COONa was selected as the appropriate carbon source for cell growth and nitrogen removal, the concentrations of carbon and nitrogen sources were also examined. Ammonia could be oxidized aerobically to nitrite by the mixed culture, and the intermediate nitrite was then reduced to dinitrogen gas. No nitrite was detected during the process. 0.212 g of ammonia/l could be removed in 30 h and nitrate could not be utilized aerobically by the mixed culture. Nitrite could be degraded aerobically as well as anaerobically. Very little ammonia was degraded anaerobically, but the ability to degrade ammonia could be recovered even after oxygen had been supplied for 42 h.     

The effects of different electron donors on anaerobic nitrogen transformations and denitrification processes in Lake Taihu sediments

Nitrogen transformations in anaerobic sediments and leachate in Lake Taihu were simulated in the laboratory. Ammonium, nitrate and nitrite were analyzed after incubation under anaerobic conditions. Different reductive states and pH values were obtained by using different electron donors, such as glucose, sucrose, potato starch and sodium acetate. Chemical nitrogen transformation mechanisms were discussed relative to physico-chemical properties of lake sediment. Results demonstrated that nitrogen transformations in anaerobic conditions supplemented with different electron donors varied, and supplementation with certain electron donors may enhance nitrogen removal from anaerobic sediments. Among the four electron donors studied, higher nitrogen removal efficiencies were observed with acetate and starch. Saccharides, such as glucose, sucrose and starch, stimulate nitrate reduction to nitrite, while acetate stimulates nitrate reduction to ammonium.     

Anaerobic degradation of 2-aminobenzoate (anthranilic acid) by denitrifying bacteria

In the presence of oxygen many aminoaromatic compounds polymerize to form recalcitrant macromolecules. To circumvent undesirable oxidation reactions, the anaerobic biodegradation of a simple member of this class of compounds was investigated. Two strains of bacteria were isolated which degrade 2-aminobenzoate anaerobically under denitrifying conditions, with nitrate as the terminal electron acceptor. Both organisms, which were assigned to the genus Pseudomonas, oxidized 2-aminobenzoate completely to CO2 and NH4+. Nitrate was reduced to nitrite. When nitrate was deplete from the growth medium the accumulated nitrite was reduced to nitrogen. The results establish a model system for the anaerobic, rapid, and complete oxidation of an aminoaromatic compound.     

Trinitrotoluene (TNT) as a sole nitrogen source for a sulfate-reducing bacteriumDesulfovibrio sp. (B strain) isolated from an anaerobic digester

A sulfate-reducing bacterium (SRB),Desulfovibrio sp. (B strain), isolated from a continuous anaerobic digester (Boopathy and Daniels, Current Microbiology, 23:327–332, 1991) was found to use 2,4,6-trinitrotoluene (TNT) as sole nitrogen source. This bacterium also used nitrate, nitrite, and ammonium as nitrogen source. A long lag period was noticed when TNT or nitrite was used as nitrogen source. Nitrate, nitrite and TNT also served as electron acceptor in the absence of sulfate for this bacterium. Under nitrogen-limiting condition, 100% removal of TNT was observed within 8 days of incubation. The main intermediate observed was diaminonitrotoluene, which was further converted to toluene via triaminotoluene by reductive deamination process. Under nitrogen-rich conditions (presence of ammonium), TNT was converted to diaminonitrotoluene, and toluene was not produced. This isolate did not degrade TNT all the way to CO₂. This study demonstrated the possibility of using this isolated to decontaminate the soil and water contaiminated with TNT under anaerobic conditions.