Selenium reduction by a denitrifying consortium

A denitrifying bacterial consortium obtained from the Pullman, Washington wastewater treatment facility was enriched under denitrifying conditions and its ability to reduce selenite and selenate was studied. Replicate experiments at two different experimental conditions were performed. All experiments were performed under electron-acceptor limiting conditions, with acetate as the carbon source and nitrate the electron acceptor. In the first set of experiments, selenite was present, whereas, in the second set, selenate was added. A significant lag period of approximately 150 h was necessary before selenite or selenate reduction was observed. During this lag period, nitrate and nitrite use was observed. Once selenite or selenate reduction had started, nitrate and nitrite reduction was concomitant with selenium species reduction. Trace amounts of selenite were detected during the selenate reduction study. Analysis of the data indicates that, once selenium species reduction was induced, the rate of reduction was proportional to the selenium species concentration and to the biomass concentration. Furthermore, at similar biomass and contaminant concentrations, selenite reduction is approximately four times faster than selenate reduction. Copyright 1999 John Wiley & Sons, Inc.     

The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium

Thauera selenatis grows anaerobically with selenate, nitrate or nitrite as the terminal electron acceptor; use of selenite as an electron acceptor does not support growth. When grown with selenate, the product was selenite; very little of the selenite was further reduced to elemental selenium. When grown in the presence of both selenate and nitrate both electron acceptors were reduced concomitantly; selenite formed during selenate respiration was further reduced to elemental selenium. Mutants lacking the periplasmic nitrite reductase activity were unable to reduce either nitrite or selenite. Mutants possessing higher activity of nitrite reductase than the wild-type, reduced nitrite and selenite more rapidly than the wild-type. Apparently, the nitrite reductase (or a component of the nitrite respiratory system) is involved in catalyzing the reduction of selenite to elemental selenium while also reducing nitrite. While periplasmic cytochrome C ₅₅₁ may be a component of the nitrite respiratory system, the level of this cytochrome was essentially the same in mutant and wild-type cells grown under two different growth conditions (i.e. with either selenate or selenate plus nitrate as the terminal electron acceptors). The ability of certain other denitrifying and nitrate respiring bacteria to reduce selenite will also be described.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.

Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity.     

利用硝酸盐和亚硝酸盐同步富集厌氧甲烷氧化微生物的比较实验

【背景】反硝化厌氧甲烷氧化(Denitrifying anaerobic methane oxidation,DAMO)是以硝酸盐或亚硝酸盐为电子受体以甲烷为电子供体的厌氧氧化过程,对认识全球碳氮循环、削减温室气体排放和开发废水脱氮新技术等方面具有重要意义。【目的】认识以硝酸盐和亚硝酸盐为电子受体的DAMO微生物富集过程和结果的差异性。【方法】在序批式反应器(Sequencing batch reaetor,SBR)内接种混合物,分别以硝酸盐和亚硝酸盐为电子受体连续培养800 d,定期检测反应器基质浓度变化、计算转化速率;利用16S rRNA基因系统发育分析研究功能微生物的多样性,利用实时荧光定量PCR技术定量测定功能微生物。【结果】以亚硝酸盐为电子受体的1、3号反应器富集到了DAMO细菌,未检测到DAMO古菌;以硝酸盐为电子受体的2号反应器富集到了DAMO细菌和古菌的混合物;3个反应器的脱氮速率经过初始低速期、快速提升期,最终达到稳定,但2号快速提升期开始时间比1、3号晚了80 d左右,达到稳定的时间更长,稳定最大速率为1、3号的44.7%、40.3%。【结论】硝酸盐和亚硝酸盐对富集产物有决定性影响;以硝酸盐为电子受体富集得到的DAMO古菌和细菌协同体系可以长期稳定共存,DAMO古菌可能是协同体系中脱氮速率的限制性因素。     

Reduction of Selenium Oxyanions in Wastewater Using Two Bacterial Strains

The biological reduction of selenium oxyanions is capable of reducing both selenate and selenite to insoluble elemental selenium. In this process, however, bacteria inevitably require expensive chemicals such as yeast extract in almost all cases. Therefore, the reduction of selenium oxyanions with inexpensive alcohol would be more practical. A Pseudomonas sp. strain 4C‐C isolated from a sludge in a wastewater treatment facility was able to reduce selenate to selenite using ethanol as an electron donor for its anaerobic respiration, but could not reduce selenite to elemental selenium. Paracoccus denitrificans JCM‐6892, on the other hand, was observed to be able to reduce selenite to elemental selenium in the presence of ethanol, but not selenate to selenite. Therefore, a mixture containing a suspension of Pseudomonas sp. strain 4C‐C and P. denitrificans JCM‐6892 cells allowed selenate to be reduced to insoluble elemental selenium via selenite in the presence of ethanol and was also capable of reducing nitrate to nitrogen gas. Aiming at simplicity of the recovery process of insoluble elemental selenium, a polymeric gel immobilized mixture of the two bacterial strains was examined using ethanol as an electron donor. The immobilized mixture could therefore reduce not only selenate to elemental selenium, but also nitrate to nitrogen gas in a single step. The gel that immobilized the microbial mixture changed its color during the process to bright red and no red elemental selenium was left in the wastewater. This indicates that the reduced elemental selenium was completely absorbed in the gel. This simple bacterial combination would therefore be effective in the presence of ethanol to reduce selenium oxyanions in various wastewaters containing selenium and the other oxyanions.     

Enhanced Toluene Degradation Under Chlorate-Reducing Conditions by Bioaugmentation of Sand Columns with Chlorate- and Toluene-Degrading Enrichments

Chlorate was examined as a potential electron acceptor for enhancing toluene degradation. Most chlorate respiring bacteria (CRB) use nitrate as an electron acceptor, and toluene is known to be degraded under denitrifying conditions. Therefore, it was hypothesized that there would be bacteria that could degrade toluene using chlorate as an electron acceptor, and that chlorate could be used to stimulate toluene degradation. Repeated tests and different approaches in batch tests failed to produce an enrichment capable of toluene degradation supported by chlorate reduction. However, the addition of chlorate increased the overall rate of toluene degradation in bioaugmented columns that were fed chlorate vs. a control column. Toluene removal at an influent toluene concentration of 11 mg/L was 93±5%, which was larger by a factor of 1.95 than toluene removal in a nonbioaugmented control column. Following the discontinued feed of chlorate, toluene removal decreased to 69±4%, demonstrating that chlorate could be used to produce a 1.36-fold increase in toluene removal.     

Effects of nitrate and acetate availability on chloroform production during carbon tetrachloride destruction

Fed batch experiments were performed to test the effects of electron donor and electron acceptor availability on the production of chloroform (CF) during carbon tetrachloride (CT) destruction by a denitrifying bacterial consortium. In one series of tests, acetate (electron donor) was present in excess while nitrate and nitrite (electron acceptor) were limiting. In the other series of tests, acetate was the limiting nutrient, and nitrate and nitrite were in excess. Under nitrate limiting conditions, 50% (+/-17%) of the CT transformed by the microorganisms was converted to CF. However, under acetate limiting conditions, only 4% (+/-4%) of the CT that was degraded appeared as CF. Previous research had suggested that denitrifying bacteria can degrade CT via two competing pathways. One of these pathways produces CF as the predominant end product. The second pathway produces CO(2) as the primary end product. The results shown here suggest that the first pathway is dominant when nitrate and nitrite are depleted while the second pathway, which produces little CF, dominates when nitrate or nitrite are available.     

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.     

Kinetics of hydrogen-dependent denitrification under varying pH and temperature conditions

It is important to determine the effect of changing environmental conditions on the microbial kinetics for design and modeling of biological treatment processes. In this research, the kinetics of nitrate and nitrite reduction by autotrophic hydrogen-dependent denitrifying bacteria and the possible role of acetogens were studied in two sequencing batch reactors (SBR) under varying pH and temperature conditions. A zero order kinetic model was proposed for nitrate and nitrite reduction and kinetic coefficients were obtained at two temperatures (25 +/- 1 and 12 +/- 1 degrees C), and pH ranging from 7 to 9.5. Nitrate and nitrite reduction was inhibited at pH of 7 at both temperatures of 12 +/- 1 and 25 +/- 1 degrees C. The optimum pH conditions for nitrate and nitrite reduction were 9.5 at 25 +/- 1 degrees C and 8.5 at 12 +/- 1 degrees C. Nitrate and nitrite reduction rates were compared, when they were used separately as the sole electron acceptor. It was shown that nitrite reduction rates consistently exceeded nitrate reduction rates, regardless of temperature and pH. The observed transitional accumulation of nitrite, when nitrate was used as an electron acceptor, indicated that nitrite reduction was slowed down by the presence of nitrate. No activity of acetogenic bacteria was observed in the hydrogenotrophic biomass and no residual acetate was detected, verifying that the kinetic parameters obtained were not influenced by heterotrophic denitrification and accurately represented autotrophic activity.     

Anaerobic degradation of alkylated benzenes in denitrifying laboratory aquifer columns

Toluene and m-xylene were rapidly mineralized in an anaerobic laboratory aquifer column operated under continuous-flow conditions with nitrate as an electron acceptor. The oxidation of toluene and m-xylene was coupled with the reduction of nitrate, and mineralization was confirmed by trapping 14CO2 evolved from 14C-ring-labeled substrates. Substrate degradation also took place when nitrous oxide replaced nitrate as an electron acceptor, but decomposition was inhibited in the presence of molecular oxygen or after the substitution of nitrate by nitrite. The m-xylene-adapted microorganisms in the aquifer column degraded toluene, benzaldehyde, benzoate, m-toluylaldehyde, m-toluate, m-cresol, p-cresol, and p-hydroxybenzoate but were unable to metabolize benzene, naphthalene, methylcyclohexane, and 1,3-dimethylcyclohexane. Isotope-dilution experiments suggested benzoate as an intermediate formed during anaerobic toluene metabolism. The finding that the highly water-soluble nitrous oxide served as electron acceptor for the anaerobic mineralization of some aromatic hydrocarbons may offer attractive options for the in situ restoration of polluted aquifers.     

Anaerobic degradation of alkylated benzenes in denitrifying laboratory aquifer columns.

Toluene and m-xylene were rapidly mineralized in an anaerobic laboratory aquifer column operated under continuous-flow conditions with nitrate as an electron acceptor. The oxidation of toluene and m-xylene was coupled with the reduction of nitrate, and mineralization was confirmed by trapping 14CO2 evolved from 14C-ring-labeled substrates. Substrate degradation also took place when nitrous oxide replaced nitrate as an electron acceptor, but decomposition was inhibited in the presence of molecular oxygen or after the substitution of nitrate by nitrite. The m-xylene-adapted microorganisms in the aquifer column degraded toluene, benzaldehyde, benzoate, m-toluylaldehyde, m-toluate, m-cresol, p-cresol, and p-hydroxybenzoate but were unable to metabolize benzene, naphthalene, methylcyclohexane, and 1,3-dimethylcyclohexane. Isotope-dilution experiments suggested benzoate as an intermediate formed during anaerobic toluene metabolism. The finding that the highly water-soluble nitrous oxide served as electron acceptor for the anaerobic mineralization of some aromatic hydrocarbons may offer attractive options for the in situ restoration of polluted aquifers.     

Biogeochemical changes induced in uranium mining waste pile samples by uranyl nitrate treatments under anaerobic conditions

Response of the subsurface soil bacterial community of a uranium mining waste pile to treatments with uranyl nitrate over different periods of time was studied under anaerobic conditions. The fate of the added U(VI) without supplementation with electron donors was investigated as well. By using 16S rRNA gene retrieval, we demonstrated that incubation with uranyl nitrate for 4 weeks resulted in a strong reduction in and even disappearance of some of the most predominant bacterial groups of the original sample. Instead, a strong proliferation of denitrifying and uranium-resistant populations of Rahnella spp. from Gammaproteobacteria and of Firmicutes occurred. After longer incubations for 14 weeks with uranyl nitrate, bacterial diversity increased and populations intrinsic to the untreated samples such as Bacteroidetes and Deltaproteobacteria propagated and replaced the above-mentioned uranium-resistant groups. This indicated that U(VI) was immobilized. Mössbauer spectroscopic analysis revealed an increased Fe(III) reduction by increasing the incubation time from four to 14 weeks. This result signified that Fe(III) was used as an electron acceptor by the bacterial community established at the later stages of the treatment. X-ray absorption spectroscopic analysis demonstrated that no detectable amounts of U(VI) were reduced to U(IV) in the time frames of the performed experiments. The reason for this observation is possibly due to the low level of electron donors in the studied oligotrophic environment. Time-resolved laser-induced fluorescence spectroscopic analysis demonstrated that most of the added U(VI) was bound by organic or inorganic phosphate phases both of biotic origin.     

Pilot-Scale Selenium Bioremediation of San Joaquin Drainage Water with Thauera selenatis

This report describes a simple method for the bioremediation of selenium from agricultural drainage water. A medium-packed pilot-scale biological reactor system, inoculated with the selenate-respiring bacterium Thauera selenatis, was constructed at the Panoche Water District, San Joaquin Valley, Calif. The reactor was used to treat drainage water (7.6 liters/min) containing both selenium and nitrate. Acetate (5 mM) was the carbon source-electron donor reactor feed. Selenium oxyanion concentrations (selenate plus selenite) in the drainage water were reduced by 98%, to an average of 12 (plusmn) 9 (mu)g/liter. Frequently (47% of the sampling days), reactor effluent concentrations of less than 5 (mu)g/liter were achieved. Denitrification was also observed in this system; nitrate and nitrite concentrations in the drainage water were reduced to 0.1 and 0.01 mM, respectively (98% reduction). Analysis of the reactor effluent showed that 91 to 96% of the total selenium recovered was elemental selenium; 97.9% of this elemental selenium could be removed with Nalmet 8072, a new, commercially available precipitant-coagulant. Widespread use of this system (in the Grasslands Water District) could reduce the amount of selenium deposited in the San Joaquin River from 7,000 to 140 lb (ca. 3,000 to 60 kg)/year.     

The influence of nitrate concentration upon the end-products of nitrate dissimilation by bacteria in anaerobic salt marsh sediment

Abstract Experiments were carried out with slurries of saltmarsh sediment to which varying concentrations of nitrate were added. The acetylene blocking technique was used to measure denitrification by accumulation of nitrous oxide, while reduction of nitrate to nitrite and ammonium was also measured. There was good recovery of reduced nitrate and at the smallest concentration of nitrate used (250 μM) there was approximately equal reduction to either ammonium or nitrous oxide (denitrification). Nitrite was only a minor end-product of nitrate reduction. As the nitrate concentration was increased the proportion of the nitrate which was denitrified to nitrous oxide increased, to 83% at the greatest nitrate concentration used (2 mM), while reduction to ammonium correspondingly decreased. This change was attributed either to a greater competitiveness by the denitrifiers for nitrate as the ratio of electron donor to electron acceptor decreased; or to the increased production of nitrite rather than ammonium by fermentative bacteria under high nitrate, the nitrite then being reduced to nitrous oxide by denitrifying bacteria.     

Removal of soluble selenium by a selenate-reducing bacterium Bacillus sp. SF-1

In order to develop a biological process for removal of selenium from industrial wastewater, Bacillus sp. strain SF-1 was isolated from selenium-contaminated sediment. The bacterium reduces selenate to selenite and subsequently to nontoxic insoluble elemental selenium using lactate as an electron donor and selenate as an electron acceptor in an anaerobic condition. Elemental selenium transformed from soluble selenium was deposited both inside and outside of the cells. Since the selenate reduction rate of the strain SF-1 was higher than the selenite reduction rate, selenite was transiently accumulated. In an experiment of the repeated soluble selenium reduction by strain SF-1, 0.5 mM of selenate was sequentially treatable with a cycle of one day. Thus, our sequential system for removal of soluble selenium is very useful.     

Transformation of selenate and selenite to elemental selenium byDesulfovibrio desulfuricans

Summary Desulfovibrio desulfuricans (DSM 1924) can be adapted to grow in the presence of 10 mM selenate or 0.1 mM selenite. This growth occurred in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor. As determined by electron microscopy with energy-dispersive X-ray analysis, selenate and selenite were reduced to elemental selenium which accumulated inside the cells. Selenium granules resulting from selenite metabolism were cytoplasmic while granules of selenium resulting from selenate reduction appeared to be in the periplasmic region. The accumulation of red elemental selenium in the media following stationary phase resulted from cell lysis with the liberation of selenium granules. Growth did not occur with either selenate or selenite as the electron acceptor and¹³C nuclear magnetic resonance indicated that neither selenium oxyanion interfered with fumarate respiration. At 1 M selenate and 100 M selenite, reduction byD. desulfuricans was 95% and 97%, respectively. The high level of total selenate and selenite reduced indicated the suitability ofD. desulfuricans for selenium detoxification.     

Identification and Characterization of an Aeromonas salmonicida (syn Haemophilus piscium) Strain That Reduces Selenite to Elemental Red Selenium

A bacterium that reduces toxic and mobile selenite to insoluble elemental selenium (Se⁰) was isolated from a laboratory scale permeable reactive biobarrier. Biochemical tests and 16S rRNA gene sequence alignment identified the isolate as Aeromonas salmonicida. Two colony types were isolated, one more resistant to selenite than the other. Both grew on agar plates containing 16 mM selenite, although the colony diameter was reduced to 8% of controls with the small colony type and to 18% with the large colony type. Further study was done with the large colony type. In anaerobic culture, this bacterium was able to use nitrate as a term electron acceptor but not selenate or selenite. In aerobic culture, when no nitrate was present, early log phase cells removed selenite at a rate of 2.6 ± 0.42 μmol SeO₃⁻²/mg protein/day. Reduction was retarded by 25 mM nitrate. Mutants with a diminished ability to reduce selenite to Se⁰ also had a reduced ability to reduce nitrate to nitrous oxide. This bacterium, or perhaps its enzymes or DNA, might be used to remove selenite from contaminated groundwaters.     

亚硝酸盐对污水生物除磷影响的研究进展

亚硝酸盐作为生物硝化和反硝化的中间产物, 存在于污水生物脱氮除磷系统中。对于生物强化除磷工艺亚硝酸盐既是电子受体用于反硝化除磷, 同时又是抑制剂影响生物除磷过程。本文综述了聚磷菌在厌氧、好氧和缺氧环境中的代谢机理, 在此基础上分别从好氧除磷和反硝化除磷两方面介绍了亚硝酸盐对污水生物除磷影响的研究, 同时概述了亚硝酸盐对生物除磷的抑制机理, 并对该领域的研究提出了个人见解。     

Failure of an enriched nitrite-DPAO population to use nitrate as an electron acceptor

The metabolism of denitrifying polyphosphate accumulating organisms (DPAO) is not completely known. Recent reports suggest the existence of two types of DPAOs: those that can use nitrate and nitrite as electron acceptors (nitrate-DPAO) and those that can only use nitrite (nitrite-DPAO). Then, the survival of nitrite-DPAO in nitrate reducing environments is due to the existence of flanking denitrifying species, which reduce nitrate to nitrite. This works aims at a better understanding of the nitrite-DPAO population. For this aim, a nitrite-DPAO population was previously selected in a SBR using nitrite as electron acceptor. Then, nitrate utilisation by nitrite-DPAO was studied within a short-term period (4 days) and within a long-term period (50 days) with simultaneous nitrite and nitrate additions. The results obtained clearly indicate that nitrite-DPAO fail to use nitrate as electron acceptor even after 50 days of periodic dosing of nitrate and agree with the dual DPAO theory. Moreover, this failure casts doubts on the feasibility of nitrite based EBPR systems (i.e. partial nitrification + nitrite-DPAO) because these systems will not be able to denitrify an occasional nitrate inlet, which will remain in the effluent.