Identification and Isolation of a Castellaniella Species Important during Biostimulation of an Acidic Nitrate- and Uranium-Contaminated Aquifer

Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.     

Autotrophic, Hydrogen-Oxidizing, Denitrifying Bacteria in Groundwater, Potential Agents for Bioremediation of Nitrate Contamination

Addition of hydrogen or formate significantly enhanced the rate of consumption of nitrate in slurried core samples obtained from an active zone of denitrification in a nitrate-contaminated sand and gravel aquifer (Cape Cod, Mass.). Hydrogen uptake by the core material was immediate and rapid, with an apparent K_m of 0.45 to 0.60 μM and a V_max of 18.7 nmol cm^-3 h^-1 at 30°C. Nine strains of hydrogen-oxidizing denitrifying bacteria were subsequently isolated from the aquifer. Eight of the strains grew autotrophically on hydrogen with either oxygen or nitrate as the electron acceptor. One strain grew mixotrophically. All of the isolates were capable of heterotrophic growth, but none were similar to Paracoccus denitrificans, a well-characterized hydrogen-oxidizing denitrifier. The kinetics for hydrogen uptake during denitrification were determined for each isolate with substrate depletion progress curves; the K_ms ranged from 0.30 to 3.32 μM, with V_maxs of 1.85 to 13.29 fmol cell^-1 h^-1. Because these organisms appear to be common constituents of the in situ population of the aquifer, produce innocuous end products, and could be manipulated to sequentially consume oxygen and then nitrate when both were present, these results suggest that these organisms may have significant potential for in situ bioremediation of nitrate contamination in groundwater.     

好氧反硝化菌的分离鉴定及特性研究

从土壤中分离到1株能以硝酸钠为氮源进行好氧反硝化作用的细菌,命名为Rhodococcussp.DN,分离菌株革兰氏染色为阳性,球状或杆状,菌落颜色为橙红色。该细菌能以乙酰胺为惟一碳源和氮源,能进行氨化和硝化作用并产生亚硝酸。部分长度的16S rDNA序列分析表明,所分离的细菌与Rhodococcus ruber的16S rDNA序列具有99%相似性。     

Potential for In Situ Bioremediation of a Low-pH,High-Nitrate Uranium-Contaminated Groundwater

The potential for stimulating microbial U(VI) reduction as an in situ bioremediation strategy for uranium-contaminated groundwater was evaluated in uranium-contaminated sediment from the FRC, Oak Ridge, TN. Sediment was at low pH (pH 4) and contained high (55 mM) concentrations of nitrate. The addition of organic electron donors resulted in a slow removal of ca. 20% of the nitrate over 120 days with a concurrent increase in pH. Uranium precipitated during nitrate reduction. This precipitation of U(VI) was not due to its reduction to U(IV) because over 90% of the uranium in the sediments remained as U(VI). Studies in which the pH of the sediments was artificially raised suggested that an increase in pH alone could not account for the precipitation of the U(VI) during nitrate reduction. Metal-reducing bacteria were recovered from the sediments in enrichment cultures, but molecular analysis of the sediment demonstrated that the addition of electron donors did not stimulate the growth of these metal reducers. Thus, although U(VI) was precipitated from the groundwater with the simple addition of electron donors, most of the uranium in the sediments was in the form of U(VI), and thus was not effectively immobilized.     

Denitrifying ability of thirteen Rhizobium meliloti strains

The denitrifying ability of thirteen strains of Rhizobium meliloti was tested. Most of the strains were able to reduce nitrate to nitrous oxide or dinitrogen. However, they failed to use nitrate as electron acceptor for ATP generation or growth at low oxygen tensions. Under micro-aerobic conditions, free-living cells of R. meliloti 102-F-51 strain exhibited a constitutive nitrate reductase activity independent of the presence of nitrate. On the other hand, nitrite reductase activity was dependent not only on low levels of oxygen but also on the presence of a high nitrate concentration in the medium. Denitrification activity proceeded immediately once a threshold level of nitrite was accumulated in the medium or in cells incubated with 1mM nitrite. However, a lag period was required when cells were incubated with nitrate.     

Design and Validation of ureC-based Primers for Groundwater Detection of Urea-Hydrolyzing Bacteria

Polymerase chain reaction primers based on the ureC gene are described for use in detecting diverse groundwater urea-hydrolyzing bacteria. Six degenerate primers were designed and evaluated for their ability to detect the gene encoding the large catalytic subunit of urease, ureC. Five combinations of these primers were tested pair-wise and displayed an overlapping detection range for bacterial isolates. Pair L2F/L2R exhibited the greatest detection range for described bacterial species and for bacterial isolates from groundwater samples belonging to the bacterial divisions Firmicutes, Actinobacteria, and the α , β , and γ subdivisions of Proteobacteria. Primers L2F/L2R exhibited a greater detection range than previously described ureC-specific primers, and amplified novel ureC sequences from groundwater isolates in the genera Hydrogenophaga, Acidovorax, Janthinobacterium, and Arthrobacter. A comparative phylogenetic analysis of ureC and 16S rRNA genes was performed to determine the utility of groundwater ureC sequence information as a phylogenetic marker for ureolytic species. Our results were consistent with previous analyses of urease genes which demonstrated that the ureC gene has undergone lateral transfer and is not a robust phylogenetic marker. However, the ureC-specific primers, L2F/L2R, demonstrate a broad detection range for ureolytic species, and can serve to enhance functional diversity analyses of ureolytic bacteria.     

Identification of Denitrifying Bacteria Diversity in an Activated Sludge System by using Nitrite Reductase Genes

Nitrite reduction is the key step in the denitrification reaction with two predominant types of nitrite reductase genes: nirS and nirK. The diversity of denitrifying bacteria in a municipal wastewater treatment plant is described by using both these genes. Of the cultured colonies, 22.5% contained the NirS gene and 12.5% the nirK gene. These nitrite reductase-containing colonies could be further divided into five different types by using both restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysis. Phylogenetic analysis showed that these five types of denitrifying bacteria were phylogenetically diverse. Finally, one nirS gene was obtained and compared with the published sequences.     

脱氮功能菌去除市政废水中氮素的研究

为了探讨实验室筛选获得的氨氧化细菌CM-NRO14和反硝化细菌CM-NRD3联合去除市政废水中氮素的应用价值,采用了两级A/O工艺进行菌株去除废水中氮素的小试实验,最后将菌株用于废水脱氮工程中。结果表明,脱氮功能菌实现了短程硝化-反硝化,氨氮去除率在98%以上,总氮去除率在75%以上, COD (化学需氧量)去除率大于90%,出水各项指标均低于城镇污水处理厂污染物排放一级(A)标准。脱氮功能菌在去除市政废水中氮素方面有很高的应用价值,可用于城镇污水处理厂提标改造等。     

Mesocosms Mimic Natural Meadows as regards Greenhouse Gas Fluxes and Potential Activities of Nitrifying and Denitrifying Bacteria

The objective of this study was to determine whether a planted mesocosm mimics a natural habitat in terms of N₂O and CH₄ fluxes, soil characteristics and potential nitrification and denitrification activities. We compared mesocosms in unchambered open-field plots and in open-top chambers with nonfiltered ambient air with three natural meadows that had similar soil characteristics and species composition. The N₂O fluxes in the mesocosms were very similar to the fluxes in the three natural meadows. There were no marked differences in potential nitrification and denitrification activities between the mesocosms and the natural meadows, either. Only the CH₄ fluxes differed slightly between the mesocosms and some of the natural meadows. Therefore, it seems that the mesocosms compared rather well to natural habitats. The open-top chambers modified only the soil water content, the values being higher in the unchambered plots than in the chambered plots. These results thus suggest that the open-top chamber experiment enables estimates of greenhouse gas and potential activities of nitrifying and denitrifying bacteria in unfertilized Finnish meadows, in spite of the chamber effects on the soil water content.     

利用可生物降解聚合物同时作为反硝化微生物的碳源和附着载体研究

饮用水源水中硝酸盐污染已引起世界各国的普遍关注,异养反硝化是去除水中硝酸盐的主要技术之一.利用可生物降解聚合物(biodegradable polymers,BDPs)同时作为反硝化微生物的碳源和附着生长的载体,近年来受到了人们的关注.该系统对进水水质的波动具有良好的适应能力;BDPs对人体无毒无害,不会污染出水水质.随着各种新型BDPs材料的不断涌现以及BDPs材料生产成本的降低,BDPs材料在饮用水源水生物脱氮中会得到越来越广泛的应用.对利用可生物降解聚合物进行反硝化的研究进展进行了综述.     

Origin,causes and effects of increased nitrite concentrations in aquatic environments

Literature frequently mentions increasednitrite concentrations along with itsinhibitory effect towards bacteria and aquaticlife. Nitrite accumulation has been studied fordecades, and although numerous causal factorshave already been commented on in literature,the mechanism of nitrite accumulation is notalways clear. From the broad range ofparameters and environmental factors reviewedin this paper, it is obvious that the causesand consequences of nitrite accumulation arenot yet completely understood. Among others,pH, dissolved oxygen, volatile fatty acids,phosphate and reactor operation have been foundto play a role in nitrite accumulation, whichresults from differential inhibition ordisruption of the linkage of the differentsteps in both nitrification anddenitrification. In the case of nitrification, thisdifferential inhibition could lead to thedisplacement or unlinking of the ammoniaoxidisers and nitrite oxidisers. In this paper,the idea is formulated that the nitrifierpopulation forms a role model for the totalmicrobial community. Increased nitriteconcentrations would in this aspect not onlysignal a disruption of nitrifiers, but possiblyalso of the total configuration of themicrobial community.     

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。     

Enhanced Remediation of Pentachlorophenol (PCP)-Contaminated Groundwater by Bioaugmentation with Known PCP-Degrading Bacteria

The objective of this study was to test whether bioaugmentation with known pentachlorophenol (PCP)-degrading bacteria (Sphingobium chlorophenolicum and Burkholderia cepacia) could enhance remediation of PCP-contaminated groundwater. Groundwater PCP concentrations were determined by US Environmental Protection Agency (EPA) method 3510C and gas chromatography. Gene expression for PCP-degrading enzymes: pentachlorophenol 4-monooxygenase (pcpB; S. chlorophenolicum) and chlorophenol 4-monooxygenase (TftD; B. cepacia) was determined by real-time polymerase chain reaction (RT-PCR) using gene-specific primers. Bioaugmented treatments with S. chlorophenolicum and B. cepacia showed 32% and 49% decrease (p < .05) whereas un-bioaugmented (indigenous) treatment did not show significant decrease (p > .05) in average PCP concentration, respectively, over 72 days. Decreased PCP levels correlated strongly (r = ?.82, p < .05) with increased PCP-tolerant bacteria in bioaugmented treatments, whereas no significant correlation was observed (r = ?.22, p > .05) in un-bioaugmented treatment. In addition, a decrease in PCP levels also correlated significantly with an increase in gene expression of PCP-degrading enzymes, pcpB (r = ?.77044) and TftD (r = ?.87905) (p < .05). PCP concentrations decreased and pcpB or TftD expressions were higher in bioaugmented treatments with S. chlorophenolicum (50%, 7-fold) or B. cepacia (67%, 10.7-fold), respectively, than indigenous treatment. Therefore, bioaugmentation with known PCP-degrading bacteria enhanced remediation of PCP-contaminated groundwater than indigenous bacteria alone. Results of this study may provide a more efficient and environmentally friendly technique for on-site remediation of PCP-contaminated groundwater.     

Aerobic and Anoxic Growth and Nitrate Removal Capacity of a Marine Denitrifying Bacterium Isolated from a Recirculation Aquaculture System

Bacterial biofilters used in marine recirculation aquaculture systems need improvements to enhance nitrogen removal efficiency. Relatively little is known about biofilter autochthonous population structure and function. The present study was aimed at isolating and characterizing an autochthonous denitrifying bacterium from a marine biofilter installed at a recirculation aquaculture system. Colonization of four different media in a marine fish farm was followed by isolation of various denitrifying strains and molecular classification of the most promising one, strain T2, as a novel member of the Pseudomonas fluorescens cluster. This strain exhibits high metabolic versatility regarding N and C source utilization and environmental conditions for growth. It removed nitrate through aerobic assimilatory metabolism at a specific rate of 116.2 mg NO₃-N g dw⁻¹ h⁻¹. Dissimilatory NO₃-N removal was observed under oxic conditions at a limited rate, where transient NO₂-N formed represented 22% (0.17 mg L⁻¹) of the maximum transient NO₂-N observed under anoxic conditions. Dissimilatory NO₃-N removal under anoxic conditions occurred at a specific rate of 53.5 mg NO₃-N g dw⁻¹ h⁻¹. The isolated denitrifying strain was able to colonize different materials, such as granular activated carbon (GAC), Filtralite and Bioflow plastic rings, which allow the development of a prototype bioreactor for strain characterization under dynamic conditions and mimicking fish-farm operating conditions.     

Isolation of Arsenic-Resistant Bacteria from Bengal Delta Sediments and their Efficacy in Arsenic Removal from Soil in Association with Pteris vittata

The potential of arsenic-resistant bacteria in association with Pteris vittata to reduce the level of arsenic from soil was studied. The physicochemical characteristics of contaminated paddy soil were analyzed, and 3 bacterial isolates amongst 11 were screened and were selected for further study. These three isolates were characterized by 16S rDNA sequencing and identified as Bacillus altitudinis Strain SS8 (KJ432582), Bacillus megaterium Strain SS9 (KJ432583) and Lysinibacillus sp. Strain SS11 (KJ432584). Of these, Lysinibacillus sp. Strain SS11 displayed arsenic tolerance of 3256 mg L^?1 for arsenate and 1136 mg L^?1 for arsenite. Additionally, it showed bioaccumulation capacity of 23.43 mg L^?1 for arsenate and 5.65 mg L^?1 for arsenite. It also showed resistance to other heavy metals, especially towards iron, copper and chromium. It was also observed that Pteris vittata was able to take up more arsenic and iron from soil in the presence of these bacterial strains than in their absence, leading to contaminant-free soil. Thus, this system appears to be an effective bioremediating process to remove arsenic from contaminated soil.     

一株耐氧反硝化细菌的筛选及脱氮特性研究

从鱼塘中分离到1株高效的有氧反硝化菌,经初步鉴定,为芽孢杆菌。在溶解氧(OD)达到2mg/L时,除氮率达97%,OD达到4～5mg/L,仍有一定的反硝化作用,除氮率为85%以上。与典型的好氧反硝化菌Pseudomonasstutzeri[1]相比,有更强的耐溶解氧的优势。同时初步探讨了水体中不同溶解氧、碳源、pH、温度对该芽孢杆菌W2菌株反硝化作用的影响,水体中存在一定量有机碳源有利于反硝化,当以葡萄糖为碳源,pH为7.0～7.5,温度为32℃时,W2菌株具有最佳的降解人工废水反硝化能力。实验结果表明,在好氧条件下,菌体浓度为1000个/mL时,对自然水体中高达1mg/L亚硝酸浓度也能发挥高效的反硝化作用。     

Composition and Diversity of Microbial Communities Recovered from Surrogate Minerals Incubated in an Acidic Uranium-Contaminated Aquifer

Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the β-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by β-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.     

Factors Affecting Nitrous Oxide Production: A Comparison of Biological Nitrogen Removal Processes with Partial and Complete Nitrification

Nitrous oxide (N₂O) emission from biological nitrogen removal (BNR) processes has recently received more research attention. In this study, two lab-scale BNR systems were used to investigate the effects of various operating parameters including the carbon to nitrogen (C/N) ratio, ammonia loading, and the hydraulic retention time on N₂O production. The first system was operated in a conventional BNR mode known as the Ludzack–Ettinger (LE) process, consisting of complete denitrification and nitrification reactors, while the second one was operated in a shortcut BNR (SBNR) mode employing partial nitrification and shortcut denitrification, which requires less oxygen and carbon sources. As the C/N ratio was decreased, a significant increase in N₂O production was observed only in the anoxic reactor of the LE process, indicating that N₂O was released as an intermediate of the denitrification reaction under the carbon-limited condition. However, the SBNR process did not produce significant N₂O even at the lowest C/N ratio of 0.5. When the SBNR process was subjected to increasing concentrations of ammonia, N₂O production from the aerobic reactor was rapidly increased. Furthermore, the increasing production of N₂O was observed mostly in the aerobic reactor of the SBNR process with a decline in hydraulic retention time. These experimental findings indicated that the increase in N₂O production was closely related to the accumulation of free ammonia, which was caused by an abrupt increase of the ammonium loading. Consequently, the partial nitrification was more susceptible to shock loading conditions, resulting in a high production of N₂O, although the SBNR process was more efficient with respect to nitrogen removals as well as carbon and oxygen requirements.