Isolation and characterization of quinoline-degrading bacteria from subsurface sediments.

Two gram-negative, motile bacteria isolated from deep subsurface sediments mineralized the nitrogen-containing polyaromatic hydrocarbon quinoline under aerobic conditions and transformed quinoline to soluble intermediates under anaerobic conditions. Many aromatic compounds were also able to serve as the sole source of carbon and energy under aerobic conditions. Rapid aerobic mineralization of quinoline at concentrations as low as 0.002 microgram ml-1 indicates that these organisms possess a high-affinity uptake and utilization system, which may reflect the oligotrophic nature of deep subsurface environments. Both bacteria harbored four plasmids of identical size, ranging from 50 to 440 kilobases.     

Microbiological Comparison of Surface Soil and Unsaturated Subsurface Soil from a Semiarid High Desert

Thirty-two chemoheterotrophic bacteria were isolated from unsaturated subsurface soil samples obtained from ca. 70 m below land surface in a high desert in southeastern Idaho. Most isolates were gram positive (84%) and strict aerobes (79%). Acridine orange direct counts of microbes in one subsurface sample showed lower numbers than similar counts performed on surface soils from the same location (ca. 5 × 10⁵ versus 2 × 10⁶ cells per g [dry weight] of soil), but higher numbers than those from plate counts performed on the subsurface material. Another sample taken from the same depth at another location showed no evidence of colonies under identical conditions. Soil analyses indicated that subsurface sediments versus surface soils were slightly alkaline (pH 7.9 versus 7.4), had a higher water content (25.7 versus 6.3%), and had lower organic carbon concentrations (0.05 to 0.17 versus 0.25% of soil dry weight). Analyses of biologically relevant gases from the unsaturated subsurface indicated an aerobic environment. As in other unsaturated soil environments, either a high proportion of bacteria in these subsurface sediments are not viable or they are incapable of growth on conventional media under aerobic conditions. The presence and numbers of bacteria in these deep sediments may be influenced by colonization opportunities afforded by periodic percolation of surface water through fractures in overlying strata.     

Potential for Aerobic Methane Oxidation in Carboniferous Coal Measures

Carbon (C), geologically sequestered in coal, is gradually released to the atmosphere as CH₄ and CO₂. Recent anthropogenic activity (coal mining) has rapidly increased the rate of C reallocation from coal deposits into the atmosphere, which has deleterious effect on the climate as both gases are effective infrared absorbers. In the current study we demonstrate that the coal bearing sedimentary rocks possess potential of biological methane oxidation. Viable methanotrophic bacteria, capable of methane oxidation at ambient air and a range of methane concentrations were found in coalbearing formations of the Upper Silesian (USCB) and Lublin Coal Basins (LCB). Factors controlling activity of the aerobic methanotrophic bacteria in the deep subsurface such as, depth, methane concentration, available electron acceptors, moisture and nutrients availability were investigated along with paleoenvironmental factors (temperature changes during and after burial and paleohydrological infiltration). The distribution and activity of the methanotrophic bacteria in the deep subsurface were found to be influenced by geological conditions among which evolution of paleotemperatures and paleohydrological conditions play a predominant role. The data presented along with analysis of molecular composition of the coalbed gases in various coal basins worldwide has led to the conclusion that aerobic methanotrophy may be a widespread process, which, to our knowledge, so far has not been included in investigations concerning C cycling in the subsurface.     

喹啉废水反硝化反应器中优势菌的代谢功能分析

采用纯培养技术对一个降解喹啉的反硝化反应器筛选到的26株优势菌株进行反硝化能力以及好氧降解喹啉能力研究,其中的反硝化菌还测定了反硝化条件下的喹啉降解能力。结果发现Bacillus、Staphylococcus、Pseudomonas、Brucella、Delftia等5个属的10株菌具有反硝化能力,Rhodococcus属的9株细菌能够好氧降解喹啉,揭示了反硝化喹啉降解反应器中主要细菌类型的代谢特性,发现在缺氧反硝化反应器中存在多样的代谢类型的细菌。     

Microbial transformation of heterocyclic molecules in deep subsurface sediments

Recently attempts have been made to establish the presence and to determine the metabolic versatility of microorganisms in the terrestrial deep subsurface at the Savannah River Plant, Aiken, SC, USA. Sediment samples obtained at 20 different depths of up to 526 m were examined to determine carbon mineralization under aerobic, sulfate-reducing, and methanogenic conditions. The evolution of¹⁴CO₂ from radiolabelled glucose was observed under aerobic conditions in all sediments, whereas pyridine was transformed in 50% of the 20 sediments and indole was metabolized in 85% of the sediments. Glucose mineralization in certain sediments was comparable to that in the surface environment. Sulfate was reduced in only five sediments, and two were carbon limited. Methane production was detected in ten sediments amended with formate only after long-term incubations. The transformation of indole and pyridine was only rarely observed under sulfate-reducing conditions and was never detected in methanogenic incubations. This study provides information concerning the metabolic capability of both aerobic and anaerobic microorganisms in the deep subsurface and may prove useful in determining the feasibility of microbial decontamination of such environments.     

Diversity among bacteria isolated from the deep subsurface

Culturable bacteria from the deep subsurface (179 m) at Cerro Negro, New Mexico were isolated and characterized. The average number of viable aerobic bacteria was estimated to be 5×10⁵g^–1 of sediment, but only about 0.1% of these could be recovered on agar medium when incubated under aerobic conditions. Of 158 strains isolated from this depth, 92 were characterized by cellular fatty acid profiles (FAME), 36 by analysis of partial 16S rDNA sequences, and 44 by rep-PCR genome fingerprint analysis using three different sets of oligonucleotide primers (REP, BOX, or ERIC). These analyses showed the majority of isolates (67%) were Gram-positive bacteria and primarily members of genera with a high %G+C DNA. The remaining isolates were -subdivisionProteobacteria (19%) and members of the flavobacteria group (14%). The diversity indices based on these different methods of characterization were very high suggesting this subsurface habitat harbors a highly diverse microbial community.     

Survival of subsurface microorganisms exposed to UV radiation and hydrogen peroxide.

Aerobic and microaerophilic subsurface bacteria were screened for resistance to UV light. Contrary to the hypothesis that subsurface bacteria should be sensitive to UV light, the organisms studied exhibited resistance levels as efficient as those of surface bacteria. A total of 31% of the aerobic subsurface isolates were UV resistant, compared with 26% of the surface soil bacteria that were tested. Several aerobic, gram-positive, pigmented, subsurface isolates exhibited greater resistance to UV light than all of the reference bacterial strains tested except Deinococcus radiodurans. None of the microaerophilic, gram-negative, nonpigmented, subsurface isolates were UV resistant; however, these isolates exhibited levels of sensitivity similar to those of the gram-negative reference bacteria Escherichia coli B and Pseudomonas fluorescens. Photoreactivation activity was detected in three subsurface isolates, and strain UV3 exhibited a more efficient mechanism than E. coli B. The peroxide resistance of four subsurface isolates was also examined. The aerobic subsurface bacteria resistant to UV light tolerated higher levels of H2O2 than the microaerophilic organisms. The conservation of DNA repair pathways in subsurface microorganisms may be important in maintaining DNA integrity and in protecting the organisms against chemical insults, such as oxygen radicals, during periods of slow growth.     

A phylogenetic analysis of micro-organisms isolated from subsurface environments

Three methods were used to provide information on the identity and phylogenetic relatedness of 19 aerobic, chemoheterotrophic bacteria isolated from topsoil and deep subsurface sediments at a site in South Carolina. These methods were (i) analysis of selected physiological traits, (ii) restriction endonuclease analysis (REA) of genomic DNA, and (iii) analysis of 16S ribosomal RNA sequences. When the 16S rRNA sequences were compared with those for 12 standard strains, two topsoil isolates and six subsurface strains formed a tight group with the high-G+C Gram-positive bacteria and appeared to be most closely related to Arthrobacter globiformis— a coryneform-actinomycete bacterium with unusually effective survival capabilities. The rest of the subsurface isolates were scattered among the standard strains from the Proteobacteria— including the pseudomonads and Agrobacterium tumefaciens— or the low-G+C Gram-positive bacteria.     

Effect of Starvation on Induction of Quinoline Degradation for a Subsurface Bacterium in a Continuous-Flow Column

Differences in the induction response and the initial two reactions of quinoline degradation between short-term (2 days)- and long-term (60 to 80 days)-starved cells of a subsurface Pseudomonas cepacia strain were examined by using continuous-flow columns. The ability of bacteria that are indigenous to oligotrophic environments to respond to a contaminant was assessed by using long-term starvation to induce a cell physiology that simulates the in situ physiology of the bacteria. With quinoline concentrations of 39 and 155 μM, long-term-starved cells converted quinoline to degradation products more efficiently than did short-term-starved cells. Quinoline concentrations of 155 μM and, to a greater extent, 775 μM had an inhibitory effect on induction in long-term-starved cells. However, only the length of the induction process was affected with these quinoline concentrations; degradation of quinoline at the steady state for long-term-starved cells was equal to or better than that for short-term-starved cells. The induction time for short-term-starved cells did not increase progressively with increasing quinoline concentration. Experiments with starved cells are important for the development of accurate predictive models of contaminant transport in the subsurface because starvation, which induces a cell physiology that simulates the in situ physiology of many bacteria, may affect the induction process.     

Solid–Aqueous Phase Partitioning of Radionuclides by Complexing Compounds Excreted by Subsurface Bacteria

Radionuclides are present in numerous aerobic and anaerobic subsurface environments due to nuclear weapons testing, leakage from process and storage facilities, and discharge of radioactive waste. The partitioning of radionuclides between liquid and solid phases by complexing compounds excreted by subsurface bacteria was studied. The solid–aqueous phase partitioning of pico- to submicromolar amounts of ⁵⁹Fe, ¹⁴⁷Pm, ²³⁴Th, and ²⁴¹Am was analyzed in the presence of quartz sand and exudates from three species of subsurface bacteria: Pseudomonas fluorescens, Pseudomonas stutzeri, and Shewanella putrefaciens. All were grown under aerobic conditions, and P. stutzeri and S. putrefaciens were grown under anaerobic conditions as well. The supernatants of the aerobic and anaerobic cultures were collected and radionuclide was added. Quartz sand, with a Brunauer, Emmett, and Teller (BET) surface area of 0.1 m² g^–1, was added to the supernatant radionuclide mix, and the pH was adjusted to approximately 8. After centrifuging, the amount of radionuclide in the liquid phase of the samples and controls was analyzed using scintillation. Relative to the control, aerobic supernatants maintained more than 50% of the added ⁵⁹Fe, ²³⁴Th, and ²⁴¹Am. The highest amount of metal present in the liquid phase of the anaerobic supernatants was found in the case of ²⁴¹Am, with 40% more ²⁴¹Am in samples than in controls. Both aerobic and anaerobic supernatants tested positive for complexing compounds when analyzed using the Chrome Azurol S assay. The great amounts of radionuclides in the liquid phases of samples were likely due to complexation with such compounds. Bacterially excreted complexing compounds hence seem able to influence the solid–aqueous phase partitioning of radionuclides. This could influence the mobility of radionuclides in contaminated subsurface environments.     

Ecology, physiology, and phylogeny of deep subsurface Sphingomonas sp

Several new species of the genus Sphingomonas including S. aromaticivorans, S. stygia, and S. subterranea that have the capacity for degrading a broad range of aromatic compounds including toluene, naphthalene, xylenes, p-cresol, fluorene, biphenyl, and dibenzothiophene, were isolated from deeply-buried (>200 m) sediments of the US Atlantic coastal plain (ACP). In S. aromaticivorans F199, many of the genes involved in the catabolism of these aromatic compounds are encoded on a 184-kb conjugative plasmid; some of the genes involved in aromatic catabolism are plasmid-encoded in the other strains as well. Members of the genus Sphingomonas were common among aerobic heterotrophic bacteria cultured from ACP sediments and have been detected in deep subsurface environments elsewhere. The major source of organic carbon for heterotrophic metabolism in ACP deep aquifers is lignite that originated from plant material buried with the sediments. We speculate that the ability of the subsurface Sphingomonas strains to degrade a wide array of aromatic compounds represents an adaptation for utilization of sedimentary lignite. These and related subsurface Sphingomonas spp may play an important role in the transformation of sedimentary organic carbon in the aerobic and microaerobic regions of the deep aquifers of the ACP. Received 12 May 1999/ Accepted in revised form 25 July 1999     

Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review

Bioremediation of aromatic hydrocarbons in groundwater and sediments is often limited by dissolved oxygen. Many aromatic hydrocarbons degrade very slowly or not at all under anaerobic conditions. Nitrate is a good alternative electron acceptor to oxygen, and denitrifying bacteria are commonly found in the subsurface and in association with contaminated aquifer materials. Providing both nitrate and microaerophilic levels of oxygen may result in oxidation of the stable benzene rings in aromatic contaminants and allow for the intermediates of this oxidation to degrade via denitrification. The effects of using mixed electron acceptors on biodegradation of subsurface contaminants is unclear. Below some critical oxygen threshold, aerobic biodegradation is inhibited, however high levels of oxygen inhibit denitrification. The mechanisms which regulate electron transfer to oxygen and nitrate are complex. This review: 1) describes the factors which may affect the utilization of oxygen and nitrate as dual electron acceptors during biodegradation; 2) summarizes the incidence of dual use of nitrate and oxygen (aerobic denitrification); and 3) presents evidence of the effectiveness of bioremediation under mixed oxygen/nitrate conditions. Received 08 November 1995/ Accepted in revised form 09 June 1996     

Related assemblages of sulphate-reducing bacteria associated with ultradeep gold mines of South Africa and deep basalt aquifers of Washington State

We characterized the diversity of sulphate-reducing bacteria (SRB) associated with South African gold mine boreholes and deep aquifer systems in Washington State, USA. Sterile cartridges filled with crushed country rock were installed on two hydrologically isolated and chemically distinct sites at depths of 3.2 and 2.7 km below the land surface (kmbls) to allow development of biofilms. Enrichments of sulphate-reducing chemolithotrophic (H2) and organotrophic (lactate) bacteria were established from each site under both meso- and thermophilic conditions. Dissimilatory sulphite reductase (Dsr) and 16S ribosomal RNA (rRNA) genes amplified from DNA extracted from the cartridges were most closely related to the Gram-positive species Desulfotomaculum thermosapovorans and Desulfotomaculum geothermicum, or affiliated with a novel deeply branching clade. The dsr sequences recovered from the Washington State deep aquifer systems affiliated closely with the South African sequences, suggesting that Gram-positive sulphate-reducing bacteria are widely distributed in the deep subsurface.     

深部生物圈古菌的研究进展与展望

古菌作为深部生物圈中常见的原核生物,广泛分布于各类海洋沉积生境中,在沉积物生物地球化学循环中发挥着重要作用.由于不同的古菌类群对环境条件存在生理适应性差异,它们分别在近岸沿海和开阔大洋沉积物中构成了厌氧微生物生态系统和好氧微生物生态系统.本文通过对近岸与远洋、沉积物与上覆水体两个不同维度的古菌群落结构进行比较,以及对出...     

Experimental criteria for interpretation of bacterial sensitivity to dioxidine determined by diffusion from the disks

Antibacterial activity of dioxidine against aerobic and facultative anaerobic organisms under conditions of anaerobiosis i. e. conditions really observed for example in abscess cavities or necrotic tissues is 30 to 100 times as high as that under aerobic conditions. There is a relationship between sensitivity of bacteria to dioxidine under aerobic and anaerobic conditions which is expressed by the regression equation. Therefore, comparison of the MICs determined under anaerobic conditions with the growth inhibition zones formed by disks with the drug under aerobic conditions is possible. The MIC of dioxidine was determined under anaerobic conditions for 179 clinical strains of aerobic and facultative anaerobic bacteria and the growth inhibition zones of the same bacteria under aerobic conditions were evaluated with the use of disks containing 100, 75, 50, 25, 20, and 15 micrograms of the drug. The border line. MIC differentiating between resistant and sensitive strains was chosen to be equal to 4 micrograms/ml. Differentiation of the strains into sensitive and resistant ones by the values of the growth inhibition zones was performed with the method of error minimization described by C. Metzler and R. De Haan in 1974. Disks containing 25 micrograms of the drug allowed one to differentiate the strains under aerobic conditions into sensitive and resistant ones: the growth inhibition zones greater than 11 mm corresponded to the sensitive strains (the MIC smaller than 4 micrograms/ml) and the growth inhibition zones smaller than 11 mm corresponded to the resistant strains (the MIC greater than 4 micrograms/ml).     

Molybdenum-dependent degradation of quinoline by Pseudomonas putida Chin IK and other aerobic bacteria

Eighteen different aerobic bacteria were isolated which utilized quinoline as sole source of carbon, nitrogen, and energy. Attempts were unsuccessful at isolating anaerobic quinoline-degrading bacteria. The optimal concentration of quinoline for growth was in the range of 2.5 to 5 mM. Some organisms excreted 2-hydroxyquinoline as the first intermediate. Hydroxylation of quinoline was catalyzed by a dehydrogenase which was induced in the presence of quinoline or 2-hydroxyquinoline. Quinoline dehydrogenase activity was dependent on the availability of molybdate in the growth medium. Growth on quinoline was inhibited by tungstate, an antagonist of molybdate. Partially purified quinoline dehydrogenase from Pseudomonas putida Chin IK indicated the presence of flavin, iron-sulfur centers, and molybdenum-binding pterin. M _r of quinoline dehydrogenase was about 300 kDa in all isolates investigated.Abbreviations APS ammonium peroxodisulfate - DCPIP 2,6-dichlorophenol-indophenol - EEO electroendosmosis - MTT thiazolyl blue - PES phenazine ethosulfate - TEMED N,N,N,N-tetramethyl-ethylenediamine     

反应器进水管生物膜中喹啉降解菌的分离鉴定及降解特性

【背景】喹啉是一类高毒、致癌且难降解的含氮杂环化合物,本实验室建立了一个长期高效运行的反硝化喹啉降解生物反应器。【目的】从反应器进水管富集的生物膜中筛选有氧条件下降解喹啉的菌株。【方法】通过以喹啉为唯一碳源的培养基来富集、分离、纯化菌株;利用16S rRNA基因的序列分析鉴定分离株的系统发育地位;比较不同pH及温度条件下菌株的喹啉降解特性。【结果】经鉴定,4株分离物Q1、Q3、Q7和Q8分别属于Sphingobium、Massilia、Rhodococcus和Dyadobacter属。降解实验表明,以上菌株均能在48 h内实现50 mg/L喹啉的完全去除,但各自表现出不同的降解特性,其中Q1、Q3和Q8在降解过程中都检测到了喹啉降解产物2-羟基喹啉的积累。降解喹啉的Sphingobium、Massilia和Dyadobacter属菌株尚未见报道。【结论】从喹啉降解生物反应器的进水管内分离的4株喹啉降解菌可为设计处理含喹啉工业废水的反应器提供新菌种资源,对于完善喹啉生物降解机理研究具有实际意义。     

Anaerobic and aerobic degradation of pyridine by a newly isolated denitrifying bacterium.

New denitrifying bacteria that could degrade pyridine under both aerobic and anaerobic conditions were isolated from industrial wastewater. The successful enrichment and isolation of these strains required selenite as a trace element. These isolates appeared to be closely related to Azoarcus species according to the results of 16S rRNA sequence analysis. An isolated strain, pF6, metabolized pyridine through the same pathway under both aerobic and anaerobic conditions. Since pyridine induced NAD-linked glutarate-dialdehyde dehydrogenase and isocitratase activities, it is likely that the mechanism of pyridine degradation in strain pF6 involves N-C-2 ring cleavage. Strain pF6 could degrade pyridine in the presence of nitrate, nitrite, and nitrous oxide as electron acceptors. In a batch culture with 6 mM nitrate, degradation of pyridine and denitrification were not sensitively affected by the redox potential, which gradually decreased from 150 to -200 mV. In a batch culture with the nitrate concentration higher than 6 mM, nitrite transiently accumulated during denitrification significantly inhibited cell growth and pyridine degradation. Growth yield on pyridine decreased slightly under denitrifying conditions from that under aerobic conditions. Furthermore, when the pyridine concentration used was above 12 mM, the specific growth rate under denitrifying conditions was higher than that under aerobic conditions. Considering these characteristics, a newly isolated denitrifying bacterium, strain pF6, has advantages over strictly aerobic bacteria in field applications.     

Radiation sensitization by thiol-binding agents of radio-resistant and radiosensitive Escherichia coli and the oxygen effect

Dose-survival curves of four strains of E. coli-B/r her- try-, Bs-1, B/r CSH, and 15T-were obtained in the presence and in the absence of non-toxic levels of several thiol-binding agents-N-ethylmaleimide (NEM), iodoacetamide (IA), and hydroxymercuribenzoate (HMB). The degree of radiosensitization by these agents was estimated from the increase in slope of the dose-survival curve, under anoxic and aerobic conditions. Chemical sensitization of both radioresistant and radiosensitive strains of bacteria with the thiol-binding enzyme poisons NEM, IA, and HMB has been observed. NEM sensitized only under anoxic conditions, IA sensitized under both anoxic and aerobic conditions, although to a much greater extent aerobically, and HMB sensitized only under aerobic conditions. The formation of long-lived radiolytic products toxic to bacteria is observed in sensitization by IA. When sensitization occurs, the magnitude of the dose-modifying factor is equal to or greater than the oxygen enhancement ratio. Inhibition of an energy-requiring enzymic repair process does not seem to be involved as a primary mechanism of sensitization. Interference by thiol-binding agents with a "rapid repair" process which is different from the usual "enzymic repair," and which operates in radio-sensitive as well as in radioresistant bacteria, is suggested.     

Distribution of aerobic bacteria,protozoa, algae,and fungi in deep subsurface sediments

The distribution of microorganisms in deep subsurface profiles was determined at three sites at the Savannah River Plant, Aiken, South Carolina. Acridine orange direct counts (AODC) of bacteria were highest in surface soil samples and declined to the 10⁶ to 10⁷ per gram range in the subsurface, but then did not decline further with depth. In the subsurface, AODC values varied from layer to layer, the highest being found in samples from sandy aquifer formations and the lowest in clayey interbed layers. Sandy aquifer sediments also contained the highest numbers of viable bacteria as determined by aerobic spread plate counts (CFU) on a dilute heterotrophic medium. In some of these samples bacterial CFU values approached 100% of the AODC values. Viable protozoa (amoebae and flagellates, but no ciliates) were found in samples with high bacterial CFU values. A variety of green algae, phytoflagellates, diatoms, and a few cyanobacteria were found at low population densities in samples from two of the three boreholes. Low numbers of fungi were evenly distributed throughout the profiles at all three sites. Microbial population density estimates correlated positively with sand content and pore‐water pH, and negatively with clay content and pore‐water metal concentration. A large diversity of prokaryotic and eukaryotic microorganisms was found in samples with high population densities. A survey of bacterial strains isolated from subsurface samples revealed associations of gram‐positive bacteria with high clay sediments and gram‐negative bacteria with sandy sediments. The ability to deposit lipophilic storage material (presumably poly‐ß‐hydroxybutyrate) was found in a high proportion of isolates from sandy sediments, but only rarely in isolates from high clay sediments.