Growth of Bacteria Degrading Naphthalene and Salicylate at Low Temperatures

A total of 58 bacterial strains degrading naphthalene and salicylate were isolated from soil samples polluted with oil products, collected in different regions of Russia during winter and summer. The isolates were assessed for their ability to grow at low temperatures (4, 8, and 15°C); bacteria growing at 4°C in the presence of naphthalene or salicylate accounted for 65 and 53%, respectively, of the strains isolated. The strains differed in the temperature dependence of their growth rates. It was demonstrated that the type of expression of the Nah⁺ phenotype at low temperatures depended on the combination of host bacterium and plasmid.     

Identification of Acetate- or Methanol-Assimilating Bacteria under Nitrate-Reducing Conditions by Stable-Isotope Probing

Stable-isotope probing (SIP) was used to identify acetate- or methanol-assimilating bacteria under nitrate-reducing conditions in activated sludge. A sludge sample obtained from wastewater treatment systems was incubated in a denitrifying batch reactor fed with synthetic wastewater containing [¹³C]acetate or [¹³C]methanol as the main carbon source and nitrate as the electron acceptor. We analyzed how growth of bacterial populations was stimulated by acetate or methanol as the external carbon source in nitrogen-removal systems. Most of the acetate- or methanol-assimilating bacteria identified by SIP have been known as denitrifiers in wastewater treatment systems. When acetate was used as the carbon source, 16S rRNA gene sequences retrieved from ¹³C-labeled DNA were closely related to the 16S rRNA genes of Comamonadaceae (e.g., Comamonas and Acidovorax) and Rhodocyclaceae (e.g., Thauera and Dechloromonas) of the Betaproteobacteria, and Rhodobacteraceae (e.g., Paracoccus and Rhodobacter) of the Alphaproteobacteria. When methanol was used as the carbon source, 16S rRNA gene sequences retrieved from ¹³C-DNA were affiliated with Methylophilaceae (e.g., Methylophilus, Methylobacillus, and Aminomonas) and Hyphomicrobiaceae. Rarefaction curves for clones retrieved from ¹³C-DNA showed that the diversity levels for methanol-assimilating bacteria were considerably lower than those for acetate-assimilating bacteria. Furthermore, we characterized nitrite reductase genes (nirS and nirK) as functional marker genes for denitrifier communities in acetate- or methanol-assimilating populations and detected the nirS or nirK sequence related to that of some known pure cultures, such as Alcaligenes, Hyphomicrobium, and Thauera. However, most of the nirS or nirK sequences retrieved from ¹³C-DNA were clustered in some unidentified groups. On the basis of 16S rRNA gene clone libraries retrieved from ¹³C-DNA, these unidentified nir sequences might be identified by examining the nir gene in candidates for true denitrifiers (e.g., the families Comamonadaceae, Hyphomicrobiaceae, Methylophilaceae, and Rhodobacteraceae).     

Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of l-guluronic acid, d-glucose, and d-glycero-d-mannoheptose. ¹³C-labeled EPS and ¹³C-labeled cellulose were purified from bacterial cultures grown on [¹³C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from ¹³C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.     

Identification of Benzo[a]pyrene-Metabolizing Bacteria in Forest Soils by Using DNA-Based Stable-Isotope Probing

DNA-based stable-isotope probing (DNA-SIP) was used in this study to investigate the uncultivated bacteria with benzo[a]pyrene (BaP) metabolism capacities in two Chinese forest soils (Mt. Maoer in Heilongjiang Province and Mt. Baicaowa in Hubei Province). We characterized three different phylotypes with responsibility for BaP degradation, none of which were previously reported as BaP-degrading microorganisms by SIP. In Mt. Maoer soil microcosms, the putative BaP degraders were classified as belonging to the genus Terrimonas (family Chitinophagaceae, order Sphingobacteriales), whereas Burkholderia spp. were the key BaP degraders in Mt. Baicaowa soils. The addition of metabolic salicylate significantly increased BaP degradation efficiency in Mt. Maoer soils, and the BaP-metabolizing bacteria shifted to the microorganisms in the family Oxalobacteraceae (genus unclassified). Meanwhile, salicylate addition did not change either BaP degradation or putative BaP degraders in Mt. Baicaowa. Polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) genes were amplified, sequenced, and quantified in the DNA-SIP ¹³C heavy fraction to further confirm the BaP metabolism. By illuminating the microbial diversity and salicylate additive effects on BaP degradation across different soils, the results increased our understanding of BaP natural attenuation and provided a possible approach to enhance the bioremediation of BaP-contaminated soils.     

Characteristics of Soil Organic Matter and Sorption of Phenanthrene,Naphthalene 1,3,5-TCB and o-Xylene

Nonlinear isotherm behavior has been reported for the sorption of hydrophobic organic compounds (HOCs) in soil/water systems, but the mechanisms are unclear. The model of “soft” and “hard” carbon domains has been extensively cited in the sorption literature to account for nonlinear sorption behaviors, but the structural compositions of soil organic matter (SOM) are not well understood. The objective of this study was to examine the characteristics of SOM and the effect of SOM heterogeneity on sorption isotherm by elemental analysis, organic petrographic examination, scanning electron microscopy, ¹³C nuclear magnetic resonance and studying the sorption behaviors of phenanthrene, naphthalene, 1,3,5-trichlorobenzene and o-xylene in soil and its isolated fractions, humic acid (HA) and humin (denser particulates and lighter particulates). DP mainly contained low maturation and high paraffinic carbon huminite. LP was composed of inertinite, huminite, vitrinite and exinite, with smaller particle size and higher maturation than DP. Humic acid approached the lignite coal rank.

All isotherms were nonlinear, and nonlinearity increased in the following order: HA > DP > soil > BE > LP. The sorption of HOCs in soil was primarily regulated by SOM. Humic acid seemed to be the soft carbon domain and insoluble condensed organic matter (humin) the hard carbon domain. Isotherm nonlinearity was negatively correlated with hydrophobicity and molecular size, while sorption capacity increased with increase of these parameters. Aliphatic structures of SOM, as observed for LP, could also contribute to both isotherm nonlinearity and large sorption capacity.     

Isolation,Characterization of a Strain Capable of Degrading Imazethapyr and Its Use in Degradation of the Herbicide in Soil

One strain of bacterium IM-4, capable of degrading imazethapyr (IMZT), was isolated from the IMZT-contaminated soil. The isolate was identified as Pseudomonas sp. according to its physiological characteristics, biochemical tests, and 16S rRNA gene phylogenetic analysis. This strain could utilize IMZT as the sole carbon and energy source. About 73.4% of the 50 mg l⁻¹ initially added IMZT was degraded after 7 days of inoculation with strain IM-4. This strain also showed the capability to degrade other imidazolinone herbicides such as imazapyr, imazapic, and imazamox. The inoculation strain IM-4 to soil treated with IMZT resulted in a higher degradation rate than in noninoculated soil regardless if the soil was sterilized or nonsterilized. Inoculation of strain IM-4 could also mitigate the phytotoxic effects of IMZT on the growth of maize.     

Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H₂-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [¹³C]propionate (<10 mM) and the oxidation of ~30 μmol of ¹³C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were ¹³C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” ¹³C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of ¹³C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [¹³C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.     

Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing

DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [¹³C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase α subunits (BphA) from bacteria that incorporated [¹³C]into DNA in 3-day incubations of the soils with [¹³C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.Polychlorinated biphenyls (PCBs) are very stable chloroorganic compounds with the general formula C₁₂H_10-xCl_x. Mixtures of PCBs have been used as coolants and lubricants in transformers, capacitors, and other electrical equipment as they do not burn easily and are good insulators. It is estimated that some 1.5 million tons of PCBs were produced up to 1988 worldwide (11; http://www.atsdr.cdc.gov/cercla; http://www.epa.gov/epawaste/hazard/tsd/pcbs/pubs/about.htm). Although production of these compounds was stopped, due to their long-term persistence, many sites all over the world are still contaminated with PCBs. Moreover, not only do PCBs threaten human health in the vicinity of the contaminated area, but lower PCB congeners volatilize and migrate to places far from where they were originally released (2, 3, 16). Also, their metabolic products have environmental significance; activities of both plants and microorganisms result in formation of different intermediates and final products whose toxicity can in some cases be even higher than that of the original toxicant (24, 26; http://www.atsdr.cdc.gov/cercla).Physical-chemical methods used for the removal of PCBs often cause further natural disturbance and pollution; in contrast, biological methods of removal (i.e., bioremediation) are less expensive and more environmentally sound and thus have aroused much interest (7). These methods include the use of microorganisms and also exploitation of plants (i.e., phytoremediation) (19) and the cooperation of plants with microorganisms in the rhizosphere (i.e., rhizoremediation) (21). These bioremediation options also include the use of genetically modified bacteria (6) and/or plants (18, 23). PCBs were only recently introduced into the environment, and no completely efficient pathways for the aerobic bacterial degradation of all of these compounds have evolved (34); however, lower chlorinated PCB congeners can be degraded via the pathway that is used by aerobic bacteria to degrade biphenyl (35). Therefore, metabolism of biphenyl as a potential cometabolite of PCBs was the subject of this study.The biphenyl degradation pathway is the same in all aerobic bacteria, and enzymes of this pathway degrade biphenyl in four steps into benzoate and 2-hydroxypenta-2,4-dienoate (21). The first enzyme of the pathway, biphenyl dioxygenase, has broad substrate specificity and thus permits degradation of biphenyl-related compounds (9). Substrates for biphenyl dioxygenase comprise, in addition to biphenyl itself, other diphenyl or benzene skeletons with several substituents, including halogens and bicyclic or tricyclic fused heterocyclic aromatics (35). These substrates also include certain natural compounds, including some plant flavonoids, phenols, or terpenes (10). Bacteria capable of metabolizing biphenyl are thus pervasive members of many microbial communities in vegetated soil.As reported previously (20), there are two main problems with introduction of a new population of degrading or genetically modified microorganisms to enhance the biodegradation of PCBs in a contaminated environment: legislative barriers and the inability of strains added to the soil to survive. Therefore, the use of microorganisms for bioremediation of contaminated sites is not likely to be successful. Hence, understanding the biodegradative processes in the natural communities is necessary for planning remediation strategies. Identification of members of the community potentially responsible for the degradative process has recently been enabled by DNA-based stable isotope probing (SIP), as reviewed previously; therefore, this technique has become an efficient tool in microbial ecology (33). In this study, by tracking the transfer of ¹³C from [¹³C]biphenyl into bacterial DNA, it was possible to identify biphenyl-metabolizing bacteria in PCB-contaminated soil. To analyze how the bacterial diversity can be changed by introduction of a plant and subsequent cultivation in a greenhouse, bacteria in the rhizosphere of horseradish (Armoracia rusticana) cultivated in a contaminated soil were studied.     

Seasonal Biotransformation of Naphthalene, Phenanthrene, and Benzo[a]pyrene in Surficial Estuarine Sediments

Transformation rates of naphthalene, phenanthrene, and benzo[a]pyrene in oxidized surficial sediments of a polluted urban estuary, Boston Harbor, Mass., were determined over a period of 15 months. Three sites characterized by muddy sediments were selected to represent a >300-fold range of ambient polycyclic aromatic hydrocarbon (PAH) concentration. Transformation rates were determined by a trace-level radiolabel PAH assay which accounted for PAH mineralization, the formation of polar metabolites, residue, and recovered parental PAHs in sediment slurries. Transformation rates of the model PAHs increased with increasing ambient PAH concentrations. However, turnover times for a given PAH were similar at all sites. The turnover times were as follows: naphthalene, 13.2 to 20.1 days; phenanthrene, 7.9 to 19.8 days, and benzo[a]pyrene, 53.7 to 82.3 days. At specific sites, rates were significantly affected by salinity, occasionally affected by temperature, but not affected by pH over the course of the study. Seasonal patterns of mineralization were observed for each of the PAHs at all sites. The timing of seasonal maxima of PAH mineralization varied from site to site. Seasonal potential heterotrophic activities as measured by acetate and glutamate mineralization rates did not always coincide with PAH mineralization maxima and minima, suggesting that the two processes are uncoupled in estuarine sediments.     

一株石油烃降解菌分子鉴定及特性分析

从受污海水中分离筛选了具有石油烃降解能力的降解菌Bac1020,经PCR扩增得到1 497 bp长16S rDNA序列,通过Blast比对,与主要石油烃降解菌属16S rDNA序列构建系统发生树,鉴定其为不动杆菌(Acinetobactersp.)。降解菌(Acinetobactersp.)在72 h内生长稳定,对石油烃的降解率随时间的延长而不断增加。建立了快速筛选及鉴定石油烃降解菌的方法,应用于海洋石油烃污染的生物降解。     

Use of a Packed-Column Bioreactor for Isolation of Diverse Protease-Producing Bacteria from Antarctic Soil

Seventy-five aerobic heterotrophs have been isolated from a packed-column bioreactor inoculated with soil from Antarctica. The column was maintained at 10°C and continuously fed with a casein-containing medium to enrich protease producers. Twenty-eight isolates were selected for further characterization on the basis of morphology and production of clearing zones on skim milk plates. Phenotypic tests indicated that the strains were mainly psychrotrophs and presented a high morphological and metabolical diversity. The extracellular protease activities tested were optimal at neutral pH and between 30 and 45°C. 16S ribosomal DNA sequence analyses showed that the bioreactor was colonized by a wide variety of taxons, belonging to various bacterial divisions: α-, β-, and γ-Proteobacteria; the Flexibacter-Cytophaga-Bacteroides group; and high G+C gram-positive bacteria and low G+C gram-positive bacteria. Some strains represent candidates for new species of the genera Chryseobacterium and Massilia. This diversity demonstrates that the bioreactor is an efficient enrichment tool compared to traditional isolation strategies.     

Identification of Anthraquinone-Degrading Bacteria in Soil Contaminated with Polycyclic Aromatic Hydrocarbons

Quinones and other oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are toxic and/or genotoxic compounds observed to be cocontaminants at PAH-contaminated sites, but their formation and fate in contaminated environmental systems have not been well studied. Anthracene-9,10-dione (anthraquinone) has been found in most PAH-contaminated soils and sediments that have been analyzed for oxy-PAHs. However, little is known about the biodegradation of oxy-PAHs, and no bacterial isolates have been described that are capable of growing on or degrading anthraquinone. PAH-degrading Mycobacterium spp. are the only organisms that have been investigated to date for metabolism of a PAH quinone, 4,5-pyrenequinone. We utilized DNA-based stable-isotope probing (SIP) with [U-¹³C]anthraquinone to identify bacteria associated with anthraquinone degradation in PAH-contaminated soil from a former manufactured-gas plant site both before and after treatment in a laboratory-scale bioreactor. SIP with [U-¹³C]anthracene was also performed to assess whether bacteria capable of growing on anthracene are the same as those identified to grow on anthraquinone. Organisms closely related to Sphingomonas were the most predominant among the organisms associated with anthraquinone degradation in bioreactor-treated soil, while organisms in the genus Phenylobacterium comprised the majority of anthraquinone degraders in the untreated soil. Bacteria associated with anthracene degradation differed from those responsible for anthraquinone degradation. These results suggest that Sphingomonas and Phenylobacterium species are associated with anthraquinone degradation and that anthracene-degrading organisms may not possess mechanisms to grow on anthraquinone.     

Improving Mathematical Model in Biodegradation of PAHs Contaminated Soil Using Gram-Positive Bacteria

In published literature there are limited studies on the estimation of kinetic parameters of polycyclic aromatic hydrocarbons (PAHs) in soil. In addition, neither the kinetic studies were performed with Gram-positive bacteria nor conducted under non-indigenous condition in order to understand their removal performance. Thus, a mathematical model describing biodegradation of phenanthrene-contaminated soil by Corynebacterium urealyticum, bacterium isolated from municipal sludge, was developed in this study. The model includes three kinetic parameters that were determined using TableCurve 2D software, namely qmax (maximum substrate utilization rate per unit mass of bacteria), X (biomass concentration) and Ks (substrate concentration at one half the maximum substrate utilization rate). These parameters were evaluated and verified in five different initial phenanthrene concentrations. Highest degradation rate was determined to be 79.24 mg kg^?1 day^?1 at 500 mg kg^?1 initial phenanthrene concentrations. This high concentration shows that bacteria perform better in contaminated sand compared to liquid media. High r² values, ranging from 0.92 to 0.99, were obtained excluding 1000 mg/kg phenanthrene. The kinetic parameters, i.e., qmax and Ks, increased with the phenanthrene concentration and thus suggest that bacteria degrade at a higher degradation rate. This model successfully described the biodegradation profiles observed at different initial phenanthrene concentrations. The established model can be used to simulate the duration of phenanthrene degradation using only the value of the initial PAHs concentration.     

Identification of Hydrocarbon-Degrading Bacteria in Soil by Reverse Sample Genome Probing

Bacteria with limited genomic cross-hybridization were isolated from soil contaminated with C5+, a mixture of hydrocarbons, and identified by partial 16S rRNA sequencing. Filters containing denatured genomic DNAs were used in a reverse sample genome probe (RSGP) procedure for analysis of the effect of an easily degradable compound (toluene) and a highly recalcitrant compound (dicyclopentadiene [DCPD]) on community composition. Hybridization with labeled total-community DNA isolated from soil exposed to toluene indicated enrichment of several Pseudomonas spp., which were subsequently found to be capable of toluene mineralization. Hybridization with labeled total-community DNA isolated from soil exposed to DCPD indicated enrichment of a Pseudomonas sp. or a Sphingomonas sp. These two bacteria appeared capable of producing oxygenated DCPD derivatives in the soil environment, but mineralization could not be shown. These results demonstrate that bacteria, which metabolize degradable or recalcitrant hydrocarbons, can be identified by the RSGP procedure.     

Population Dynamics of Two Toluene Degrading Bacterial Species in a Contaminated Stream

Toluene uptake by a benthic biofilm community was previously shown to vary seasonally from 0.03 m hr⁻¹ in winter to 0.2 m hr⁻¹ in summer in a solvent-contaminated stream of the Aberjona watershed. We used quantitative PCR to estimate the population dynamics of previously isolated species of toluene-degrading Xanthobacter autotrophicus and Mycobacterium sp. in both toluene-contaminated and uncontaminated reaches of the stream, and to estimate their relative roles in overall biodegradation rate. Quantification using specific 16S rDNA primers for X. autotrophicus and Mycobacterium sp. showed that populations of both species were much larger in the toluene-contaminated than the toluene-free reach, in agreement with earlier culture-based investigations. A relatively brief bloom of X. autotrophicus occurred in the contaminated reach in the summer, while Mycobacterium sp. populations occurred at elevated densities for more than 5 months. Calculations showed that Mycobacterium, previously thought to be less important than Xanthobacter in annual toluene degradation based on single time-point CFU estimates, appears actually more important because of this longer persistence.     

Isolation and Characterization of a Subsurface Bacterium Capable of Growth on Toluene, Naphthalene, and Other Aromatic Compounds

A bacterium, designated F199, utilized toluene, naphthalene, dibenzothiophene, salicylate, benzoate, p-cresol, and all isomers of xylene as a sole carbon and energy source. This bacterium was isolated from Middendorf sediments, a Cretaceous age formation that underlies the Southeast Coastal Plain in South Carolina, at a depth of approximately 410 m. F199 is a gram-positive, irregular-shaped bacterium that has a varied cell morphology that is dependent on culture medium type and growth stage. F199 required microaerobic conditions (40 to 80 μM O₂) for growth on hydrocarbons, glucose, acetate, and lactate in mineral salts medium but not for growth on rich media. [¹⁴C]naphthalene mineralization by F199 was induced by either naphthalene or toulene; however, [¹⁴C]toluene mineralization by this strain was induced by toluene but not naphthalene. F199 was also found to harbor two plasmids larger than 100 kb. Restricted F199 plasmid and genomic DNA did not hybridize with toluene (pWW0) or naphthalene (NAH7) catabolic plasmid DNA probes. The presence in the Middendorf formation of bacteria with the capacity for degrading a variety of aromatic compounds suggests that indigenous microorganisms may have potential for in situ degradation of organic contaminants.     

Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO₃⁻-N mg of mixed-liquor volatile suspended solids (MLVSS)⁻¹ h⁻¹ to a steady-state value of 0.06 mg of NO₃⁻-N mg of MLVSS⁻¹ h⁻¹ over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [¹³C]methanol to biomark the DNA of the denitrifiers. The extracted [¹³C]DNA and [¹²C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [¹³C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [¹²C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [¹⁴C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.     

Genome Sequence of Pseudomonas putida Strain SJTE-1, a Bacterium Capable of Degrading Estrogens and Persistent Organic Pollutants

Pseudomonas putida strain SJTE-1 can utilize 17β-estradiol and other environmental estrogens/toxicants, such as estrone, and naphthalene as sole carbon sources. We report the draft genome sequence of strain SJTE-1 (5,551,505 bp, with a GC content of 62.25%) and major findings from its annotation, which could provide insights into its biodegradation mechanisms.     

Genome Sequence of Pseudomonas aeruginosa Strain SJTD-1, a Bacterium Capable of Degrading Long-Chain Alkanes and Crude Oil

Pseudomonas aeruginosa strain SJTD-1 can utilize long-chain alkanes, diesel oil, and crude oil as sole carbon sources. We report the draft genome sequence of strain SJTD-1 (6,074,058 bp, with a GC content of 66.83%) and major findings from its annotation, which could provide insights into its petroleum biodegradation mechanism.     

Treatment of Groundwater Contaminated with PAHs, Gasoline Hydrocarbons, and Methyl tert-butyl Ether in a Laboratory Biomass-Retaining Bioreactor

In this study, we investigated the treatability of co-mingled groundwater contaminated with polycyclic aromatic hydrocarbons (PAHs), gasoline hydrocarbons, and methyl tert-butyl ether (MtBE) using an ex-situ aerobic biotreatment system. The PAHs of interest were naphthalene, methyl-naphthalene, acenaphthene, acenaphthylene, and carbazole. The gasoline hydrocarbons included benzene, toluene, ethyl benzene, and p-xylene (BTEX). Two porous pot reactors were operated for a period of 10 months under the same influent contaminant concentrations. The contaminated groundwater was introduced into the reactors at a flow rate of 4 and 9 l/day, resulting in a hydraulic retention time (HRT) of 32 and 15 h, respectively. In both reactors, high removal efficiencies were achieved for the PAHs (>99%), BTEX and MtBE (>99.7%). All the PAHs of interest and the four BTEX compounds were detected at concentrations less than 1 μg/l throughout the study duration. Effluent MtBE from both reactors was observed at higher levels; nevertheless, its concentration was lower than the 5 μg/l Drinking Water Advisory for MtBE implemented in California.