Role of extracellular polymeric substances in the surface chemical reactivity of Hymenobacter aerophilus, a psychrotolerant bacterium.

Bacterial surface layers, such as extracellular polymeric substances (EPS), are known to play an important role in metal sorption and biomineralization; however, there have been very few studies investigating how environmentally induced changes in EPS production affect the cell's surface chemistry and reactivity. Acid-base titrations, cadmium adsorption assays, and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the surface reactivities of Hymenobacter aerophilus cells with intact EPS (WC) or stripped of EPS (SC) and purified EPS alone. Linear programming modeling of titration data showed SC to possess functional groups corresponding to phosphoryl (pKa approximately 6.5), phosphoryl/amine (pKa approximately 7.9), and amine/hydroxyl (pKa approximately 9.9). EPS and WC both possess carboxyl groups (pKa approximately 5.1 to 5.8) in addition to phosphoryl and amine groups. FT-IR confirmed the presence of polysaccharides and protein in purified EPS that can account for the additional carboxyl groups. An increased ligand density was observed for WC relative to that for SC, leading to an increase in the amount of Cd adsorbed (0.53 to 1.73 mmol/liter per g [dry weight] and 0.53 to 0.59 mmol/liter per g [dry weight], respectively). Overall, the presence of EPS corresponds to an increase in the number and type of functional groups on the surface of H. aerophilus that is reflected by increased metal adsorption relative to that for EPS-free cells.

Bacterial biodegradation of aliphatic sulfides under aerobic carbon- or sulfur-limited growth conditions.

AIMS: To isolate bacteria capable of cleaving aliphatic carbon-sulfur bonds as potential biological upgrading catalysts for the reduction of molecular weight and viscosity in heavy crude oil. METHODS AND RESULTS: Thirty-one bacterial strains isolated from enrichment cultures were able to biotransform model compounds representing the aliphatic sulfide bridges found in asphaltenes. Using gas chromatography and mass spectrometry, three types of attack were identified: alkyl chain degradation, allowing use as a carbon source; nonspecific sulfur oxidation; and sulfur-specific oxidation and carbon-sulfur bond cleavage, allowing use as a sulfur source. Di-n-octyl sulfide degradation produced octylthio- and octylsulfonyl-alkanoic acids, consistent with terminal oxidation followed by beta-oxidation reactions. Utilization of dibenzyl sulfide or 1,4-dithiane as a sulfur source was regulated by sulfate, indicating a sulfur-specific activity rather than nonspecific oxidation. Finally, several isolates were also able to use dibenzothiophene as a sulfur source, and this was the preferred organic sulfur substrate for one isolate. CONCLUSIONS: The use of commercially available alkyl sulfides in enrichment cultures gave isolates that followed a range of metabolic pathways, not just sulfur-specific attack. SIGNIFICANCE AND IMPACT OF THE STUDY: These results give new insight into biodegradation of organosulfur compounds from petroleum and for biotreatment of such compounds in chemical munitions.

Characterization of Sphingomonas sp. Ant 17, an aromatic hydrocarbon-degrading bacterium isolated from Antarctic soil.

Sphingomonas sp. strain Ant 17 was isolated from fuel-contaminated soil collected at Scott Base, Ross Island, Antarctica. We anticipated that Ant 17 would be a good model organism for studying cold climate bioremediation, and therefore determined its biodegradation capabilities and tolerance of potentially growth-limiting environmental conditions. Sphingomonas sp. Ant 17 degrades the aromatic fraction of several different crude oils, jet fuel, and diesel fuel at low temperatures and without nutrient amendment. It utilizes or transforms a broad range of pure aromatic substrates, including hydrocarbons, heterocycles, and aromatic acids and alcohols. Ant 17 grows at temperatures of 1 degree C to 35 degrees C and mineralizes radiolabeled phenanthrene over a range of more than 24 degrees C. This psychrotolerant isolate appears to utilize hydrocarbons more efficiently at low temperatures than would be predicted by mesophilic enzyme kinetics. The optimum pH for growth was 6.4 at 22 degrees C, with extended lag phases observed in more alkaline media. However, there was less effect of pH on lag phase at lower temperatures. Ant 17 displayed greater tolerance to UV irradiation and freeze-thaw cycles than the hydrocarbon-degrading isolate Sphingomonas sp. WPO-1, which may reflect adaptation to its Antarctic soil environment. However, it was more sensitive than expected to desiccation and to low concentrations of NaCl and CaCl(2). Ant 17 was phenotypically stable and lacked detectable plasmids, suggesting a chromosomal location for genes encoding aromatic degradation enzymes. Its broad aromatic substrate range and tolerance of low and fluctuating temperature and low nutrients make Sphingomonas sp. Ant 17 a valuable microbe for examining fuel spill bioremediation in cold soils.

Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens LP6a.

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.

Microbial life beneath a high arctic glacier.

The debris-rich basal ice layers of a high Arctic glacier were shown to contain metabolically diverse microbes that could be cultured oligotrophically at low temperatures (0.3 to 4 degrees C). These organisms included aerobic chemoheterotrophs and anaerobic nitrate reducers, sulfate reducers, and methanogens. Colonies purified from subglacial samples at 4 degrees C appeared to be predominantly psychrophilic. Aerobic chemoheterotrophs were metabolically active in unfrozen basal sediments when they were cultured at 0.3 degrees C in the dark (to simulate nearly in situ conditions), producing (14)CO(2) from radiolabeled sodium acetate with minimal organic amendment (> or =38 microM C). In contrast, no activity was observed when samples were cultured at subfreezing temperatures (< or =-1.8 degrees C) for 66 days. Electron microscopy of thawed basal ice samples revealed various cell morphologies, including dividing cells. This suggests that the subglacial environment beneath a polythermal glacier provides a viable habitat for life and that microbes may be widespread where the basal ice is temperate and water is present at the base of the glacier and where organic carbon from glacially overridden soils is present. Our observations raise the possibility that in situ microbial production of CO(2) and CH(4) beneath ice masses (e.g., the Northern Hemisphere ice sheets) is an important factor in carbon cycling during glacial periods. Moreover, this terrestrial environment may provide a model for viable habitats for life on Mars, since similar conditions may exist or may have existed in the basal sediments beneath the Martian north polar ice cap.

Hydrocarbon-degrading filamentous fungi isolated from flare pit soils in northern and western Canada.

Sixty-four species of filamentous fungi from five flare pits in northern and western Canada were tested for their ability to degrade crude oil using gas chromatographic analysis of residual hydrocarbons following incubation. Nine isolates were tested further using radiorespirometry to determine the extent of mineralization of model radiolabelled aliphatic and aromatic hydrocarbons dissolved in crude oil. Hydrocarbon biodegradation capability was observed in species representing six orders of the Ascomycota. Gas chromatography indicated that species capable of hydrocarbon degradation attacked compounds within the aliphatic fraction of crude oil, n-C12-n-C26; degradation of compounds within the aromatic fraction was not observed. Radiorespirometry, using n-[1-14C]hexadecane and [9-14C]phenanthrene, confirmed the gas chromatographic results and verified that aliphatic compounds were being mineralized, not simply transformed to intermediate metabolites. This study shows that filamentous fungi may play an integral role in the in situ biodegradation of aliphatic pollutants in flare pit soils.

Degradation of hydrocarbons in crude oil by the ascomycete Pseudallescheria boydii (Microascaceae).

Four unique strains of Pseudallescheria boydii were isolated from oil-soaked soils in British Columbia and Alberta and compared to strains from cattle dung and raw sewage. Considerable variability in morphology, colony appearance, colony diameter, and temperature tolerance occurred among the strains. They also varied in the sporogenous states produced in culture; all strains had a Scedosporium anamorph and either the Graphium anamorph or cleistothecial teleomorph. Conspecificity of the six isolates was inferred from their morphology and supported by restriction fragment length polymorphism profiles of the internally transcribed spacer region of rDNA and comparing these to Petriella sordida, a similar taxon in the Microascaceae. Three of the strains isolated from oil-contaminated soil and the strain from sewage were tested for their ability to utilize hydrocarbons by incubation with Prudhoe Bay Crude oil as the sole carbon source. Gas chromatographic analysis of the residual oil revealed that the strains isolated from oil-contaminated soil degraded the linear aliphatics. The strain from sewage, previously shown by others to utilize the volatile n-alkanes (i.e., ethane, propane, and butane), did not utilize the liquid saturate compounds. None of the strains was observed to degrade compounds in the aromatic fraction. Pseudallescheria boydii may be an important agent for in situ bioremediation of saturates in oil-contaminated sites.

Effect of nitrate injection on the microbial community in an oil field as monitored by reverse sample genome probing.

The reverse sample genome probe (RSGP) method, developed for monitoring the microbial community in oil fields with a moderate subsurface temperature, has been improved by (i) isolation of a variety of heterotrophic bacteria and inclusion of their genomes on the oil field master filter and (ii) use of phosphorimaging technology for the rapid quantitation of hybridization signals. The new master filter contains the genomes of 30 sulfate-reducing, 1 sulfide-oxidizing, and 16 heterotrophic bacteria. Most have been identified by partial 16S rRNA sequencing. Use of improved RSGP in monitoring the effect of nitrate injection in an oil field indicated that the sulfide-oxidizing, nitrate-reducing isolate CVO (a Campylobacter sp.) becomes the dominant community component immediately after injection. No significant enhancement of other community members, including the sulfate-reducing bacteria, was observed. The elevated level of CVO decayed at most sampling sites within 30 days after nitrate injection was terminated. Chemical analyses indicated a corresponding decrease and subsequent increase in sulfide concentrations. Thus, transient injection of a higher potential electron acceptor into an anaerobic subsurface system can have desirable effects (i.e., reduction of sulfide levels) without a permanent adverse influence on the resident microbial community.

Environmental gasoline-utilizing isolates and clinical isolates of Pseudomonas aeruginosa are taxonomically indistinguishable by chemotaxonomic and molecular techniques.

A total of 42 Pseudomonas aeruginosa strains was isolated previously from clinical sources (27 strains) and from a gasoline-contaminated aquifer (15 strains). Selected strains were subjected to taxonomic tests involving chemical and molecular biological techniques, including membrane fatty acid analysis, phage-sensitivity, growth temperature range, presence of plasmids, and PCR-amplification and sequencing of a species-specific 16S-23S rDNA internal transcribed spacer region. The clinical and environmental isolates formed a coherent taxonomic group with few distinguishing characteristics. Of the phenotypes observed, a consistent difference was the ability of the aquifer strains to utilize gasoline supplied in the gas phase as sole carbon source and, conversely, the inability of the clinical strains to do so. Fourteen of the 15 environmental strains possessed similar-sized cryptic plasmids. The clinical isolates either lacked detectable plasmids or contained plasmids of a different size. The observation that the clinical and environmental isolates of P. aeruginosa were taxonomically indistinguishable is discussed in terms of its relevance to environmental-regulatory guidelines because P. aeruginosa, a known opportunistic pathogen, is a prime candidate for use in bioremediation processes involving deliberate release of this organism to the environment.

Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens.

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.

Characterization of the diversity of sulfate-reducing bacteria in soil and mining waste water environments by nucleic acid hybridization techniques.

Nucleic acid hybridization techniques were used to characterize the sulfate-reducing bacterial communities at seven waste water and two soil sites in Canada. Genomic DNA was obtained from liquid enrichment cultures of samples taken from these nine sites. The liquid enrichment protocol favored growth of the sulfate-reducing bacterial component of the communities at these sites. The genomic DNA preparations were analyzed with (i) a specific gene probe aimed at a single genus (Desulfovibrio), (ii) a general 16S rRNA gene probe aimed at all genera of sulfate-reducing bacteria and other bacteria, and (iii) whole genome probes aimed at specific bacteria. This three-pronged approach provided information on the sulfate-reducing bacterial community structures for the nine sites. These were compared with each other and with the sulfate-reducing bacterial communities of western Canadian oil field production waters, studied previously. It was found that there is considerable diversity in the sulfate-reducing bacterial community at each site. Most sulfate-reducing bacteria isolated from distinct sites are genomically different and differ also from sulfate-reducing bacteria found in oil field production waters.

Expression of dibenzothiophene-degradative genes in two Pseudomonas species.

The genes encoding dibenzothiophene (DBT) degradation in Pseudomonas alcaligenes strain DBT2 were cloned into plasmid pC1 by other workers. This plasmid was conjugally transferred into a spontaneous variant of Pseudomonas sp. HL7b (designated HL7bR) incapable of oxidizing DBT (Dbt- phenotype). Acquisition of plasmid pC1 simultaneously restored oxidation of DBT and naphthalene to the transconjugant, although the primary DBT metabolite produced by transconjugant HL7bR(pC1) corresponded to that produced by wild-type strain DBT2 rather than that from wild-type strain HL7b. Inducers of the naphthalene pathway (naphthalene, salicylic acid, and 2-aminobenzoate) stimulated DBT oxidation in transconjugant HL7bR(pC1) when present at 0.1 mM concentrations but had no effect on wild-type strain HL7b. Higher concentrations (5 mM) of salicylic acid and naphthalene were inhibitory to DBT oxidation in all strains. DNA-DNA hybridization was not observed between plasmid pC1 and genomic DNA from strains HL7b or HL7bR, nor between authentic naphthalene-degradative genes (plasmid NAH2) and either plasmid pC1 or strain HL7b, despite the observation that the degradative genes encoded on plasmid pC1 functionally resembled broad-specificity naphthalene-degradative genes. Transconjugant HL7bR(pC1) is a mosaic of the parental types regarding DBT metabolite production, regulation, and use of carbon sources.

Mineralization of [14C]hexadecane and [14C]phenanthrene in crude oil: specificity among bacterial isolates.

Bacteria isolated from freshwater, marine, and estuarine samples were tested for the ability to produce 14CO2 from n-[1-14C]hexadecane or [9-14C]phenanthrene added to Prudhoe Bay crude oil. Of 138 isolates tested, 54 (39%) mineralized the model aliphatic compound hexadecane and 6 (4%) mineralized the model aromatic compound phenanthrene. None mineralized both compounds. There was no apparent correlation between degradative ability and genus or source. Additional hydrocarbon-degrading bacteria from diverse sources were tested and found to mineralize either hexadecane or phenanthrene. Of 61 hexadecane- and 21 phenanthrene-mineralizing bacteria tested, none mineralized both model compounds. Selected isolates and commercially available cultures were tested for mineralization of specific 14C-labelled mono-, di-, and tri-cyclic aromatics. An apparent hierarchy of degradation was observed: strains mineralizing the mono- and di-cyclic aromatics toluene and naphthalene did not mineralize biphenyl or the tricyclic aromatics anthracene and phenanthrene, whereas those strains that mineralized the tricyclic aromatics also mineralized the smaller substrates. Similarly, not all n-alkane-mineralizing isolates tested mineralized the isoprenoid pristane. A combined culture consisting of one aliphatic- and one aromatic-degrading isolate was tested for mineralization of the model compounds and for degradation of other crude oil components by gas chromatography. No synergism or antagonism was observed compared with degradation by the individual isolates.

Effect of emulsan on biodegradation of crude oil by pure and mixed bacterial cultures.

Crude oil was treated with purified emulsan, the heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population as well as nine different pure cultures isolated from various sources was tested for biodegradation of emulsan-treated and untreated crude oil. Biodegradation was measured both quantitatively and qualitatively. Recovery of CO(2) from mineralized C-labeled substrates yielded quantitative data on degradation of specific compounds, and capillary gas chromatography of residual unlabeled oil yielded qualitative data on a broad spectrum of crude oil components. Biodegradation of linear alkanes and other saturated hydrocarbons, both by pure cultures and by the mixed population, was reduced some 50 to 90% after emulsan pretreatment. In addition, degradation of aromatic compounds by the mixed population was reduced some 90% in emulsan-treated oil. In sharp contrast, aromatic biodegradation by pure cultures was either unaffected or slightly stimulated by emulsification of the oil.

Degradation of polycyclic aromatic hydrocarbons and aromatic heterocycles by a Pseudomonas species.

Enrichment cultures were established with the aromatic fraction of a crude oil and screened for aromatic-degrading pseudomonads, using a sprayed plate technique. One isolate identified as Pseudomonas sp. HL7b was chosen for further study because it oxidized several polycyclic aromatic hydrocarbons and aromatic heterocycles without an apparent lag. Using capillary gas chromatography, spectrophotometry, and radiorespirometry, it was found to be capable of mineralizing and (or) oxidizing a wide range of polycyclic aromatic hydrocarbons, S-, N-, and O-heterocyclic analogues, and alkyl polycyclic aromatic hydrocarbons, but not aliphatic hydrocarbons. The isolate displayed two colonial morphologies which correlated with variation in degradative phenotype and hydrophobicity as measured by polystyrene adherence. Four cryptic plasmids were observed in both colonial types. Pseudomonas sp. HL7b degraded dibenzothiophene co-metabolically by a recognized pathway, but this degradation was constitutive, rather than inducible as reported for other bacteria.