Cold tolerance of Pseudomonas sp. 30-3 isolated from oil-contaminated soil, Antarctica

Pseudomonas sp. 30-3 was enriched from oil-contaminated soil from Wright Valley, Antarctica using JP8 jet fuel as sole carbon source. This isolate exhibited tolerance to temperatures ranging from 0°C to 35°C when cultured in laboratory medium. In a freeze-thaw study, an 89% survival was observed when Pseudomonas sp. 30-3 was exposed to 4°C prior to freezing. PCR amplification of a 248-bp DNA fragment in Pseudomonas sp. 30-3 using capB-gene specific primers showed a 98% amino acid sequence homology with CapB of Pseudomonas fragi and 62% homology with CspA of Escherichia coli. Radiolabeling of total cellular proteins exhibited elevated expression of an 8-kDa protein at 4°C, which suggests that the CapB in Pseudomonas sp. 30-3 may play a pivotal role in survival and tolerance at cold and subzero temperatures. Tolerance to cold temperatures and the ability to degrade hydrocarbons by Pseudomonas sp. 30-3 provide support for the application of bioremediation for petroleum hydrocarbons in Antarctic soils.     

Biodegradation and bioremediation of hydrocarbons in extreme environments

Many hydrocarbon-contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, high salt concentrations, or high pressure, Hydrocarbon-degrading microorganisms, adapted to grow and thrive in these environments, play an important role in the biological treatment of polluted extreme habitats. The biodegradation (transformation or mineralization) of a wide range of hydrocarbons, including aliphatic, aromatic, halogenated and nitrated compounds, has been shown to occur in various extreme habitats. The biodegradation of many components of petroleum hydrocarbons has been reported in a variety of terrestrial and marine cold ecosystems. Cold-adapted hydrocarbon degraders are also useful for wastewater treatment. The use of thermophiles for biodegradation of hydrocarbons with low water solubility is of interest, as solubility and thus bioavailability, are enhanced at elevated temperatures. Thermophiles, predominantly bacilli, possess a substantial potential for the degradation of environmental pollutants, including all major classes. Indigenous thermophilic hydrocarbon degraders are of special significance for the bioremediation of oil-polluted desert soil. Some studies have investigated composting as a bioremediation process. Hydrocarbon biodegradation in the presence of high salt concentrations is of interest for the bioremediation of oil-polluted salt marshes and industrial wastewaters, contaminated with aromatic hydrocarbons or with chlorinated hydrocarbons. Our knowledge of the biodegradation potential of acidophilic, alkaliphilic, or barophilic microorganisms is limited.     

Search for active psychrotrophic microbial oil degraders and their characterization

The ability of 96 microbial strains degrading oil and 32 strains degrading polycyclic aromatic hydrocarbons (PAHs) to consume diesel fuel and oil at 4-6 degrees C and 24 degrees C and at elevated NaCl concentrations was studied. The temperature range, salt tolerance, ability to produce bioemulsifiers, range of substrates, and antibiotic resistance were determined. The eleven most active oil-degrading and PAH-degrading strains were genotyped by a polymerase chain reaction with BoxA1R primers and a restriction analysis of ribosomal DNA amplicons. For six strains, the degree of oil degradation at 4-6 degrees C was higher than at 24 degrees C. For the most active strains, the degree of oil degradation in liquid mineral medium ranged from 15 to 26% at 24 degrees C and from 28 to 47% at 4-6 degrees C. An artificial association of six of the strains degraded the oil by 46% at 24 degrees C.     

Comparative Mesocosm Study of Biostimulation Efficiency in Two Different Oil-Amended Sub-Antarctic Soils

Biological treatment has become increasingly popular as a remediation method for soils and groundwater contaminated with petroleum hydrocarbon, chlorinated solvents, and pesticides. Bioremediation has been considered for application in cold regions such as Arctic and sub-Arctic climates and Antarctica. Studies to date suggest that indigenous microbes suitable for bioremediation exist in soils in these regions. This paper reports on two case studies at the sub-Antarctic Kerguelen Island in which indigenous bacteria were found that were capable of mineralizing petroleum hydrocarbons in soil contaminated with crude oil and diesel fuel. All results demonstrate a serious influence of the soil properties on the biostimulation efficiency. Both temperature elevation and fertilizer addition have a more significant impact on the microbial assemblages in the mineral soil than in the organic one. Analysis of the hydrocarbons remaining at the end of the experiments confirmed the bacterial observations. Optimum temperature seems to be around 10 degrees C in organic soil, whereas it was higher in mineral soil. The benefit of adding nutrients was much stronger in mineral than in the organic soil. Overall, this study suggests that biostimulation treatments were driven by soil properties and that ex situ bioremediation for treatment of cold contaminated soils will allow greater control over soil temperature, a limiting factor in cold climates.     

Microbial metabolism of polycyclic aromatic hydrocarbons: isolation and characterization of a pyrene-degrading bacterium.

Microbiological analyses of sediments located near a point source for petrogenic chemicals resulted in the isolation of a pyrene-mineralizing bacterium. This isolate was identified as a Mycobacterium sp. on the basis of its cellular and colony morphology, gram-positive and strong acid-fast reactions, diagnostic biochemical tests, 66.6% G + C content of the DNA, and high-molecular-weight mycolic acids (C58 to C64). The mycobacterium mineralized pyrene when grown in a mineral salts medium supplemented with nutrients but was unable to utilize pyrene as a sole source of carbon and energy. The mycobacterium grew well at 24 and 30 degrees C and minimally at 35 degrees C. No growth was observed at 5 or 42 degrees C. The mycobacterium grew well at salt concentrations up to 4%. Pyrene-induced Mycobacterium cultures mineralized 5% of the pyrene after 6 h and reached a maximum of 48% mineralization within 72 h. Treatment of induced and noninduced cultures with chloramphenicol showed that pyrene-degrading enzymes were inducible in this Mycobacterium sp. This bacterium could also mineralize other polycyclic aromatic hydrocarbons and alkyl- and nitro-substituted polycyclic aromatic hydrocarbons including naphthalene, phenanthrene, fluoranthene, 3-methylcholanthrene, 1-nitropyrene, and 6-nitrochrysene. This is the first report of a bacterium able to extensively mineralize pyrene and other polycyclic aromatic hydrocarbons containing four aromatic rings.     

Microbial metabolism of polycyclic aromatic hydrocarbons: isolation and characterization of a pyrene-degrading bacterium

Microbiological analyses of sediments located near a point source for petrogenic chemicals resulted in the isolation of a pyrene-mineralizing bacterium. This isolate was identified as a Mycobacterium sp. on the basis of its cellular and colony morphology, gram-positive and strong acid-fast reactions, diagnostic biochemical tests, 66.6% G + C content of the DNA, and high-molecular-weight mycolic acids (C58 to C64). The mycobacterium mineralized pyrene when grown in a mineral salts medium supplemented with nutrients but was unable to utilize pyrene as a sole source of carbon and energy. The mycobacterium grew well at 24 and 30 degrees C and minimally at 35 degrees C. No growth was observed at 5 or 42 degrees C. The mycobacterium grew well at salt concentrations up to 4%. Pyrene-induced Mycobacterium cultures mineralized 5% of the pyrene after 6 h and reached a maximum of 48% mineralization within 72 h. Treatment of induced and noninduced cultures with chloramphenicol showed that pyrene-degrading enzymes were inducible in this Mycobacterium sp. This bacterium could also mineralize other polycyclic aromatic hydrocarbons and alkyl- and nitro-substituted polycyclic aromatic hydrocarbons including naphthalene, phenanthrene, fluoranthene, 3-methylcholanthrene, 1-nitropyrene, and 6-nitrochrysene. This is the first report of a bacterium able to extensively mineralize pyrene and other polycyclic aromatic hydrocarbons containing four aromatic rings.     

Cold acclimation increases basal heart rate but decreases its thermal tolerance in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss, Walbaum) were acclimated to 4 degrees C and 17 degrees C for more than 4 weeks and heart rate was determined in the absence and presence of adrenaline to see how thermal adaptation influences basal heart rate and its beta-adrenergic control in a eurythermal fish species. The basal heart rate in vitro was higher in cold-acclimated than warm-acclimated rainbow trout at temperatures below 17 degrees C. On the other hand, adaptation to cold decreased thermal tolerance of heart rate so that the maximal heart rates were achieved at 17 degrees C (75 +/- 4 bpm) and 24 degrees C (88 +/- 2 bpm) in cold-acclimated and warm-acclimated trout, respectively. Beta-adrenergic response of the heart was enhanced by cold-adaptation, since adrenaline (100 nmol l(-1)) caused stronger stimulation of heart rate in cold-acclimated (29 +/- 14%) than in warm-acclimated fish (10 +/- 1%; P = 0.03). Furthermore, adrenaline strongly opposed the temperature-dependent deterioration of force production in cold-acclimated trout but not in warm-acclimated trout. The results indicate that adaptation to cold increases basal heart rate but decreases its thermal tolerance in rainbow trout. Cold acclimation up-regulates the beta-adrenergic system, and beta-adrenoceptor activation seems to provide cardioprotection against high temperatures in the cold-adapted rainbow trout.     

Degradation of hydrocarbons and alcohols at different temperatures and salinities by Rhodococcus erythropolis DCL14

Rhodococcus erythropolis DCL14 cells were able to metabolise C5-C16 hydrocarbons and C1-C12 alcohols as sole carbon and energy sources, both at 15 and 28 degrees C. Metabolic activity was also observed at 1.00%, 1.95% and 2.50% sodium chloride. Almost complete degradation of n-, iso- and cyclo-alkanes and aromatic compounds present in fuel oil was achieved after 9 months, 60% being consumed in the first three months. The results from the conditions tested here suggest that this type of bacterium could be involved in bioremediation processes in marine environments such as the Atlantic, Pacific and Indian Ocean.     

Rapid cold hardening in the predatory mite Euseius (Amblyseius) finlandicus (Acari: Phytoseiidae)

A rapid cold hardening response was studied in diapause and non-diapause females of the predatory mite Euseius finlandicus. When laboratory reared diapause and non-diapause females were transferred and maintained from the rearing temperature of 20 degrees C for 2 h to -11.5 degrees C and -10 degrees C, 10 to 20% survived respectively. However, conditioning of diapause females for 4 h at a range of temperatures from 0 to 10 degrees C before their exposure for 2 h to -11.5 degrees C, increased survival to approximately 90%. Similarly, conditioning of non-diapause females for 4 h at 5 degrees C before their exposure for 2 h to -10 degrees C increased survival to 90%. A similar rapid cold hardening response in both diapause and non-diapause females was also induced through gradual cooling of the mites, at a rate of approximately 0.4 degrees C per min. The rapid increase in cold tolerance after prior conditioning of the mites to low temperatures, was rapidly lost when they returned to a higher temperature of 20 degrees C. Rapid cold hardening extended the survival time of diapause and non-diapause females at sub-zero temperatures. The cost of rapid cold hardening in reproductive potential after diapause termination was negligible. In non-diapause females, however, the increase in cold tolerance gained through gradual cooling could not prevent cold shock injuries, as both fecundity and survival were reduced.     

Survival of clinical and poultry-derived isolates of Campylobacter jejuni at a low temperature (4 degrees C)

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans, and contamination of poultry has been implicated in illness. The bacteria are fastidious in terms of their temperature requirements, being unable to grow below ca. 31 degrees C, but have been found to be physiologically active at lower temperatures and to tolerate exposure to low temperatures in a strain-dependent manner. In this study, 19 field isolates of C. jejuni (10 of clinical and 9 of poultry origin) were studied for their ability to tolerate prolonged exposure to low temperature (4 degrees C). Although substantial variability was found among different strains, clinical isolates tended to be significantly more likely to remain viable following cold exposure than poultry-derived strains. In contrast, the relative degree of tolerance of the bacteria to freezing at -20 degrees C and freeze-thawing was strain specific but independent of strain source (poultry versus clinical) and degree of cold (4 degrees C) tolerance.     

Characterization of psychrotolerant heterotrophic bacteria from Finnish Lapland

A total of 331 aerobic heterotrophic bacterial strains were isolated from various ecosystems of Finnish Lapland (68-69 degrees N) including forest soil, arctic alpine-tundra soil, stream water, lake and mire sediments, lichen and snow algae. Whole cell fatty acid and 16S rRNA gene sequence analysis and microscopy indicated that the isolates were dominated by Gram-negative bacteria, while only 20 Gram-positive strains were isolated. Based on 16S rRNA gene sequences the isolates were members of alpha-, beta-, gamma-Proteobacteria, Gram-positives with low G+C content, Actinobacteria and the Cytophaga/Flexibacter/Bacteroides group. More than one-third of the isolates could be tentatively identified as Pseudomonas spp. which were particularly abundant in the alpine-tundra soils where they represented 60% of all isolates. Other frequently isolated Gram-negative taxa were Burkholderia sp., Collimonas sp., Pedobacter sp., Janthinobacter sp., Duganella sp., Dyella sp. and Sphingomonas sp. Growth temperature ranges and hydrolytic enzyme activities of selected ca.100 strains were screened. The strains were psychrotolerant growing generally at temperatures ranging from 0 to 30 degrees C, as 82% of the isolates grew at 0 degrees C while only 7% grew at 35 degrees C. Protease and lipase activities at 5 degrees C were detected in more than half of the strains while approximately 20% of the strains possessed amylase and/or cellulase activities.     

Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26 degrees C) and low (4 degrees C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26 degrees C and 4 degrees C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26 degrees C and 4 degrees C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO(2) fixation and O(2) evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.     

Bioremediation Potential of Terrestrial Fuel Spills

A bioremediation treatment that consisted of liming, fertilization, and tilling was evaluated on the laboratory scale for its effectiveness in cleaning up a sand, a loam, and a clay loam contaminated at 50 to 135 mg g of soil⁻¹ by gasoline, jet fuel, heating oil, diesel oil, or bunker C. Experimental variables included incubation temperatures of 17, 27, and 37°C; no treatment; bioremediation treatment; and poisoned evaporation controls. Hydrocarbon residues were determined by quantitative gas chromatography or, in the case of bunker C, by residual weight determination. Four-point depletion curves were obtained for the described experimental variables. In all cases, the disappearance of hydrocarbons was maximal at 27°C and in response to bioremediation treatment. Poisoned evaporation controls underestimated the true biodegradation contribution, but nevertheless, they showed that biodegradation makes only a modest contribution to gasoline disappearance from soil. Bunker C was found to be structurally recalcitrant, with close to 80% persisting after 1 year of incubation. The three medium distillates, jet fuel, heating oil, and diesel oil, increased in persistence in the listed order but responded well to bioremediation treatment under all test conditions. With bioremediation treatment, it should be possible to reduce hydrocarbons to insignificant levels in contaminated soils within one growing season.     

Temperature effects on Korean entomopathogenic nematodes, Steinernema glaseri and S. longicaudum, and their symbiotic bacteria

We investigated the temperature effects on the virulence, development, reproduction, and motility of two Korean isolates of entomopathogenic nematodes, Steinernema glaseri Dongrae strain and S. longicaudum Nonsan strain. In addition, we studied the growth and virulence of their respective symbiotic bacterium, Xenorhabdus poinarii for S. glaseri and Xenorhabdus sp. for S. longicaudum, in an insect host at different temperatures. Insects infected with the nematode-bacterium complex or the symbiotic bacterium was placed at 13 degrees C, 18 degrees C, 24 degrees C, 30 degrees C, or 35 degrees C in the dark and the various parameters were monitored. Both nematode species caused mortality at all temperatures tested, with higher mortalities occurring at temperatures between 24 degrees C and 30 degrees C. However, S. longicaudum was better adapted to cold temperatures and caused higher mortality at 18 degrees C than S. glaseri. Both nematode species developed to adult at all temperatures, but no progeny production occurred at 13 degrees C or 35 degrees C. For S. glaseri, nematode progeny production was best at inocula levels above 20 infective juveniles/host at 24 degrees C and 30 degrees C, but for S. longicaudum, progeny production was generally better at 24 degrees C. Steinernema glaseri showed the greatest motility at 30 degrees C, whereas S. longicaudum showed good motility at 24 degrees C and 30 degrees C. Both bacterial species grew at all tested temperatures, but Xenorhabdus sp. was more virulent at low temperatures (13 degrees C and 18 degrees C) than X poinarii.     

Isolation and taxonomic and catabolic characterization of a 3,6-dimethylphenanthrene-utilizing strain of Sphingomonas sp

A bacterial strain capable of utilizing 3,6-dimethylphenanthrene (3,6-DMP) as its sole source of carbon and energy was isolated from a creosote-contaminated soil. The isolate was identified as a strain of Sphingomonas sp. and was designated strain JS1. Utilization of 3,6-DMP was demonstrated by an increase in bacterial biomass concomitant with a decrease in 3,6-DMP in a liquid mineral medium with this compound as its sole source of carbon and energy. Strain JS1 showed a high specificity in the use of the most abundant alkylderivatives of crude oils, such as alkylnaphthalenes and other alkylphenanthrenes, as the sole source of carbon and energy. It can also use several polycyclic aromatic hydrocarbons of three and four rings and their alkylated derivatives as growth substrates or transform them. The identification of several intermediate metabolites points to extensive metabolic activity, including the following: (i) aromatic ring oxidation and cleavage, (ii) methyl group oxidations, and (iii) methylenic oxidations. The metabolic actions of Sphingomonas sp. JS1 on the aromatic fraction extracted from a creosote-contaminated soil are also examined.     

<Emphasis Type="Italic">Acinetobacter</Emphasis> sp. strain Ths,a novel psychrotolerant and alkalitolerant bacterium that utilizes hydrocarbon

A novel psychrotolerant, alkalitolerant bacterium, strain Ths, was isolated from a soil sample immersed in hot spring water containing hydrocarbons and grown on a chemically defined medium containing n-tetradecane as the sole carbon source. The isolate grew at 0 degrees C but not at temperatures higher than 45 degrees C; its optimum growth temperature was 27 degrees C. It grew in the pH range of 7-9. The strain utilized C(13)-C(30) n-alkane and fluorene at pH 9 and 4 degrees C. To our knowledge, this is the first report on the bacterium that utilizes a wide range of hydrocarbons at a high pH and a low temperature. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Ths is closely related to genomic species 6 ATCC 17979 (99.1% similarity), genomic species BJ13/TU14 ATCC 17905 (97.8% similarity), genomic species 9 ATCC 9957 (97.6% similarity) belonging to the genus Acinetobacter and to Acinetobacter calcoaceticus JCM 6842(T) (97.5% similarity). DNA-DNA hybridization revealed that the isolate has 62, 25, 18 and 19% relatedness, respectively, to genomic species 6 ATCC 17979, genomic species BJ13/TU14 ATCC 17905, genomic species 9 ATCC 9957 and A. calcoaceticus, respectively.     

Aromatic hydrocarbon-degrading bacteria from soil near Scott Base, Antarctica

Hydrocarbons persist in Antarctic soils when fuel oils such as JP8 jet fuel are spilled. For clean-up of hydrocarbon-contaminated soils in Antarctica, bioremediation has been proposed using hydrocarbon-degrading microbes indigenous to Antarctic soils. A number of alkane-degrading bacteria have been isolated previously from Antarctic soils. In this paper we describe the direct isolation of aromatic hydrocarbon-degrading bacteria from oil-contaminated Antarctic soil. Isolates that grew on JP8 jet fuel were characterised for their ability to degrade aromatic and aliphatic hydrocarbons and for growth at a range of temperatures. All isolates were gram-negative, oxidase-positive, rod-shaped bacteria. Representative strains were identified using 16S rDNA sequence analysis as either Sphingomonas spp. or Pseudomonas spp. Aromatic-degrading bacteria from Antarctic soils were psychrotolerant and appear similar to those found worldwide. Accepted: 27 September 1999     

Low-Temperature-Induced DnaA Protein Synthesis Does Not Change Initiation Mass in Escherichia coli K-12

Expression of the dnaA gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14 degrees C than at 37 degrees C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30 degrees C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of dnaA gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.     

Low temperature bioremediation of oil-contaminated soil using biostimulation and bioaugmentation with a Pseudomonas sp. from maritime Antarctica

AIMS: To identify native Antarctic bacteria capable of oil degradation at low temperatures. METHODS AND RESULTS: Oil contaminated and pristine soils from Signy Island (South Orkney Islands, Antarctica) were examined for bacteria capable of oil degradation at low temperatures. Of the 300 isolates cultured, Pseudomonas strain ST41 grew on the widest range of hydrocarbons at 4 degrees C. ST41 was used in microcosm studies of low temperature bioremediation of oil-contaminated soils. Microcosm experiments showed that at 4 degrees C the levels of oil degradation increased, relative to the controls, with (i) the addition of ST41 to the existing soil microbial population (bioaugmentation), (ii) the addition of nutrients (biostimulation) and to the greatest extent with (iii) a combination of both treatments (bioaugmentation and biostimulation). Addition of water to oil contaminated soil (hydration) also enhanced oil degradation, although less than the other treatments. Analysis of the dominant species in the microcosms after 12 weeks, using temporal temperature gradient gel electrophoresis, showed Pseudomonas species to be the dominant soil bacteria in both bioaugmented and biostimulated microcosms. CONCLUSIONS: Addition of water and nutrients may enhance oil degradation through the biostimulation of indigenous oil-degrading microbial populations within the soil. However, bioaugmentation with Antarctic bacteria capable of efficient low temperature hydrocarbon degradation may enhance the rate of bioremediation if applied soon after the spill. SIGNIFICANCE AND IMPACT OF THE STUDY: In the future, native soil bacteria could be of use in bioremediation technologies in Antarctica.