Low-temperature microbial degradation of crude oil products differing in the extent of condensation

Out of the 30 strains capable of oil degradation at 4-6 degrees C, four were selected by the ability to degrade 40% of the oil substrate present in the growth medium: Rhodococcus spp. DS-07 and DS-21 and Pseudomonas spp. DS-09 and DS-22. We studied the activity of these strains as degraders of oil products of various condensation degrees (crude oil, masut, petroleum oils, benzene resins and ethanol-benzene resins) at 4-6 degrees C. The maximum degrees of degradation of masut and ethanol-benzene resins were observed in Pseudomonas spp. DS-22 (17.2% and 5.2%, respectively). The maximum degradation of petroleum oils and benzene resins was observed in Rhodococcus spp. DS-07 (40% and 16.6%, respectively). The strains provide a basis for developing biodegrader preparations applicable to bioremediation of oil-polluted sites under the conditions of cold climate.     

Intensification of Microbial Degradation of Crude Oil and Oil Products in the Presence of Perfluorodecalin

The possibility of using perfluorinated organic compounds for growing microorganisms and degrading xenobiotics has been demonstrated for the first time with perfluorodecalin (PFD), a gas-transporting component of the blood substitute Perftoran. This is particularly promising for intensifying microbial degradation of oil and oil products and the production of biodegrader biomass in synthetic mineral media. The addition of PFD to a mineral medium with crude oil and masut increased by 4.5–10.2 times the maximum concentrations and growth rates of all bacterial strains under study (Pseudomonas, Rhodococcus, and Bacillus genera). The degree of oil product consumption was increased 8.7–12.7 times.     

Selection of Microorganisms Capable of Degrading Petroleum and Its Products at Low Temperatures

Of 150 cultures capable of degrading petroleum at +6°C, 40 strains growing in a liquid mineral nutrient medium containing petroleum (2%) as the sole source of carbon were selected. Of them, 13 cultures displaying a petroleum degradation rate exceeding 25% were selected. Abilities of these cultures and their associations to utilize fuel oil and its components—oils and benzene resins—were studied. A culture exhibiting degradation rates of fuel oil, its oils, benzene resins, and petroleum amounting to 17, 26, 10, and 51%, respectively, was selected. This culture can be used for cleanup of petroleum pollution under cold climatic conditions.     

Change of oil-degrading activity in microorganisms stored under laboratory conditions

Change of the oil-degrading activity was studied in psychrophilic microbial strains Rhodococcus spp. DS-07, DS-21 and Pseudomonas spp. DS-09, DS-22 maintained on various media: rich and synthetic with a selective agent. After 2.5 years of storage on rich medium, the oil-degrading activity decreased by 50–60%, whereas this decrease was insignificant in the medium with oil. Passages to selective medium with oil after the storage partly restored the activity. It was found that storage of oil-degrading microorganisms caused loss of biodegradation plasmids. Their recovery and long-term preservation demand the presence of the selective agent in the medium.     

Aerobic biodegradation of nonylphenol by cold-adapted bacteria

Three strains capable of mineralizing nonylphenol as sole carbon source were isolated from a sample of contaminated soil and characterized as two Pseudomonas spp. and a Stenotrophomonas sp. The two Pseudomonas spp. expressed characteristics typical of psychrophiles growing optimally of 10 °C and capable of growing at 0 °C. The Stenotrophomonas sp. was more likely psychrotrophic because it had an optimal temperature between 14 and 22 °C although it was not capable of growing at 4 °C. At 14 °C, one of the Pseudomonas spp. exhibited the highest rate of degradation of nonylphenol (4.4 mg l^–1 d^–1), when compared with axenic or mixed cultures of the isolates. This study represents, to the best of our knowledge, the first reported case of cold-adapted microorganisms capable of mineralizing nonylphenol.     

Selection of microorganisms capable of degrading petroleum and petroleum products at decreased temperatures

Of 150 cultures capable of degrading petroleum at +6 degrees C, 40 strains growing in the liquid mineral nutrient medium containing petroleum (2%) as a sole source of carbon were selected. Of them, 13 cultures displaying a petroleum degradation rate exceeding 25% were selected. Abilities of these cultures and their associations to utilize fuel oil and its components--oils and benzene resins--were studied. The culture exhibiting degradation rates of fuel oil, its oils, benzene resins, and petroleum amounting to 17, 26, 10, and 51%, respectively, was selected. This culture can be used for cleanup of petroleum pollution under cold climatic conditions.     

Effect of dissolved oxygen regime on growth dynamics of Pseudomonas spp during benzene degradation

We investigated the effect of different oxygen regimes on growth patterns of Pseudomonas spp. during benzene degradation in microcosm batch studies. Benzene degradation was induced by limiting oxygen available for microbial activity, which consists of three initial-dissolved oxygen (DO) levels of oxic, hypoxic, and anoxic conditions. Batch experiments were performed for cell growth and benzene degradation by inoculating three strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) in mineral salt medium containing aqueous benzene. Results showed that all strains were capable to grow and degrade benzene under all oxygen regimes but in a different manner. The highest cell growth of P. aeruginosa and P. fluorescens was achieved under oxic and anoxic condition, respectively, but there was no substantial difference on benzene degradation between the oxygen treatments with about 25% reduction for both strains. P. putida showed a facultative process for both cell growth and benzene degradation. This reveals that care should be taken in selection of microorganisms with regard to environmental studies since they exhibit different responses for given environmental conditions such as DO levels.     

The diversity of extradiol dioxygenase (edo) genes in cresol degrading rhodococci from a creosote-contaminated site that express a wide range of degradative abilities

Analysis of the bacterial population of soil surface samples from a creosote-contaminated site showed that up to 50% of the culturable micro-organisms detected were able to utilise a mixture of cresols. From fifty different microbial isolates fourteen that could utilise more than one cresol isomer were selected and identified by 16S rRNA analysis. Eight isolates were Rhodococcus strains and six were Pseudomonas strains. In general, the Rhodococcus strains exhibited a broader growth substrate range than the Pseudomonas strains. The distribution of various extradiol dioxygenase (edo) genes, previously associated with aromatic compound degradation in rhodococci, was determined for the Rhodococcus strains by PCR detection and Southern-blot hybridization. One strain, Rhodococcus sp. I1 exhibited the broadest growth substrate range and possessed five different edogenes. Gene disruption experiments indicated that two genes (edoC and edoD) were associated with isopropylbenzene and naphthalene catabolism respectively. The other Rhodococcus strains also possessed some of the edo genes and one (edoB) was present in all of the Rhodococcus strains analysed. None of the rhodococcal edo genes analysed were present in the Pseudomonas strains isolated from the site. It was concluded that individual strains of Rhodococcus possess a wide degradative ability and may be very important in the degradation of complex mixtures of substrates found in creosote.     

Composition of Soil Microbial Communities Enriched on a Mixture of Aromatic Hydrocarbons

Soil contaminated with C5+, which contained benzene (45%, wt/wt), dicyclopentadiene (DCPD) plus cyclopentadiene (together 20%), toluene (6%), styrene (3%), xylenes (2%), naphthalene (2%), and smaller quantities of other compounds, served as the source for isolation of 55 genomically distinct bacteria (standards). Use of benzene as a substrate by these bacteria was most widespread (31 of 44 standards tested), followed by toluene (23 of 44), xylenes (14 of 44), styrene (10 of 44), and naphthalene (10 of 44). Master filters containing denatured genomic DNAs of all 55 standards were used to analyze the community compositions of C5+ enrichment cultures by reverse sample genome probing (RSGP). The communities enriched from three contaminated soils were similar to those enriched from three uncontaminated soils from the same site. The compositions of these communities were time dependent and showed a succession of Pseudomonas and Rhodococcus spp. before convergence on a composition dominated by Alcaligenes spp. The dominant community members detected by RSGP were capable of benzene degradation at all stages of succession. The enrichments effectively degraded all C5+ components except DCPD. Overall, degradation of individual C5+ hydrocarbons followed first-order kinetics, with the highest rates of removal for benzene.     

Degradation of crude oil by an arctic microbial consortium

The ability of a psychrotolerant microbial consortium to degrade crude oil at low temperatures was investigated. The enriched arctic microbial community was also tested for its ability to utilize various hydrocarbons, such as long-chain alkanes (n-C₂₄ to n-C₃₄), pristane, (methyl-)naphthalenes, and xylenes, as sole carbon and energy sources. Except for o-xylene and methylnaphthalenes, all tested compounds were metabolized under conditions that are typical for contaminated marine liquid sites, namely at pH 6–9 and at 4–27°C. By applying molecular biological techniques (16S rDNA sequencing, DGGE) nine strains could be identified in the consortium. Five of these strains could be isolated in pure cultures. The involved strains were closely related to the following genera: Pseudoalteromonas (two species), Pseudomonas (two species), Shewanella (two species), Marinobacter (one species), Psychrobacter (one species), and Agreia (one species). Interestingly, the five isolated strains in different combinations were unable to degrade crude oil or its components significantly, indicating the importance of the four unculturable microorganisms in the degradation of single or of complex mixtures of hydrocarbons. The obtained mixed culture showed obvious advantages including stability of the consortium, wide range adaptability for crude oil degradation, and strong degradation ability of crude oil.     

Characterization of a pyridine-degrading branched Gram-positive bacterium isolated from the anoxic zone of an oil shale column

Summary From the anoxic zone of an oil shale leachate column three pyridine-degrading bacterial strains were isolated. Two strains were Gram-negative facultative anaerobic rods and one strain was a branched Gram-positive bacterium. The branched Gram-positive strain had the best pyridine-degrading ability. This organism was aerobic, non-motile, catalase positive, oxidase negative, and had no flagellum. The G+C content of the DNA was 66.5 mol%. The major menaquinone was MK-8(H₂). The main cellular fatty acids were saturated and monounsaturated straight chains. This organism contained mycolic acid, meso-diaminopimelic acid, arabinogalactan and glycolyl residues in the cell wall. Due to morphological, physiological and chemotaxonomic characteristics this strain was placed in the genus Rhodococcus. The optimum culture conditions were as follows: temperature 32° C, pH 8.0 and 0.1% v/v of pyridine as sole carbon, energy and nitrogen source. Utilization of pyridine by a batch fermentor culture of Rhodococcus sp. was characterized by a specific growth rate of 0.13 h^–1, growth yield of 0.61 mg cell·mg pyridine^–1 and a doubling time of 5.3 h^–1. Offprint requests to: S.-T. Lee     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Design of bacterial defined mixed cultures for biodegradation of specific crude oil fractions, using population dynamics analysis by DGGE

Hydrocarbon-degrading bacteria isolated from oil-polluted soils, were used to design three defined mixed cultures (DMC) for biodegradation of Maya crude oil fractions. The first degrading culture, DMC A was made up with 10 strains. Design of DMC B (six strains) and DMC C (three strains) was based on DGGE profiles obtained throughout biodegradation assays of different petroleum fractions. Biodegradation of the aliphatic fraction (10 000 mg l⁻¹) and an aromatic–polar mixture (5000 mg l⁻¹) was evaluated for the DMC B. Biodegradation of total hydrocarbons (10 000 mg l⁻¹) and its fractions was evaluated for DMC B and DMC C. During biodegradation assays, O₂ consumption and CO₂ production were assessed by respirometry, while population dynamics of predominant strains was based on PCR-DGGE profiles of partial 16S rDNA. Aliphatic fraction was completely biodegraded by DMC B, while degradation of the aromatic–polar mixture was 12.5% and for total hydrocarbons 40.5%. DMC B was able to degrade the aromatic fraction (31%) and even the polar fraction (19.6%) present in total hydrocarbons. DMC C degraded the aromatic and polar fractions (5.6% and 2%, respectively) present in total hydrocarbons. DGGE profiles of the DMCs indicated that Pseudomonas sp., Gordonia rubripertincta and a non-identified strain were predominant and probably responsible of the hydrocarbons biodegradation. The use of DGGE-fingerprinting to track microbial populations, allowed selecting strains to design efficient oil-degrading defined mixed cultures.     

Survey of petroleum-degrading bacteria in coastal waters of Sunderban Biosphere Reserve

A survey of petroleum-degrading bacteria was carried out in the Indian part of deltaic Sunderbans to evaluate the distribution of the naturally occurring petroleum-degrading aerobic bacteria. Bacteriological analysis of surface water samples collected from five different locations in the Hooghly–Matla river mouth showed that, depending on the location, 0.08–2.0% of the heterotrophic bacteria culturable in marine agar medium could degrade crude petroleum hydrocarbons as the sole source of carbon. In the entire study area, the number of heterotrophic bacteria ranged from 1 × 10³ to 3.8 × 10⁵ c.f.u/ml, amongst which 2.7 × 10¹ to 6 × 10³ c.f.u/ml were petroleum degraders. There was a maximum number of petroleum-degrading bacteria in the waters of Haldia Port and its surrounding areas, where the water is highly polluted by hydrocarbon discharges from a nearby oil refinery and from the ships docking at the port. Among the isolates, identified on the basis of their Gram reaction, morphological and biochemical tests including the use of API20E strips, Pseudomonas, Mycobacterium, Klebsiella, Acinetobacter, Micrococcus, and Nocardia were the most common petroleum degraders. Other heterotrophic bacteria included several species of Escherichia, Klebsiella, non-oil-degrading Pseudomonas, Vibrio, Streptococcus, Staphylococcus and Bacillus. Following preliminary selection, five strains, showing best growth in medium with oil fraction as sole carbon source, were chosen for estimation of the efficiency of crude oil biodegradation. The selected strains belonged to Pseudomonas (two strains), Mycobacterium (two strains), and Nocardia (one strain). These strains degraded 47–78% of Arab-Mix crude oil over a period of 20 days. The best oil-degrading isolate, a strain of Pseudomonas aeruginosa, (BBW1), was found to degrade and multiply more rapidly in crude oil than the rest. BBW1 showed profuse growth in Bushnell Haas medium containing crude oil (as sole source of carbon) at high concentrations ranging from 0.2 to 20% (v/v), with optimum at 10%. As much as 75% of the oil was degraded within 72 h of incubation with the bacteria. Physicochemical analysis showed considerable decrease in initial boiling point and carbon residue of the degraded oil. The ability to degrade crude oil was found to be associated with a single 70-kb plasmid, pBN70. Resistance to the metals Mn²⁺ (50 mM), Mg²⁺ (200 mM), Zn²⁺ (6 mM), Ni²⁺ (10 mM) and antibiotics like ampicillin (10 g/ml), cephalexin (30 g/ml), nitrofurantoin (300 g/ml) and penicillin (10 U/ml) were plasmid-mediated.     

The destruction of methicillin by soil bacteria

Summary The stability in the soil of a new penicillin (methicillin) which is resistant to staphylococcal penicillinase, has been investigated. The results revealed its inactivation in both sterile and non-sterile soils of p _H 7.4–7.6, with indication of biological inactivation in the latter.Three strains identified as Pseudomonas spp., were isolated by enrichment technique from the soil, and were found able to inactivate methicillin through production of an exocellular enzyme destructable at 90°C. Such an enzyme proved to be a type of penicillinase that inactivated benzyl penicillin more actively than methicillin.     

A hyper-thermostable, alkaline lipase from Pseudomonas sp. with the property of thermal activation

A hyper-thermostable, alkaline lipase from a newly-isolated, mesophilic Pseudomonas sp. was optimal at pH 11 and at 90 °C. It had a half-life of more than 13 h at 90 °C. It was activated by 30% when heated at 90 °C for 2 h. The enzyme had a greater affinity for mustard oil (K _m=40 mg ml^–1) than for olive oil (K _m=140 mg ml^–1).     

Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation

Among 25 crude oil-degrading bacteria isolated from a marine environment, four strains, which grew well on crude oil, were selected for more study. All the four isolated had maximum growth on 2.5% of crude oil and strain BC (Pseudomonas) could remove crude oil by 83%. The drop collapse method and microtiter assay show that this strain produces more biosurfactant, and its biofilm formation is higher compared to other strains. Bacterial adhesions to crude oil for strains CS-2 (Pseudomonas), BC, PG-5 (Rhodococcus) and H (Bacillus) were 30%, 46%, 10% and 1%, respectively. Therefore, strain H with a low production of biosurfactant and biofilm formation had showed the least growth on these compounds. PCR analysis of these four strains showed that all isolates had alk-B genes from group (III) alkane hydroxylase. All isolate strains could utilize cyclohexan, octane, hexadecane, octadecan and diesel fuel oil; however, the microtiter plate assay showed that strain BC had more growth, respiration and biofilm formation on octadecan.     

Effects of temperature and holding time on production of renewable fuels from pyrolysis of Chlorella protothecoides

To investigate the influence of temperature andholding time on the pyrolyzate yields of Chlorella protothecoides, the microalgal cells weresubjected to pyrolysis at 200, 300, 400, 500 and 600 ^°Cfor 5, 20, 60 and 120 min, separately. High oil yields above 40% dry weight cells wereobtained both at relatively low temperature (300 ^°C)with relatively long holding times (20–120min) and relatively high temperatures (400–500 ^°C)with relatively short holding times (5–20min). The maximum oil yield of 52.0% was achieved at500 ^°C for 5 min. The gas yield was generallyincreased with the increasing temperature and holdingtime. It could reach 63.3–76.0% at 600 ^°C.High pyrolytic rates of 72–87% were obtained at allexperiments except at 200 ^°C for 5–20 min or300 ^°C for 5 min. Thermogravimetric analysisindicated that the main thermal degradation of thismicroalga occurred at 200–520 ^°C. The resultsimply that C. protothecoides is a good candidatefor the production of renewable fuels by pyrolysis.     

Storage of pollen grains of Crotalaria retusa in oils

Summary Attempts were made to store pollen grains of Crotalaria retusa L. in a mineral oil (paraffin oil) and two vegetable oils (soybean oil and olive oil). Under laboratory conditions pollen grains not stored in oil lost in vitro germinability within 15–30 days, while those stored in oils maintained some degree of germinability even after 60 days. Pollen samples stored in oils at –20° C did not show any decline in germinability or pollen tube vigour even after 6 months of storage. The results amply demonstrate the feasibility of using oils for short- and long-term pollen storage.