Oluwafemi S. Obayori

Microbial population changes in tropical agricultural soil experimentally contaminated with crude petroleum

Impacts of crude petroleum pollution on the soil environment and microbial population dynamics as well as recovery rates of an abandoned farmland was monitored for seven months spanning the two major seasons in Nigeria with a view to establishing process conditions necessary for development of effective strategies for bioremediation. The physico-chemistry of the control and contaminated soils differed just significantly (P soil showed significant diversity in structure and number of flora .There was an initial drop in microbial population densities at the onset of pollution but, a gradual increase was observed thereafter. Higher counts of microflora were obtained for April, May, June and July samples which coincided with the onset and peak of wet season. A rapid and significant reduction in residual oil concentration was observed during this period. Overall, nearly 100% of the crude oil pollutant was degraded within the 28-week study period. The residual oil concentration gave a high but negative correlation coefficient (r = - 0.84 to -0.90) with total heterotrophic and hydrocarbon-utilizing populations. On application of data generated to model equations, approximately 60.5 weeks would elapse before the contaminated soil could recover from the impact of the oil. Our results show that a natural population readily able to degrade crude oil is present in the soil chosen for this study. However, it may be necessary to monitor the level of inorganic nutrients and adjust some appropriately to enhance biodegradation of the organic pollutant.

Biodegradation of petroleum hydrocarbons in the presence of nickel and cobalt.

Bioremediation of environments co‐contaminated with hydrocarbons and heavy metals often pose a challenge as heavy metals exert toxicity to existing communities of hydrocarbon degraders. Multi‐resistant bacterial strains were studied for ability to degrade hydrocarbons in chemically defined media amended with 5.0 mM Ni2+, and Co2+. The bacteria, Pseudomonas aeruginosa CA207Ni, Burkholderia cepacia AL96Co, and Corynebacterium kutscheri FL108Hg, utilized crude oil and anthracene without lag phase at specific growth rate spanning 0.3848–0.8259 per day. The bacterial populations grew in hydrocarbon media amended with nickel (Ni) and cobalt (Co) at 0.8393–1.801 days generation time (period of exponential growth, t = 15 days). The bacteria degraded 96.24–98.97, and 92.94–96.24% of crude oil, and anthracene, respectively, within 30 days without any impedance due to metal toxicity (at 5.0 mM). Rather, there was reduction of Ni and Co concentrations in the axenic culture 30 days post‐inoculation to 0.08–0.12 and 0.11–0.15 mM, respectively. The metabolic functions of the bacteria are active in the presence of toxic metals (Ni and Co) while utilizing petroleum hydrocarbons for increase in biomass. These findings are useful to other baseline studies on decommissioning of sites co‐contaminated with hydrocarbons and toxic metals.

Biodegradation of Fresh and Used Engine Oils by Pseudomonas aeruginosa LP5

Pseudomonas aeruginosa strain LP5, a hydrocarbon degrader isolated from petroleum contaminated soil based on its ability to grow on pyrene, was used to degrade two different grades of fresh and used engine oil (SAE 40W and SAE 20W 50) in liquid cultures. The organism degraded more than 90 percent of all oil types within 21 days with values of 95%, 93%, 96% and 92% for fresh SAE 40W, used SAE 40W, fresh SAE 20W 50 and used SAE 20W 50 respectively. Similarly, growth rates were slightly higher in the fresh oils with values of 0.17, 0.13, 0.14 and 0.13 d-1 recorded respectively for fresh SAE 40W, used SAE 40W, fresh SAE 20W 50 and used SAE 20W 50. Significantly higher initial rates of degradation of 177.42 mg l-1d-1 and 207.14 mgl-1d-1 were recorded for fresh SAE 40 and SAE 20W 50 in the first 21 days compared to the values for the used oil counterparts, which were 73.23 mgl-1d-1 for SAE 40 and 74.37 mgl-1d-1 for SAE 20W 50. In the used oils, lower peaks that were not present at Day 0 reappeared on Day-21. All peaks greater than C20 also disappeared. In all cases, it was the medium fraction ranges of C14, C15, and C17 that remained with discernible peaks on Day 21, albeit at very low heights of less than 10% of Day 0 values. Thus whereas degradation rates differ on oil types and fresh oil appear more amenable to early degradation, the organism Pseudomonas aeruginosa LP5 showed remarkable potential for use in degradation of both fresh and used engine oils.

Degradation of weathered crude oil (Escravos Light) by bacterial strains from hydrocarbons-polluted site

7cfu/ml, while for LG2 it was 1.63 x 10 7 cfu/ml. In the two cases slight but consistent pH drops to < 6.51 were observed. LG1 had a growth rate of 0.38 d -1 and doubling time 1.83 d. The corresponding values for LG2 during the first log phase were 0.58 d -1 and 1.19 d, respectively, while in the second log phase the values were 0.14 d -1 and 5.13 d Gas chromatographic analysis showed that chrysene was degraded by LG and LG2 by 92.91 and 80.25%, respectively, anthracene by 92.21 and 91.53%, respectively, and Benzo (b) fluoranthene was degraded by 77.19 and 98.41%, respectively. LG2 had a higher degradative capability of the polyaromatic fractions with 80.15% while LG I was lower at 52.33%. Statistical analysis showed that there was a significant difference (p < 0.05) in the degradation of various aromatic components of the oil by LG1 and LG2.

Extensive Biodegradation of Nigerian Crude Oil (Escravos Light) by Newly Characterized Yeast Strains

Abstract Because microbial degradation is known to be an efficient process in the in situ decontamination of oil-bearing environments, it is believed that development of effective bioremediation strategies will be aided by microbial sourcing of novel and competent hydrocarbon degraders with a broad and unusual substrate spectrum. Thus, in keeping with this objective, two Candida strains (MN1 and MC1) isolated after a repeated batch enrichment technique were tested for their biodegradation potentials on Nigerian crude oil, Escravos light. Axenic cultures of strains MN1 and MC1 grew at a rate of 1.623 and 0.586 d−1, respectively, in mineral salts medium supplemented with 8.4 g L−1 of crude oil. Whereas strain MN1 degraded aliphatic fractions by 97.6% and the aromatics by 74.61%, the corresponding values obtained for MC1 were 97.2% and 67.29% during the 14-day incubation period. The gas chromatography (GC) fingerprinting of aliphatic fractions showed major degradation of heptadecane (C17), octadecane (C18), nonadecane (C19), eicosane (C20), undodecane (C21), tricosane (C23), hexacosane (C26), octacosane (C28), and nonacosane (C29) in less than 6 days, whereas nearly 100% of these fractions including the isoprenoid molecules was metabolized in 14 days. Among the aromatic fractions that were nearly eliminated during the cultivation period were naphthalene, phenanthrene, fluoranthrene, chrysene, benzo(a)anthracene, benzo(b)fluoranthrene, and benzo(a)pyrene. Interestingly, substrate uptake studies showed that both strains grew very well on petroleum cuts, biphenyl, phenol, xylene, and quite a number of polycyclic aromatic hydrocarbons including pyrene, phenanthrene, and anthracene.

Biodegradation of Used Engine Oil by a Methylotrophic Bacterium, Methylobacterium Mesophilicum Isolated from Tropical Hydrocarbon-contaminated Soil

A Gram-negative facultatively methylotrophic bacterium putatively identified as Methylobacterium mesophilicum strain RD1 displayed extensive degradative ability on used engine oil (SAE 40W) in liquid cultures. The rate of degradation of used engine oil (1274.85 mg L−1) by the isolate, for the first 12 days and the last 9 days were 65 and 40 mg L−1 d−1, respectively. Gas chromatographic (GC) analyses of residual used engine oil revealed that 61.2% and 89.5% of the initial concentration of the used engine oil were degraded within 12 and 21 days. GC fingerprints of the used engine oil after 12 days of incubation showed total disappearance of C15, C23, C24, C25, and C26 hydrocarbon fractions as well as drastic reductions of C13, C14, C16, and PAHs fractions such as C19-Anthracene and C22-Pyrene. At the end of 21 days’ incubation, total disappearance of C17-pristane, C22-pyrene, one of the C19-anthracene, and significant reduction of C18-phytane (96.8%) fractions were observed. In addition, less than 10% of Day 0 values of medium fraction ranges C13, C14, and C16 were discernible after 21 days. This study has established the potential of Methylobacterium mesophilicum strain RD1 in degradation of aliphatic, aromatic, and branched alkane components of used engine oils.

Fluorene biodegradation potentials of Bacillus strains isolated from tropical hydrocarbon-contaminated soils

Two fluorene-degrading Gram-positive Bacillus strains, putatively identified as Bacillus subtilis BM1 and Bacillus amyloliquefaciens BR1 were isolated from hydrocarbon- and asphalt-contaminated soils in Lagos, Nigeria. The polluted soils have a relatively high total hydrocarbon content (16888.9 and 9923.1 mg/kg, respectively), very low concentrations of macronutrients and the total organic carbon was less than 4%. The two strains tolerated NaCl concentration of up to 7% while strain BR1 exhibited moderate growth at 10%. Shared resistance to ceftriazone and cotrimozaxole were exhibited by both strains while only strain BM1 was resistant to both amoxycilin and streptomycin. The rate of degradation of fluorene (50 mg/L) by the two isolates, after 30 days of incubation were 0.09 and 0.08 mg/L/h for strains BM1 and BR1, respectively. Gas chromatographic analyses of residual fluorene, revealed that 56.9 and 46.8% of 50 mg/L fluorene was degraded in 12 days by strains BM1 and BR1. However, after 21 days on incubation, 86 and 82% of 50 mg/L fluorene were degraded by strains BM1 and BR1, respectively. To the best of our knowledge, this is the first report highlighting flourene degradation potential of Bacillus strains isolated from tropical African environment. Keywords: Biodegradation, fluorene, hydrocarbon-contaminated soils, Bacillus spp. African Journal of Biotechnology , Vol 13(14), 1554-1559

Degradation of Spiked Pyrene and Non-pyrene Hydrocarbons in Soil Microcosms by Pseudomonas Species Isolated From Petroleum Polluted Soils

The abilities of three Pseudomonads, Pseudomonas sp. strain LP1, Pseudomonas aeruginosa LP5 and P. aeruginosa LP6 to survive and enhance the degradation of pyrene and non-pyrene hydrocarbons in soil were tested in field-moist microcosms. All three organisms were able to survive and maintain high densities > × 107 in soil. In sterilized soils inoculated with bacterial isolates, 37.34%, 50.30%, and 42.21% were degraded by LP1, LP5, and LP6, respectively. The rates of pyrene degradation in soil microcosms were 0.046, 0.041, and 0.061 mg kg−1 h−1 for LP1, LP5, and LP6, respectively. A mixture of the three isolated degraded 7.73% was degraded in sterilized soil and 87.65% in native unsterilized soil (NS). The isolates also degraded non-pyrene hydrocarbon in the soils by more than 80%. The potentials these pseudomonads isolates for use as seed for bioremediation was successfully demonstrated.

Degradation of cyclohexane and cyclohexanone by Bacillus lentus strain LP32

A Gram-positive bacterium, Bacillus lentus LP32, originally isolated on the basis of its ability to utilise pyrene as sole source of carbon was found to be able to grow luxuriantly on alicyclic compounds as sole substrates. It showed poor growth on anthracene, naphthalene, 1-naphthol and phenanthrene. Growth rate on cyclohexane was 1.32 d -1 , while doubling time was 0.76 d. The corresponding values for growth on cyclohexanone were 0.77 d -1 and 1.29 d, respectively. Within 10 days, the amount of cyclohexane in culture reduced from 317.62 to 102.55 mgl -1 , then to 23.04 mgl -1 on day 18. On cyclohexanone, substrate concentration decreased from 287.56 mgl -1 to 101.66 mgl -1 in 10 days before declining to 24.21 mgl -1 on day 18. The rate of degradation when growing on cyclohexane was 23.50 mgl -1 d -1 in the first 10 days and 9.93 mgl -1 d -1 between day 10 and day 18, with 67.71% degradation in 10 days and overall percentage degradation of 92.43%. On cyclohexanone, the corresponding values were 18.59 and 9.68 mg l -1 d -1 as well as 64.65 and 91.58%, respectively. This organism is a potential candidate for bioremediation purpose. Keywords : Degradation, cyclohexane, cyclohexanone, alicyclic compounds.

Hydrocarbon Degradation and Biosurfactant Production by an Acenaphthene-degrading Pseudomonas Species

ABSTRACT An acenaphthene-degrading bacterium putatively identified as Pseudomonas sp. strain KR3 and isolated from diesel-contaminated soil in Lagos, Nigeria was investigated for its degradative and biosurfactant production potentials on crude oil. Physicochemical analysis of the sampling site indicates gross pollution of the soil with high hydrocarbon content (2100 mg/kg) and detection of various heavy metals. The isolate grew luxuriantly on crude oil, engine oil and acenaphthene. It was resistant to septrin, amoxicillin and augmentin but was susceptible to pefloxacin, streptomycin and gentamycin. It was also resistant to elevated concentration of heavy metals such as 1–15 mM lead, nickel and molybdenum. On acenaphthene, the isolate exhibited specific growth rate and doubling time of 0.098 day−1 and 3.06 days, respectively. It degraded 62.44% (31.2 mg/l) and 91.78% (45.89 mg/l) of 50 mg/l acenaphthene within 12 and 21 days. On crude oil, the specific growth rate and doubling time were 0.375 day−1 and 1.85 days with corresponding percentage degradation of 33.01% (903.99 mg/l) and 87.79% (2403.71 mg/l) of crude oil (2738.16 mg/l) within 9 and 18 days. Gas chromatographic analysis of residual crude oil at the end of 18 days incubation showed significant reductions in the aliphatic, alicyclic and aromatic fractions with complete disappearance of benzene, propylbenzene, pristane, phytane, and nC18-octadecane fractions of the crude oil. The isolate produced growth-associated biosurfactant on crude oil with the highest emulsification index (E24) value of 72% ± 0.23 on Day 10 of incubation. The partially purified biosurfactant showed zero tolerance for salinity and had its optimal activity at 27°C and pH 2.0.