scholarly journals Molecular Identification of Crude Oil-Degrading Bacteria and Screening for Catechol 2, 3 Dioxygenase (C23O) Gene

2020 ◽  
pp. 1-14
Author(s):  
Olukunle, Oluwatoyin Folake
Keyword(s):  
Crude Oil ◽  
Degenerate Primers ◽  
Polluted Soils ◽  
Gene Encoding ◽  
Bacterial Gene ◽  
Colony Pcr ◽  
Conserved Sequence ◽  
Degrading Bacteria ◽  
Efficiency And Effectiveness ◽  
Rivers State

Aims: To identify crude oil-degrading bacteria isolated from polluted soils and waters and screen the presence of catechol 2, 3 dioxygenase (C23O) gene encoding oil-degradation in the strains with the highest degradative activity. Study Design: Laboratory-experimental design was used in this study. Place and Duration of Study: Crude oil polluted soils and waters were collected from Awoye, Mese and Oluwa villages in Ondo State, Nigeria and three different flow stations (Agbada-Aluu shell, Obite, and Bonny) in Rivers State, Nigeria. Methodology: The identities of the isolates were confirmed by extracting their total genomic DNA using standard DNA protocols while a portion of 16S bacterial gene of their DNA was amplified by polymerase chain reaction (PCR) using the primers E9F and U1510R and sequenced using Sanger method. Degenerate primers were used to isolate and identify the gene encoding C23O, responsible for the degradation of oil. Molecular cloning of the gene was done by transforming into Escherichia coli DHα. The correct inserts from the selected clones were performed by colony PCR. The isolated gene was sequenced with a Dye terminator sequencing kit and the product was analyzed with Prism DNA sequencer. Results: The results obtained from the conserved sequence of the 16S rRNA coupled with the nucleotide sequence revealed ten (10) crude oil-degrading bacteria, with CFfab 14 and CFfab 12 having the highest and lowest degrading activity of  78.92 ± 0.9 Unit/mL/h and 43.89 ± 1.3 Unit/mL/h on day 3 respectively. Conclusion: The gene C23O responsible for the production of catechol 2, 3 dioxygenase was isolated from strains CFfab 5, CFfab 14 and CFfab 15. The nucleotide base sequence of the gene was determined to be 238 bp. It is expected that in bioremediation, indigenous microorganisms from polluted environments should be screened for the possible existence of this unique gene sequence for effectiveness. Further studies could be conducted on the possibility of cloning this C23O gene into other bacteria for more efficiency and effectiveness in the bioremediation process.

scholarly journals Bioremediation Potential of Native Hydrocarbons Degrading Bacteria in Crude Oil Polluted Soil

2017 ◽  
Vol 74 (1) ◽  
pp. 19 ◽  
Author(s):  
Mariana MARINESCU ◽  
Anca LACATUSU ◽  
Eugenia GAMENT ◽  
Georgiana PLOPEANU ◽  
Vera CARABULEA
Keyword(s):  
Crude Oil ◽  
Contaminated Soil ◽  
Petroleum Hydrocarbon ◽  
Polluted Soil ◽  
Total Petroleum Hydrocarbon ◽  
Polluted Soils ◽  
Degrading Bacteria ◽  
Bacterial Inoculum ◽  
Native Crude ◽  
Petroleum Pollutants

Bioremediation of crude oil contaminated soil is an effective process to clean petroleum pollutants from the environment. Crude oil bioremediation of soils is limited by the bacteria activity in degrading the spills hydrocarbons. Native crude oil degrading bacteria were isolated from different crude oil polluted soils. The isolated bacteria belong to the genera Pseudomonas, Mycobacterium, Arthrobacter and Bacillus. A natural biodegradable product and bacterial inoculum were used for total petroleum hydrocarbon (TPH) removal from an artificial polluted soil. For soil polluted with 5% crude oil, the bacterial top, including those placed in the soil by inoculation was 30 days after impact, respectively 7 days after inoculum application, while in soil polluted with 10% crude oil,  multiplication top of bacteria was observed in the determination made at 45 days after impact and 21 days after inoculum application, showing once again how necessary is for microorganisms habituation and adaptation to environment being a function of pollutant concentration. The microorganisms inoculated showed a slight adaptability in soil polluted with 5% crude oil, but complete inhibition in the first 30 days of experiment at 10% crude oil.

scholarly journals Bioremediation Efficiency of Bacillus amyloliquefaciens and Pseudomonas aeruginosa with the Nutrient Amendment on Crude Oil Polluted the Soil

2019 ◽  
pp. 1-13
Author(s):  
David N. Ogbonna ◽  
Renner R. Nrior ◽  
Festus E. Ezinwo
Keyword(s):  
Crude Oil ◽  
Bacillus Amyloliquefaciens ◽  
Heterotrophic Bacteria ◽  
Polluted Soil ◽  
Total Petroleum Hydrocarbon ◽  
Polluted Soils ◽  
Fish Waste ◽  
Goat Manure ◽  
Nutrient Amendment ◽  
Rivers State

Aim: To assess the Bioremediation efficiency of Bacillus amyloliquefaciens and Pseudomonas aeruginosa strain CL 9 with nutrient amendment using bio-stimulating agents such as Fish waste and Goat manure on crude oil polluted soils in Rivers State, Nigeria. Study Design: The study employs experimental design, statistical analysis of the data and interpretation. Place and Duration of Study: A portion of Rivers State University demonstration farmland in Nkpolu-Oroworukwo, Mile 3 Diobu area of Port Harcourt, Rivers State was used for this study. The piece of land is situated at Longitude 4°48’18.50’’N and Latitude 6o58’39.12’’E measuring 5.4864 m x 5.1816 m with a total area of 28.4283 m2. Bioremediation monitoring lasted for 56 days, analysis carried out weekly (per 7 days interval). Methodology: Seven (7) experimental plots were employed using a Randomized Block Design each having dimensions of 100 x 50 x 20 cm (Length x Breadth x Height) were formed and mapped out on agricultural soil and left fallow for 6 days before contamination on the seventh day; after which it was allowed for 21 days for proper contamination and exposure to natural environmental factors to mimic crude oil spill site. Thereafter bio stimulating agents usually referred to as nutrient amendment organics in this study (fish waste and goat manure) and bio-augmenting microorganisms were applied. Soil profile before and after contamination was assayed while parameters like Nitrate, Sulphate, Phosphate, Total Organic Carbon (TOC) and Total Petroleum Hydrocarbon (TPH), were monitored throughout the experimental period. Microbial analyses such as Total Heterotrophic Bacteria (THB), Total Heterotrophic Fungi (THF), Hydrocarbon Utilizing Bacteria (HUB) and Hydrocarbon Utilizing Fungi (HUF) were recorded. Bioremediation efficiency was estimated from percentage (%) reduction of Total Petroleum Hydrocarbon (TPH) from day 1 to the residual hydrocarbon at day 56 of bio augmented/ biostimulation plots with the control. Results: Results revealed amount of remediated hydrocarbon and % Bioremediation efficiency at 56 days in the different treatment plots (initial TPH contamination value of  9296.83  mg/kg) in a decreasing order as follows: PS+Bac+Pse+GF+FW (8032.825 mg/kg; 86.40%) >PS+GF+FW (6867.825 mg/kg; 73.87%) >PS+Bac+Pse (6587.825mg/kg; 70.86%) >PS+FW (6441.825mg/kg; 69.29%) >PS+GF (5909.825 mg/kg; 63.57%) >CTRL 2 (Polluted soil without amendment) (3604.825mg/kg; 38.78%). Microbiological results showed increased colonial values with increase time exposure. The results observed on day 56 indicate that Polluted soil + Bacillus + Pseudomonas (10.11 Log10 CFU/g) > Polluted soil but un-amended soil (8.76 Log10 CFU/g) > unpolluted soil (8.68 Log10 CFU/g). Comparatively, Polluted soil +Bacillus + Pseudomonas expressed higher heterotrophic bacteria of 9.77 and 9.67 Log10 CFU/g while fungal counts recorded 6.04 and 6.82 Log10 CFU/g. Conclusion: Study showed that bioremediation of crude oil-polluted soils with bacteria singly is less effective but a combination with other organic nutrients is a better palliative measure. Therefore, amendment with organic nutrients like Goat manure and Fish wastes is recommended for crude oil polluted soils due to its high nutrient content as substrates for biostimulation of indigenous and augmenting biodegrading microbes. This process could be a source of enhanced natural attenuation of oil-contaminated environments in Nigeria.

scholarly journals Effects of Dispersants and Biosurfactants on Crude-Oil Biodegradation and Bacterial Community Succession

2021 ◽  
Vol 9 (6) ◽  
pp. 1200
Author(s):  
Gareth E. Thomas ◽  
Jan L. Brant ◽  
Pablo Campo ◽  
Dave R. Clark ◽  
Frederic Coulon ◽  
...  
Keyword(s):  
Crude Oil ◽  
Early Stage ◽  
Niche Partitioning ◽  
Hydrocarbon Biodegradation ◽  
Hydrocarbonoclastic Bacteria ◽  
Bacterial Response ◽  
Oil Biodegradation ◽  
Interfacial Surface ◽  
Degrading Bacteria ◽  
Oil Spill Response

This study evaluated the effects of three commercial dispersants (Finasol OSR 52, Slickgone NS, Superdispersant 25) and three biosurfactants (rhamnolipid, trehalolipid, sophorolipid) in crude-oil seawater microcosms. We analysed the crucial early bacterial response (1 and 3 days). In contrast, most analyses miss this key period and instead focus on later time points after oil and dispersant addition. By focusing on the early stage, we show that dispersants and biosurfactants, which reduce the interfacial surface tension of oil and water, significantly increase the abundance of hydrocarbon-degrading bacteria, and the rate of hydrocarbon biodegradation, within 24 h. A succession of obligate hydrocarbonoclastic bacteria (OHCB), driven by metabolite niche partitioning, is demonstrated. Importantly, this succession has revealed how the OHCB Oleispira, hitherto considered to be a psychrophile, can dominate in the early stages of oil-spill response (1 and 3 days), outcompeting all other OHCB, at the relatively high temperature of 16 °C. Additionally, we demonstrate how some dispersants or biosurfactants can select for specific bacterial genera, especially the biosurfactant rhamnolipid, which appears to provide an advantageous compatibility with Pseudomonas, a genus in which some species synthesize rhamnolipid in the presence of hydrocarbons.

A short conserved sequence is involved in the light-inducibility of a gene encoding ribulose 1,5-bisphosphate carboxylase small subunit of pea

Nature ◽  
1985 ◽  
Vol 315 (6016) ◽  
pp. 200-204 ◽  
Author(s):  
Giorgio Morelli ◽  
Ferenc Nagy ◽  
Robert T. Fraley ◽  
Stephen G. Rogers ◽  
Nam-Hai Chua
Keyword(s):  
Small Subunit ◽  
Gene Encoding ◽  
Bisphosphate Carboxylase ◽  
Conserved Sequence ◽  
Light Inducibility

scholarly journals CRUDE OIL BIOREMEDIATION BY INDIGENOUS BACTERIA ISOLATED FROM OILY SLUDGE

2016 ◽  
Vol 78 (11-2) ◽  
Author(s):  
Nur Hafizah Azizan ◽  
Kasing Ak Apun ◽  
Lesley Maurice Bilung ◽  
Micky Vincent ◽  
Hairul Azman Roslan ◽  
...  
Keyword(s):  
Crude Oil ◽  
Enrichment Culture ◽  
Molecular Techniques ◽  
Oily Sludge ◽  
Minimal Salt Medium ◽  
Alkane Degradation ◽  
Growth Study ◽  
The Third ◽  
Cell Counts ◽  
Degrading Bacteria

Enrichment culture technique leads to the discovery of six presumptive TPH-degrading bacteria. Identification and characterization tests using morphological, biochemical and molecular techniques have successfully isolated Pseudomonas aeruginosa (UMAS1PF), Serratia marcescens (UMAS2SF) and Klebsiella spp. (UMAS3KF). All strains were able to use crude oil as sole carbon and energy source for their growth since they were able to survive in Minimal Salt medium supplemented with 1% (v/v) crude oil. Growth study showed that they produced the highest cell counts on the third or fourth day by 108 – 1011 CFU/ml. Six artificial consortium inoculums have been produced from the growth study. Gas chromatography analysis showed that all isolates had the ability to degrade aliphatic hydrocarbon with 100% degradation of nC19 – C24. Among the isolates, UMAS2SF was the best and fastest n-alkane degrader with degradation percentage between 55 – 90% of n-C14 – C18 in 14 days. This was followed by UMAS1PF and UMAS3KF with 11 – 82% and 1.3% degradation, respectively. Enhancement study showed that plot with inoculum and NPK addition successfully enhanced n-alkane degradation. Plot A2:B3+NPK degraded n-alkane the fastest followed by plot treated by C+NPK, A1:B2, B+NPK and A2:B3. Result showed that UMAS1PF was the best PAHs degrader as most of the high molecular weight PAHs was degraded. In the enhancement study, the plot amended with A2:B3 showed the highest PAHs degradation, followed by plots A1:B2, A3:B1:C2 and A1:C3 that was assigned as the third, fourth and fifth best in mineralizing PAHs, respectively.

scholarly journals Biotreatability of Crude Oil Polluted Aquatic Environment Using Indigenous Hydrocarbon Utilizing Bacteria

2020 ◽  
pp. 52-62
Author(s):  
Tudararo-Aherobo Laurelta ◽  
Okotie Sylvester ◽  
Ataikiru Tega ◽  
Stephen Avwerosuoghene
Keyword(s):  
Crude Oil ◽  
Contaminated Soils ◽  
Aquatic Environment ◽  
Bacterial Counts ◽  
A Value ◽  
Degrading Bacteria ◽  
Petroleum Resources ◽  
Oil Concentration ◽  
American Society ◽  
Hydrocarbon Utilizing Bacteria

Aim: The research aims to assess the biodegradability of crude oil polluted aquatic environment using indigenous hydrocarbon degrading bacteria. Place and Duration of Study: The research was conducted in the Environmental Management and Toxicology Laboratory, Federal University of Petroleum Resources, Effurun, Delta State. Methodology: Hydrocarbon degrading bacteria species were isolated from hydrocarbon contaminated soils, screened and used for the degradation of crude oil. 5% and 10% crude oil were used to spike the test microcosm. Physicochemical parameters such as, pH, turbidity, total petroleum hydrocarbon (TPH) and bacterial counts of the bioremediated crude oil contaminated water were monitored on Day 0, 7 and 14. The biodegradation of the crude oil was done with the various bacteria isolates singly and as a consortium. Standard methods of American Public Health Association (APHA) and American Society for Testing and Materials (ASTM) were used for the analysis. Results: The isolates identified and used for the biodegradation process were, Azomonas sp., Enterococcus sp., Klebsiella sp. and Rhizobactersp. On day 14, in the microcosms with 5% crude oil contamination, Azomonas sp. recorded the highest turbidity reading of 328 ± 2.0 NTU, while Rhizobacter sp. recorded the least with 57.67 ± 0.58 NTU. The bacterial countswere between 7.68 ± 0.002 CFU/ml and 8.05 ± 0.10x 107 CFU/ml for Rhizobacter sp. and Azomonas sp. respectively.The crude oil was also degraded most in the microcosm treated with Azomonas sp. with a residual TPH concentration of 0.0013± 0.005 mg/l.For the 10% crude oil contaminated microcosms, TPH was also biodegraded most by Azomonas sp. with a value of 0.0026 ± 0.002mg/l. Turbidity readings were between 82 ± 1.0 NTU and 375.33 ± 0.57 NTU for Rhizobacter sp. and Azomonas sp. respectively. Bacterial counts were between (7.71± 0.012)x 107CFU/ml – (8.13± 0.001) x 107CFU/ml for Rhizobacter sp. and Azomonassp. respectively. Conclusion:There wasincreased microbial countsand decrease of residual crude oil concentration, indicating degradation of the crude oil by all the isolates.However, Azomonas sp. recorded the highest TPH degradation for both the 5% and 10% crude oil contaminated microcosms.Thus, findings from the research indicate that hydrocarbon degrading bacteria exist in our environment and can be used in the remediation of aquatic polluted environment.

scholarly journals Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase

1998 ◽  
Vol 36 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Claire Poyart ◽  
Gilles Quesne ◽  
Stephane Coulon ◽  
Patrick Berche ◽  
Patrick Trieu-Cuot
Keyword(s):  
Superoxide Dismutase ◽  
Pcr Assay ◽  
Degenerate Primers ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Type Strains ◽  
Internal Fragment ◽  
Rrna Sequences ◽  
16S Rrna Sequences

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodAint ) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus,S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus,S. canis, S. cricetus, S. downei,S. dysgalactiae, S. equi subsp.equi, S. equi subsp. zooepidemicus,S. equinus, S. gordonii, S. iniae,S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis,S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis,S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodAint fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of theirsodAint fragments on the phylogenetic tree of the sodAint fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

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