OPTIMIZED PRODUCTION OF BIOSURFACTANT BY PseudomonasaeruginosaAND ITS APPLICATION IN BIOREMEDIATION OF CRUDE OIL/HEAVY METAL POLLUTED SOIL IN OKARKI, RIVER STATE

SOURCE:

Faculty: Biosences
Department: Applied Microbiology And Brewing

CONTRIBUTORS:

Anaukwu, C.G;
Ekwealor,, I,A;

ABSTRACT:

Crude oil and heavy metal pollution of the lithosphere has become a serious environmental problem in Nigeria since the past decade. They are associated with severe health and ecological problems, therefore, safe and effective techniques for reclamation of such soils are needed. In this study, optimized production of biosurfactant by Pseudomonas aeruginosaand its application in bioremediationof crude oil/heavy metal-polluted soil were carried out. Bacterial organisms capable of producing biosurfactants were isolated from spent-engine oil-polluted soil and screened for biosurfactant production on mineral salts medium. “One factor at a time” method of optimization was used to screen various nitrogen sources and waste materials for use as carbon sources for the production of biosurfactant by the organisms. Quadratic response models for optimum biosurfactant production by the isolatesbased on response surface methodology were developed.Surface tension reduction ability and emulsification activities of the biosurfactants were determined. Functional components of the biosurfactants were examined using gas chromatography-mass spectrometry (GC-MS). Physicochemical and microbiological profile of the crude oil/heavy metal-polluted soil samples collected from Okarki, Ahoada LGA, River State were analyzed. Gravimetric crude oil degradation analysis was carried out on the indigenous isolates.Bioremediation was carried out on the polluted soil for a period of 12 weeks, and techniques used include natural attenuation, bioaugmentation and biostimulation. A total of seventeen bacterial isolates were tested for biosurfactant production and the three active producers namely Pseudomonas aeruginosaCCUG, Pseudomonas aeruginosaI3 and Pseudomonas aeruginosaST11 were identified based on 16S rRNA sequencing. Sugar cane molasses and sodium nitrate were the carbon and nitrogen of choicerespectively for biosurfactant production byPseudomonas aeruginosastrains. Optimum conditions for some process variables for biosurfactant production in 250ml flask were 20g/L cane molasses, 5g/L NaNO3, 1.93ml inoculum size, 60ml medium volume for P. aeruginosa CCUG; 25g/L of molasses, 25g/L of NaNO3, 1ml inoculum and 60ml medium volume for P.aeruginosa strain I3 and 16.92g/L molasses, 15.51g/L NaNO3, 1.93ml inoculum and 41.4ml medium volume for P.aeruginosa ST11. Analysis of variance revealed that the quadratic response models were significant(p-value < 0.05).The biosurfactants produced by P. aeruginosaCCUG, I3 and ST11 reduced surface tension of water from 72mN/m to 45mN/m, 55mN/m and 42.3mN/m respectively, and showed emulsification activities with the hydrophobic substrates tested. Biosurfactants from P. aeruginosaI3 and ST11 showed positive results for glycolipid type biosurfactant while P. aeruginosaCCUG showed a negative result. GC-MS of the biosurfactants revealed the presence of various organic compounds likecyclotetrasiloxane, methyl stearate, octadecanoic acid, cyclododecanol, tert-butyl isopropyl disulphide, 9-octadecenoic acid, n-hexadecanoic acid, trimyristin and sulphuric acid. Physicochemical analysis showed that the polluted soil had a pH of 5.8± 0.01 while the control soil pH was 6.5± 0.03. The conductivity, total nitrate, total phosphate, total organic carbon and total petroleum hydrocarbon contents were relatively higher in the polluted soil. Heavy metals present in the polluted soil werearsenic (0.13±0.00 mg/kg), lead (0.34±0.00 mg/kg), mercury (1.56±0.04 mg/kg), cadmium (0.18±0.00 mg/kg), and chromium (0.57±0.00 mg/kg). Dehydrogenase enzyme activity was higher in the control soil than in the polluted soil. The culture-dependent microbiological profile showed the presence of microorganisms belonging to the genera Staphylococcus, Citrobacter, Micrococcus, Pseudomonas, Bacillus, Corynebacterium, Aspergillus and Penicillium in the polluted soil, whileStaphylococcus, Micrococcus, Bacillus, Streptococcus,Lactobacillus, Serratia, Pseudomonas, Fusarium, Aspergillus and Penicilliumwere in the control soil. Heterotrophic bacterial and fungal counts in the polluted soil were 2.57±0.01LogCfu/g and 2.38±0.07 LogCfu/g respectively, while those of the control soil were 5.65±0.03 LogCfu/g and 5.26±0.04 Log Cfu/g respectively.Metagenomic analysis showed the presence of 3.13% and 1.08% archaeal population and 96.87% and 98.92% bacteria in the polluted and control soil respectively. Among the indigenous organisms tested for crude oil degradation in shake flask,Bacillus subtilisgave the highest crude oil degradation (98.8 ± 0.7%)andMicrococcus sp. gave the least (6.7± 2.3%). During bioremediation of the polluted soil, increase in bacterial and fungal counts were observed, dehydrogenase enzyme activities were improved and the pH of the soil fluctuated throughout the period. The bioremediation results showed thatgreater than 85% of the crude oil were degradedin bioaugmentation and biostimulation treatments and approximately 50%in natural attenuation. There was significant difference in the performances of all the treatments (p-value< 0.05). Heavy metal removal ranged from 3.7 – 100% in all the treatments and it correlated positively with crude oil degradation. Crude oil degradation strongly correlated positively with bacterial count and dehydrogenase enzyme activity. Therefore, the stimulation of the indigenous organisms in the soil with biosurfactant and augmentation with crude oil degraders can be effective at removing crude oil and heavy metals from polluted soil.