Toluene (C7H8) a hydrocarbon in crude oil, is a common contaminant in soil and groundwater. In this study, the ability to degrade toluene was investigated from twelve bacteria isolates which were isolated from soil contaminated with oil. Out of 12 bacterial isolates tested, most of Pseudomonas sp. showed the capability to grow in 1 mM of toluene compared with other isolates on the third day of incubation. Based on enzyme assays towards toluene monooxygenase, Pseudomonas aeruginosa UKMP-14T and Bacillus cereus UKMP-6G were shown to have the highest ability to degrade toluene. The toluene monoxygenase activity was analysed by using two calorimetric methods, Horseradish peroxidase (HRP) and indole-indigo. Both of the methods measured the production of catechol by the enzymatic reaction of toluene monooxygenase. In the HRP assay, the highest enzyme activity was 0.274 U/mL, exhibited by Pseudomonas aeruginosa UKMP-14T. However, for indole-indigo assay, Bacillus cereus UKMP-6G produced the highest enzyme activity of 0.291 U/ml. Results from both experiments showed that Pseudomonas aeruginosa UKMP-14T and Bacillus cereus UKMP-6G were able to degrade toluene.
Titanium dioxide nanoparticles were synthesized by using sol gel method and their physico-chemical properties were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and UV-Vis spectrophotometer. The photocatalytic property of TiO2 nanoparticles was investigated by inactivation of Escherichia coli under irradiation of fluorescent lamp. The results showed that the size of TiO2 was in the range of 3 to 7 nm with high crystallinity of anatase phase. The sharp peaks in FTIR spectrum determined the purity of TiO2 nanoparticles and absorbance peak of UV-Vis spectrum showed the energy band gap of 3.2 eV. Optimum inactivation of E. coli was obtained at 1.0 g/L TiO2 nanoparticles, with 80% of E. coli population was inactivated. The light scattering effect and insufficient concentration are the factors that cause the less effective inactivation reaction for 2.5 g/L and 0.1 g/L TiO2 concentration.
Biosurfactants are amphiphilic compounds produced by microorganisms as secondary metabolite. The unique properties of biosurfactants make them possible to replace or to be added to synthetic surfactants which are mainly used in food, cosmetics and pharmaceutical industries and in environmental applications. In this study twenty hydrocarbon-degrading bacteria were screened for biosurfactant production. All of the bacterial isolates were grown in mineral salt medium (MSM) with addition of 1% (v/v) Tapis crude oil as carbon source. The presence of biosurfactant was determined by the drop-collapse test, microplate analysis, oil spreading technique, emulsification index (%Ei24) and surface tension measurement. Only one isolate, Pseudomonas aeruginosa UKMP14T, was found to be positive for all the qualitative tests and reducing the surface tension of the medium to 49.5 dynes/cm with emulsification index of 25.29%. This isolate produced biosurfactant optimally at pH9.0 and incubation temperature of 37°C. Furthermore, P. aeruginosa UKMP14T when grown in MSM with addition of 1% (v/v) glycerol and 1.3 g/L ammonium sulphate with C/N ratio 14:1 produced biosurfactant with percentage of surface tension reduction at 55% or 30.6 dynes/cm with %Ei 24 of 43%. This percentage of surface tension reduction represents an increasing reduction in surface tension of medium by 39% over the value before optimization. This study showed that P. aeruginosa UKMP14T has the ability to biodegrade hydrocarbon and
concurrently produce biosurfactant.
Microbial communities of two oil reservoirs from Malaysia, denoted as Platform Bo and Platform Pe were studied using
culture-independent approach. Environmental DNA was extracted and the universal amplified ribosomal region (UARR)
was target amplified for both prokaryotes and eukaryotes. The amplified products were purified and cloned into pTZ57R/T
vector to construct the 16S/18S rDNA library. Restriction endocucleases HhaI and MspI were used to screen the library.
From that, 125 and 253 recombinant plasmid representative clones from Platform Bo and Platform Pe, respectively, were
sent for DNA sequencing. Twenty-six operational taxonomic units (OTUs) consist of 20 genera detected at Platform Bo
and 17 OTUs consist of 13 genera detected at Platform Pe. Marinobacter and Acinetobacter species co-occurred in both
platforms whereas the rest are site-specific. Gammaproteobacteria accounted for 86.0% of the microbial community in
Platform Bo, where OTUs affiliated to Marinobacter, Pseudomonas and Marinobacterium that were the most abundant. The
major OTUs in the Platform Pe were with affinities to Achromobacter, followed by Stenotrophomonas and Serratia. The
only archaeal isolates were detected in Platform Pe, which affiliated to Thermocladium. The singletons and doubletons
accounted for about 50.0% of the OTU abundance in both platforms, which considerably significant despite their rare
occurrence.
Bioremediation of crude oil using biostimulation and/or bioaugmentation was done by simulation study in the green house under uncontrolled environment temperature. In this study, the soil with indigenous microbes was spiked with Tapis crude oil at 200 g/kg. The microbial density of the amended soils was augmented by addition of fresh inoculum of microbial consortium which consist of Pseudomonas aeruginosa UKMP-14T, Acinetobacter baumannii UKMP-12T and seed culture two strains of fungi, Trichoderma virens UKMP-1M and Trichoderma virens UKMP-2M at ratio 1:1:1:1 (v/w). The amendment soil was added with 20% (v/w) of standardize consortium inoculum, 20% (w/w) of dried empty fruit bunch (EFB) and the effect of EFB was compared with 0.7% commercial fertilizer (v/w) which contain NPK (8:8:1). Soil with indigenous microbes was used as a control. Results showed total petroleum hydrocarbon (TPH) degradation for treatment added with NPK fertilizer was 70.36%, addition with EFB bulking agent 68.86% and addition of both NPK and EFB was 100% at day 30 of incubation. The control plot, 62% of TPH degradation was achieved after 30 days incubation.
Response surface analysis was conducted to optimize the concentrations of Tapis crude oil and duration of incubation in
order to achieve optimal microbial growth and crude oil biodegradation. Central Composite Rotatable Design (CCRD)
was employed, where the design contained 13 experimental runs with different combinations of incubation time and
crude oil concentration. The cultures containing mineral salt medium (MSM) with varying crude oil concentrations were
incubated at 30°C, pH6.5 with 150 rpm agitation for 120-336 h. The inoculum contained a consortium of previously
identified as oil degrading bacteria and fungi, namely Pseudomonas aeruginosa UKMP-14T, Acinetobacter baumannii
UKMP-12T and Trichoderma sp. UKMP-1M and UKMP-2M. The analysis showed the duration of incubation plays a significant
role (p<0.05) in affecting the bacterial growth and percentage of total petroleum hydrocarbon (TPH) biodegradation,
meanwhile concentrations of Tapis crude oil has insignificant effect on the responses. Interaction of the two variables was
found to be significant in affecting all the three responses, namely bacterial population, fungal biomass and percentage
of TPH biodegradation. It was predicted through the CCRD that the percentage of biodegradation can be optimized to
reach 86% on the 270th h when 5% (v/v) crude oil was used. This predicted value was verified to be achievable and
reproducible through validation experiments.
The microbial composition in coastal water of the Port Dickson beach in Negeri Sembilan, Malaysia was analyzed using several microbial indicators for the purpose of selecting the best indicator for marine water pollution. The indicators studied were total coliform (TC), fecal coliform (FC), fecal streptococci (FS) and coliphage. Five locations were selected along the Port Dickson beaches and samplings were carried out in 1998 and 2001. The results showed an increase in the number of total coliform (TC), fecal coliform (FC) and fecal streptococci (FS) between these two sampling by 98.12%, 86.12% and 99%, respectively. The numbers of TC, FC and FS exceeded the recommended limit for recreational seawater based on U.S. EPA 1986 standard. There was a positive correlation between TC, FC and FS and negative to coliphages.
This study was carried out to determine the optimal parameters for the production of biomass of Trichoderma virens UKMP-1M, a fungus isolated from oil-polluted wastewater. The isolate showed maximum growth at day six after incubation in Mineral Salt Medium (MSM) in the presence of 3% (v/v) heavy Khefji Sour crude oil. Although it grew at pH between 5.0 and 7.0, it grew best at pH 5.5. T. virens UKMP-1M grew at temperatures between 25°C and 35°C, with its highest growth at 30°C. Aeration by agitation at 200 rpm was shown to yield the greatest biomass. Peptone at concentration of 1.5% (w/v) was determined to be a better nitrogen source than urea, potassium nitrate (KNO3), yeast extract, ammonium sulphate ((NH4)2SO4) and ammonium chloride (NH4Cl). Addition of 1% (v/v) crude oil to the MSM medium led to higher biomass production than the addition of 3%, 5%, 7% and 10% (v/v) crude oil. The result also revealed that 40% of total petroleum hydrocarbon (TPH), 100% of pristane and 74% of phytane compounds were degraded after 9 days of incubation at optimal physical and nutrient parameters.
Four species of bacteria, Acinetobacter lwoffii, Aeromonas hydrophila, Pseudomonas aeruginosa and Pseudomonas putida, were isolated from soil contaminated with hydrocarbons and selected for the determination of growth requirements and the ability to degrade petroleum hydrocarbon. The bacteria were grown in mineral salt medium (MSM) supplemented with two types of crude oil, either Sumandak or South Angsi at 1% (v/v) concentration. The optimum pH for growth of A. hydrophila and P. aeruginosa was 6.5 when grown with Sumandak and South Angsi oil. For A. lwoffii and P. putida the optimum pH for growth with Sumandak and South Angsi oil was 6.5 or 7.0, respectively. The growth of P. aeruginosa was the highest in MSM when supplemented with 1% South Angsi oil and 0.5% tryptone at pH 6.5 while, in Sumandak oil the growth was the highest when yeast extract was added. Gas chromatography analysis showed that the South Angsi crude oil components of C12 to C25 were more extensively degraded by A. lwoffii after 24 h of incubation compared to the other bacteria over the same period.