Abdulkadir E. Elshafie

Biosurfactant production by Bacillus subtilis B30 and its application in enhancing oil recovery

The fermentative production of biosurfactants by Bacillus subtilis strain B30 and the evaluation of biosurfactant based enhanced oil recovery using core-flood were investigated. Different carbon sources (glucose, sucrose, starch, date molasses, cane molasses) were tested to determine the optimal biosurfactant production. The isolate B30 produced a biosurfactant that could reduce the surface tension and interfacial tension to 26.63±0.45 mN/m and 3.79±0.27 mN/m, respectively in less than 12h in both glucose or date molasses based media. A crude biosurfactant concentration of 0.3-0.5 g/l and critical micelle dilution (CMD) values of 1:8 were observed. The biosurfactants gave stable emulsions with wide range of hydrocarbons including light and heavy crude oil. The biosurfactants were partially purified and identified as a mixture of lipopeptides similar to surfactin, using high performance thin layer chromatography and Fourier transform infrared spectroscopy. The biosurfactants were stable over wide range of pH, salinity and temperatures. The crude biosurfactant preparation enhanced light oil recovery by 17-26% and heavy oil recovery by 31% in core-flood studies. The results are indicative of the potential of the strain for the development of ex situ microbial enhanced oil recovery processes using glucose or date molasses based minimal media.

Fungi and aflatoxins associated with spices in the Sultanate of Oman.

One hundred and five samples of seven spices (cumin, cinnamon, clove, black pepper, cardamom, ginger, and coriander) were purchased from five popular companies in the Sultanate of Oman. The spices were surveyed for the mycoflora and aflatoxins. Twenty fungal species were isolated in which Aspergillus flavus , A. niger . Penicillium , Rhizopus , and Syncephalastrum racemosum were the most dominant. When colony forming units per gm (cfu/gm) of fungi were compared, significant differences were found among spices and companies. Of the seven spices studied, clove was found to be the least contaminated, while cumin was the most contaminated. None of the 15 selected samples of the spices contaminated by A. flavus were found to contain aflatoxins. Nevertheless, nine isolates (45%) of the twenty A. flavus strains screened for aflatoxins were aflatoxigenic. The moisture content of most of the spices was below the maximum standard limit. The results showed that the spices were contaminated by some fungi that might constitute health hazards for humans.

Coastal sewage discharge and its impact on fish with reference to antibiotic resistant enteric bacteria and enteric pathogens as bio-indicators of pollution.

Presence of antibiotic resistant bacteria as bio-indicators of pollution was monitored in fish (colons and gills) and in sewage treated effluent (STE) in two marine sites in the Gulf of Oman, Muscat. One site was polluted as the sewage effluent STE was directly discharged into the sea while the unpolluted site was 10 km away. A total of 42 water samples (250 mL each) were collected from both sites. In the polluted site, 14 samples were taken directly from the STE discharge point and 14 from the surrounding area near the STE discharging point (7-10 m). Water and fish samples were collected from polluted and unpolluted sites. Chlorine and nitrate levels were significantly higher in STE samples. A significant difference (P<0.005) was found in total bacterial count of fish colon and gills in the two sites. Most probable number (MPN) was significantly higher in STE and polluted seawater over unpolluted. Coliforms were detected in the polluted site only. A total of 320 target enteric normal flora of animals and pathogenic species were isolated from 10 genera taken from water and fish samples. The isolates were exposed to 14 antibiotics. Most of the isolates were resistant to at least one antibiotic with ampicillin was the dominant antibiotic. This is a clear indication that sewage effluent causes contamination of marine wildlife along coastal lines.

Fungi associated with black tea and tea quality in the Sultanate of Oman.

Forty-eight samples of four popular commercial brands of black tea ( Camellia sinensis L.) were purchased from the local markets in Muscat area, Sultanate of Oman. Tea leaves were surveyed for mycoflora. Five fungal species were isolated with A. niger as the most dominant in all the brands having percentage contamination ranging between 0.66% and 30.34%. Other fungi isolated were Aspergillus flavus, Penicillium spp. and Pacelomyces spp. but having average percentages of 0.6%, 0.84% and 0.21% respectively. Significant differences were found among the batches contaminated by A. niger. None of the 25 A. flavus strains screened for aflatoxins were found aflatoxigenic. The total ash, water-soluble ash, and mineral concentration of the samples were within the British standards and were not affected by fungal contamination. The results showed that black tea is contaminated by fungi that might constitute health hazards for humans. The post harvest contamination of tea could be eliminated or reduced if processing is conducted under more hygienic conditions.

Residual-Oil Recovery Through Injection of Biosurfactant, Chemical Surfactant, and Mixtures of Both Under Reservoir Temperatures: Induced-Wettability and Interfacial-Tension Effects

In this study, a biosurfactant produced by a Bacillus subtilis strain isolated from oil-contaminated soil from an Omani oil field was tested for its potential in enhancing oil recovery by a series of coreflooding experiments. It was found that the performance of the biosurfactant was increased by mixing with chemical surfactants, by which the maximum production went up to 50% of residual oil at a mixing ratio of (50:50). The second objective of this study was to investigate the effects of the biosurfactant on wettability alteration and to estimate its tendency to loss caused by adsorption. The influence of biosurfactant on wettability was studied by contact-angle measurements, atomic force microscopy (AFM) technique on few-layer graphene (FLG) surfaces, and Amott wettability tests on Berea sandstone cores. Contact-angle measurements showed that the wettability of the biosurfactant solution changes to more oil-wet as the angle decreased from 70.6 to 25.32° when treated with 0.25% (w/v) biosurfactant solution. Amott testing showed a change in wettability index from strongly water-wet in the untreated core toward less water-wet in biosurfactant-treated cores. These results confirmed the ability of the biosurfactant to alter the wetting conditions against different surfaces, thereby serving as a mechanism for enhancing oil recovery. The maximum loss of biosurfactant caused by adsorption was 1.2 mg/g of rock, which is comparable with reported chemical-surfactant values.

Sophorolipids Production by Candida bombicola ATCC 22214 and its Potential Application in Microbial Enhanced Oil Recovery

Biosurfactant production using Candida bombicola ATCC 22214, its characterization and potential applications in enhancing oil recovery were studied at laboratory scale. The seed media and the production media were standardized for optimal growth and biosurfactant production. The production media were tested with different carbon sources: glucose (2%w/v) and corn oil (10%v/v) added separately or concurrently. The samples were collected at 24 h interval up to 120 h and checked for growth (OD660), and biosurfactant production [surface tension (ST) and interfacial tension (IFT)]. The medium with both glucose and corn oil gave better biosurfactant production and reduced both ST and IFT to 28.56 + 0.42mN/m and 2.13 + 0.09mN/m, respectively within 72 h. The produced biosurfactant was quite stable at 13–15% salinity, pH range of 2–12, and at temperature up to 100°C. It also produced stable emulsions (%E24) with different hydrocarbons (pentane, hexane, heptane, tridecane, tetradecane, hexadecane, 1-methylnaphthalene, 2,2,4,4,6,8-heptamethylnonane, light and heavy crude oil). The produced biosurfactant was extracted using ethyl acetate and characterized as a mixture of sophorolipids (SPLs). The potential of SPLs in enhancing oil recovery was tested using core-flooding experiments under reservoir conditions, where additional 27.27% of residual oil (Sor) was recovered. This confirmed the potential of SPLs for applications in microbial enhanced oil recovery.

Biomonitoring marine habitats in reference to antibiotic resistant bacteria and ampicillin resistance determinants from oviductal fluid of the nesting green sea turtle, Chelonia mydas

During the egg-laying process, oviductal fluid was collected using a non-invasive procedure from the cloacal vent of the green turtles. Forty-two independent isolates of antibiotic-resistant bacteria from 11 genera were obtained from 20 turtles during nesting. The dominant isolate was Citrobacter (52.4%), followed by Pseudomonas, Proteus, Enterobacter, Salmonella, Escherichia coli, Shigella, Edwardsiella, Morganella, Providencia and Arcomobacter. Most of the isolates were resistant to ampicillin. Ampicillin-resistant isolates showed variations in their resistance for the following classes of β-lactamases: extended-spectrum β-lactamases (EBSLs), AmpC type β-lactamases C (AmpC), and screen-positive β-lactamase. None of the isolates produced metallo β-lactamase. Some ampicillin-resistant genes were detected by multiplex polymerase chain reaction (PCR) only. Inhibitor based test (IBT) categorized some isolates as AmpC β-lactamase producers. β-Lactamase genes were detected from a few strains. The sequencing of those genes revealed the presence of cephamycinase (CMY) and AmpC β-lactamases. The oviductal fluid was used in this study as a source of bacterial antibiotic-resistant determinants for biomonitoring marine turtles exposed to contaminated effluents. This data can be of value in understanding the decline of this endangered species as a result of exposure to marine pollution which is threatening their survival.

Viability of multiple antibiotic resistant bacteria in distribution lines of treated sewage effluent used for irrigation.

Viability of multiple antibiotic resistant bacteria (MARB) in tertiary treated sewage effluent (TTSE) used for irrigation, was investigated at the Sultan Qaboos University sewage treatment plant (STP). This water recycle system is used here as a model for the systems commonly used throughout Oman and the Gulf region. Samples of TTSE were collected weekly from four sites, 1.5 km from each other. Chlorine levels declined gradually at the three sites with increasing distance from the STP. Viable bacteria, coliforms and nitrate concentrations increased significantly while biological oxygen demand (BOD) declined after STP chlorination. Mean values of turbidity changed slightly. Trace elements values were insignificant. A total of 336 bacteria from 8 genera revealed that the dominant isolates were Enterobacter spp., Pseudomonas spp., and Aeromonas spp. Among the isolates 59.8% were multiply resistant to several antibiotics. Resistance was higher to ampicillin followed by sulphamethoxazole, carbenicillin, streptomycine and minocycline. Frequency of resistance to the 14 antibiotics varied among the isolates. The present system related to the viability of MARB in TTSE used for irrigation may have serious implications for public health and wildlife. Results of this investigation will be of value in modifying current STPs systems and thus avoiding serious health issues.

Incidence of seed-borne fungi and aflatoxins in Sudanese lentil seeds

Thirteen seed samples of lentil (Lens esculenta) were incubated on agar plate and moist filter papers (Moist Chambers) at 28 ± 2 °C for determination of the incidence of seed-borne fungi. Aflatoxins content of the seeds was measured using the bright greenish-yellow fluorescence test (BGYF) and thin-layer chromatography (TLC). Sixty-nine species and seven varieties, which belong to 24 genera of fungi, were isolated from this crop. Of these fungi, 51 species and two varieties are considered new for this crop, whereas seven genera and 13 species are new to the mycoflora of the Sudan. The genus Aspergillus (13 species and 6 varieties) which comprising 44% of the total colony count was the most prevalent genus followed by Rhizopus (2 species, 19%), Penicillium (6 species) and Fusarium (8 species) (12%), Chaetomium (3 species) and Cladosporium (5 species) (6%), where the 18 genera (1–4 species) showed very low level of incidence (19%). Of the possible pathogens of lentil plants, F. oxysporum the main cause of vascular wilt was recovered from seeds of this crop. Thin layer chromatographic analysis of chloroform extracts of 13 seed samples showed that only one samples was naturally contaminated with aflatoxins B1, B2, G1 and G2 (14.3 μg/kg).

Diversity and trapping efficiency of nematophagous fungi from Oman.

Summary. A survey of the nematophagous mycobiota biodiversity of 82 soil and leaf-litter samples in the Sultanate of Oman yielded ten species of nematode trapping fungi belonging to three genera. The species are: Arthrobotrys eudermata, A. thaumasia, A. musiformis, A. oligospora, A. oligospora var. oligospora, A. oudemansii, A. multiformis, A. javanica, Drechslerella brochopaga and Gamsylella geophyropaga. This is the first record of these species in Oman. Arthobotrys multiformis represents the second record of this species worldwide. A systematic study showed that A. oudemansii, A. multiformis and A. javanica were morphologically more variable than was so far known. In four days, A. oligospora, A. thaumasia, D. brochopaga and A. eudermata trapped all nematodes added to the Petri dishes (Panagrellus redivivus, 100 specimens per dish). No significant differences were found in the trapping efficiency among the species tested.