The Potential of Bacterial Isolates for Emulsification with a Range of Hydrocarbons

A study was undertaken to investigate the distribution of biosurfactant producing and crude oil degrading bacteria in the oil contaminated environment. This research revealed that hydrocarbon contaminated sites are the potent sources for oil degraders. Among 32 oil degrading bacteria isolated from ten different oil contaminated sites of gasoline and diesel fuel stations, 80% exhibited biosurfactant production. The quantity and emulsification activity of the biosurfactants varied. Pseudomonas sp. DS10‐129 produced a maximum of 7.5 ± 0.4 g/l of biosurfactant with a corresponding reduction in surface tension from 68 mN/m to 29.4 ± 0.7 mN/m at 84 h incubation. The isolates Micrococcus sp. GS2‐22, Bacillus sp. DS6‐86, Corynebacterium sp. GS5‐66, Flavobacterium sp. DS5‐73, Pseudomonas sp. DS10‐129, Pseudomonas sp. DS9‐119 and Acinetobacter sp. DS5‐74 emulsified xylene, benzene, n‐hexane, Bombay High crude oil, kerosene, gasoline, diesel fuel and olive oil. The first five of the above isolates had the highest emulsification activity and crude oil degradation ability and were selected for the preparation of a mixed bacterial consortium, which was also an efficient biosurfactant producing oil emulsifying and degrading culture. During this study, biosurfactant production and emulsification activity were detected in Moraxella sp., Flavobacterium sp. and in a mixed bacterial consortium, which have not been reported before.     

Interference in adhesion of bacteria and yeasts isolated from explanted voice prostheses to silicone rubber by rhamnolipid biosurfactants

AIMS: The effects and extent of adhesion of four different bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber with and without an adsorbed rhamnolipid biosurfactant layer obtained from Pseudomonasaeruginosa DS10-129 was studied. METHODS AND RESULTS: The ability of rhamnolipid biosurfactant to inhibit adhesion of micro-organisms to silicone rubber was investigated in a parallel-plate flow chamber. The anti-adhesive activity of the biosurfactant at different concentrations was significant against all the strains and depended on the micro-organism tested. The results showed an effective reduction in the initial deposition rates, and the number of bacterial cells adhering after 4 h, for all micro-organisms tested at the 4 g l(-1) undiluted rhamnolipid solution. Maximum initial reduction of adhesion rate (an average of 66%) occurred for Streptococcus salivarius GB 24/9 and Candida tropicalis GB 9/9. The number of cells adhering after 4 h on silicone rubber conditioned with biosurfactant was reduced to 48% for Staphylococcus epidermidis GB 9/6, Strep. salivarius GB 24/9, Staphylococcus aureus GB 2/1 and C. tropicalis GB 9/9 in comparison to controls. Perfusing the flow chamber with biosurfactant containing solution followed by the passage of a liquid-air interface, to investigate detachment of micro-organisms adhering to silicone rubber, produced high detachment (96%) of adhered cells for all micro-organisms studied, except for Staph. aureus GB 2/1 (67%). SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that biosurfactant represent suitable compounds that should be considered in developing future strategies to prevent the microbial colonization of silicone rubber voice prostheses.     

Engineering bacteria for production of rhamnolipid as an agent for enhanced oil recovery

Rhamnolipid as a potent natural biosurfactant has a wide range of potential applications, including enhanced oil recovery (EOR), biodegradation, and bioremediation. Rhamnolipid is composed of rhamnose sugar molecule and beta-hydroxyalkanoic acid. The rhamnosyltransferase 1 complex (RhlAB) is the key enzyme responsible for transferring the rhamnose moiety to the beta-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid. Through transposome-mediated chromosome integration, the RhlAB gene was inserted into the chromosome of the Pseudomonas aeruginosa PAO1-rhlA(-) and Escherichia coli BL21 (DE3), neither of which could produce rhamnolipid. After chromosome integration of the RhlAB gene, the constitute strains P. aeruginosa PEER02 and E. coli TnERAB did produce rhamnolipid. The HPLC/MS spectrum showed that the structure of purified rhamnolipid from P. aeruginosa PEER02 was similar to that from other P. aeruginosa strains, but with different percentage for each of the several congeners. The main congener (near 60%) of purified rhamnolipid from E. coli TnERAB was 3-(3-hydroxydecanoyloxy) decanoate (C(10)-C(10)) with mono-rhamnose. The surfactant performance of rhamnolipid was evaluated by measurement of interfacial tension (IFT) and oil recovery via sand-pack flooding tests. As expected, pH and salt concentration of the rhamnolipid solution significantly affected the IFT properties. With just 250 mg/L rhamnolipid (from P. aeruginosa PEER02 with soybean oil as substrate) in citrate-Na(2)HPO(4), pH 5, 2% NaCl, 42% of oil otherwise trapped was recovered from a sand pack. This result suggests rhamnolipid might be considered for EOR applications.     

Heterologous production of Pseudomonas aeruginosa EMS1 biosurfactant in Pseudomonas putida

A new bacterial strain isolated from activated sludge, identified as Pseudomonas aeruginosa EMS1, produced a biosurfactant when grown on acidified soybean oil as the sole carbon source. An optimum biosurfactant production of 5 g/L was obtained with the following medium composition: 2% acidified soybean oil, 0.3% NH4NO3, 0.03% KH2PO4, 0.03% K2HPO4, 0.02% MgSO4.7H2O and 0.025% CaCl2.2H2O, with shaking at 200 rpm for an incubation period of 100 h at 30 degrees C. The production of the biosurfactant was found to be a function of cell growth, with maximum production occurring during the exponential phase. Hemolysis of erythrocytes and thin-layer chromatography studies revealed that the secreted biosurfactant was rhamnolipid. To overcome the complex environmental regulation with respect to rhamnolipid biosynthesis, and to replace the opportunistic pathogen P. aeruginosa with a safe industrial strain, attempts were made to achieve rhamnolipid production in a heterologous host, Pseudomonas putida, using molecular cloning of rhlAB rhamnosyltransferase genes with the rhlRI quorum sensing system, assuming that a functional rhamnosyltransferase would catalyze the formation of rhamnosyl-6-hydroxydecanoyl-6-hydroxydecanoate (mono-rhamnolipid) in P. putida. It was shown that rhamnolipid can be produced in the heterologous strain, P. putida, when provided with the rhamnosyltransferase genes.     

Bioremediation of gasoline contaminated soil by a bacterial consortium amended with poultry litter, coir pith and rhamnolipid biosurfactant.

The aim of the present study was to find methods for enhancing rates of hydrocarbon biodegradation in gasoline contaminated soil by ex situ bioremediation. Red soil (RS) was treated with gasoline-spilled soil (GS) from a gasoline station and different combinations of amendments were prepared using (i) mixed bacterial consortium (MC), (ii) poultry litter (PL), (iii) coir pith (CP) and (iv) rhamnolipid biosurfactant (BS) produced by Pseudomonas sp. DS10-129. The study was conducted for a period of 90 days during which bacterial growth, hydrocarbon degradation and growth parameters of Phaseolus aureus RoxB including seed germination, chlorophyll content, shoot and root length were measured. Approximately 67% and 78% of the hydrocarbons were effectively degraded within 60 days in soil samples amended with RS + GS + MC + PL + CP + BS at 0.1% and 1%. Maximum percentage of seed germination, shoot length, root length and chlorophyll content in P. aureus were recorded after 60 days in the above amendments. Further incubation to 90 days did not exhibit significant improvements. Statistical analysis using analysis of variance (ANOVA) and Duncan's multiple range test (DMRT) revealed that the level of amendments, incubation time and combination of amendments significantly influenced bacterial growth, hydrocarbon degradation, seed germination and chlorophyll content at a 1% probability level. All tested additives MC, PL, CP and rhamnolipid BS had significant positive effects on the bioremediation of GS.     

Towards efficient crude oil degradation by a mixed bacterial consortium

A laboratory study was undertaken to assess the optimal conditions for biodegradation of Bombay High (BH) crude oil. Among 130 oil degrading bacterial cultures isolated from oil contaminated soil samples, Micrococcus sp. GS2-22, Corynebacterium sp. GS5-66, Flavobacterium sp. DS5-73, Bacillus sp. DS6-86 and Pseudomonas sp. DS10-129 were selected for the study based on the efficiency of crude oil utilisation. A mixed bacterial consortium prepared using the above strains was also used. Individual bacterial cultures showed less growth and degradation than did the mixed bacterial consortium. At 1% crude oil concentration, the mixed bacterial consortium degraded a maximum of 78% of BH crude oil. This was followed by 66% by Pseudomonas sp. DS10-129, 59% by Bacillus sp. DS6-86, 49% by Micrococcus sp. GS2-22, 43% by Corynebacterium sp. GS5-66 and 41% by Flavobacterium sp. DS5-73. The percentage of degradation by the mixed bacterial consortium decreased from 78% to 52% as the concentration of crude oil was increased from 1% to 10%. Temperature of 30 degrees C and pH 7.5 were found to be optima for maximum biodegradation.     

Rhamnolipid stimulates uptake of hydrophobic compounds by Pseudomonas aeruginosa

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon alpha,omega-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.     

Effect of nutritional and environmental conditions on the production and composition of rhamnolipids by P. aeruginosa UG2

The production of rhamnolipid biosurfactants by P. aeruginosa UG2 was examined under different culture conditions. Rhamnolipid yield was affected by the nature of the carbon sources, the nutrient concentrations, pH, and age of the culture. Hydrophobic substrates like corn oil, lard (rich in unsaturated and saturated fat), and long chain alcohols maximized biosurfactant production (100-165 mg/g substrate). Hydrophilic substrates like glucose, and succinic acid delivered poor yields (12-36 mg/g substrate). Rhamnolipid production was greater when N as (NH4)(2)SO4 and trace metals were added in several periodic doses rather than at the beginning of the process. Increased biosurfactant production was seen in cultures maintained at neutral pH relative to cultures allowed to develop acidic conditions (pH = 6.25). Although the level of rhamnolipid production was affected by culture conditions, the distribution of rhamnolipid subspecies did not vary between cultures. A dirhamnolipid species containing two 10 carbon alpha-hydroxy fatty acids [Rh2C10C10] was the most abundant in the mixtures (60.6 mol%), while the levels of the monorhamnolipid [RhC10C10] (20.7 mol%) and two dirhamnolipids [Rh2C10C12 and its dehydro variant Rh2C10C12-H2] (18.7 mol%) were similar. Biosurfactant mixtures produced with corn oil as sole carbon source solubilized the herbicide trifluralin [2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzamine] to a greater extent. This suggests that the presence of incompletely metabolized hydrophobic by-products acting as co-solvents can increase the solubilization capacity of biosurfactant mixtures.     

一株铜绿假单胞菌及其产生的鼠李糖脂特性研究

研究高效代谢表面活性剂的菌种,并对菌种和表面活性剂的理化性质进行分析。采用菌种16S rDNA序列分析对菌种进行鉴定,采用紫外光谱扫描、色谱柱层析、薄层层析、单糖分析对生物表面活性剂的种类进行鉴定,并对表面活性剂的理化性质进行研究。从油田采出水中筛选出一株高效代谢生物表面活性剂的菌株T-1,经16S rDNA鉴定为铜绿假单胞菌属(Pseudomonas aeruginosa)。该菌种以甘油和大豆油为碳源的培养基培养4 d后,其发酵液表面张力30.216 mN/m,EI24为100%。根据表面活性剂的红外光谱分析并结合薄层分析,可确定T-1样品中含有糖脂类物质,进一步的表面活性剂的单糖分析显示水解终产物为单一的鼠李糖,最终确定其产物为鼠李糖脂,苯酚-硫酸法测定其鼠李糖脂产率为5.2 g/L,从发酵液提取的棕黄色生物表面活性剂粗品,其表观临界胶束浓度为45 mg/L,该鼠李糖脂对环境(耐温、耐酸碱、耐盐)具有较强的适应性。该表面活性剂对恶劣环境具有较强的耐受性,可应用于微生物采油等用途。     

Improved production of biosurfactant with newly isolated Pseudomonas aeruginosa S2

An indigenous strain Pseudomonas aeruginosa S2 (P. aeruginosa S2), isolated from diesel-contaminated soil, produced extracellular surface-active material identified as rhamnolipid. Due to its excellent surface activity, rhamnolipid is known to be well-suited for stimulating the bioremediation efficiency of oil contaminated sites. To improve production yield of rhamnolipid with P. aeruginosa S2, various carbon and nitrogen sources were screened to select favorable ones leading to better biosurfactant production yield. It was found that using 4% glucose could attain better rhamnolipid yield, while 50 mM NH4NO3 appeared to be the most preferable nitrogen source. Meanwhile, the effect of carbon to nitrogen ratio (C/N ratio) on rhamnolipid yield was also investigated, and the optimal C/N ratio was identified as approximately 11.4. Moreover, response surface methodology (RSM) was applied to optimize the trace element concentration for rhamnolipid production. Results from two-level design indicate that concentrations of MgSO4 and FeSO4 were the most significant factors affecting rhamnolipid production. Using steepest ascent method and RSM analysis, an optimal medium composition was determined, giving a rhamnolipid production yield of 2.37 g/L in 100 h at 37 degrees C and 200 rpm agitation. Scale-up production of rhamnolipid in a well-controlled 5 L jar fermentor using the optimal medium and operating condition (at 37 degrees C and pH 6.8) further elevated the biosurfactant production yield to 5.31 g/L (in 97 h), which is over 2-fold higher than the best results obtained from shake-flask tests.     

Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene

The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l(-1)) and at 72 h (751 mg l(-1)) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30 degrees C and 100 rpm. It reached 8373 mg l(-1) when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.     

Isolation and Characterization of Biosurfactant-Producing Bacteria from Oil-Contaminated Soil

Two biosurfactant-producing Pseudomonas aeruginosa strains (KISR C1 and KISR B1) were isolated from Kuwaiti oil-contaminated soil, which resulted from the Gulf War. The optimum environmental conditions that supported the growth and surfactant production of both isolates were examined. The two isolates differed in their biosurfactant-stimu-lating carbon source, nitrogen concentration, and the pH of the medium. C-1 isolate produced two types of rhamnolipids with a final concentration of 98.4?g/l after spiking the nitrogen-limited medium with 10?mg/ml olive oil. The other isolate (B-1) produced only one type of rhamnolipid (5.9?g/l) after spiking the medium with crude oil. The biosurfactant produced by this strain was found to be very effective in the emulsifica-tion of crude oil. The result suggests that this isolate can potentially be used to enhance bioremediation of oil-contamination and enhanced oil recovery.     

Screening and Evaluation of Biosurfactant-Producing Strains Isolated from Oilfield Wastewater

The six biosurfactant-producing strains, isolated from oilfield wastewater in Daqing oilfield, were screened. The production of biosurfactant was verified by measuring the diameter of the oil spreading, measuring the surface tension value and emulsifying capacity against xylene, n-pentane, kerosene and crude oil. The experimental result showed three strains (S2, S3, S6) had the better surface activity. Among the three strains, the best results were achieved when using S2 strain. The diameter of the oil spreading of the biosurfactant produced by S2 strain was 14 cm, its critical micelle concentration (CMC) was 21.8 mg/l and the interfacial tension between crude oil and biosurfactant solution produced by S2 strain reduced to 25.7 mN/m. The biosurfactant produced by S2 strain was capable of forming stable emulsions with various hydrocarbons, such as xylene, n-pentane, kerosene and crude oil. After S2 strain treatment, the reduction rate of oil viscosity was 51 % and oil freezing point reduced by 4 °C.     

Screening of biosurfactant-producing Bacillus strains using glycerol from the biodiesel synthesis as main carbon source

Glycerol, a co-product of biodiesel production, was evaluated as carbon source for biosurfactant production. For this reason, seven non-pathogenic biosurfactant-producing Bacillus strains, isolated from the tank of chlorination at the Wastewater Treatment Plant at Federal University of Ceara, were screened. The production of biosurfactant was verified by determining the surface tension value, as well as the emulsifying capacity of the free-cell broth against soy oil, kerosene and N-hexadecane. Best results were achieved when using LAMI005 and LAMI009 strains, whose biosurfactant reduced the surface tension of the broth to 28.8?±?0.0 and 27.1?±?0.1?mN?m(-1), respectively. Additionally, at 72?h of cultivation, 441.06 and 267.56?mg?L(-1) of surfactin were produced by LAMI005 and LAMI009, respectively. The biosurfactants were capable of forming stable emulsions with various hydrocarbons, such as soy oil and kerosene. Analyses carried out with high performance liquid chromatography (HPLC) showed that the biosurfactant produced by Bacillus subtilis LAMI009 and LAMI005 was compatible with the commercially available surfactin standard. The values of minimum surface tension and the CMC of the produced biosurfactant indicated that it is feasible to produce biosurfactants from a residual and renewable and low-cost carbon source, such as glycerol.     

Structural characterization of rhamnolipid produced by Pseudonomas aeruginosa strain FIN2 isolated from oil reservoir water

Biosurfactant-producing microorganisms inhabiting oil reservoirs are of great potential in industrial applications. Yet, till now, the knowledge about the structure and physicochemical property of their metabolites are still limited. The aim of this study was to purify and structurally characterize the biosurfactant from Pseudomonas aeruginosa strain FIN2, a newly isolated strain from an oil reservoir. The purification was conducted by silica gel column chromatography followed by pre-RP HPLC and the structural characterization was carried out by GC–MS combined with MS/MS. The results show that the biosurfactant produced by FIN2 is rhamnolipid in nature and its four main fractions were identified to be Rha-C10-C10(46.1 %), Rha–Rha-C10-C10(20.1 %), Rha-C8-C10 (7.5 %) and Rha-C10-C12:1(5.5 %), respectively. Meanwhile, the rarely reported rhamnolipid congeners containing β-hydroxy fatty acids of C6, C9, C10:1 and C11 were also proved to be present in the rhamnolipid mixture produced. The rhamnolipid mixture exhibited a strong surface activity by lowering the surface tension of distilled water to 28.6 mN/m with a CMC value of 195 mg/l.     

Swarming of Pseudomonas aeruginosa PAO1 without differentiation into elongated hyperflagellates on hard agar minimal medium

Polar flagellated Pseudomonas aeruginosa PAO1 demonstrated extensive spreading growth in 2 days on 1.5% agar medium. Such spreading growth of P. aeruginosa PAO1 strains was absent on Luria-Bertani 1.5% agar medium, but remarkable on Davis minimal synthetic agar medium (especially that containing 0.8% sodium citrate and 1.5% Eiken agar) under aerobic 37 degrees C conditions. Analyses using isogenic mutants and complementation transformants showed that bacterial flagella and rhamnolipid contributed to the surface-spreading behavior. On the other hand, a type IV pilus-deficient pilA mutant did not lose the spreading growth activity. Flagella staining of PAO1 T cells from the frontal edge of a spreading colony showed unipolar and normal-sized rods with one or two flagella. Thus, the polar flagellate P. aeruginosa PAO1 T appears to swarm on high-agar medium by producing biosurfactant rhamnolipid and without differentiation into an elongated peritrichous hyperflagellate.     

Identification of small RNAs involved in nitrogen fixation in Anabaena sp. PCC 7120 based on RNA-seq under steady state conditions

The aim of the present study was to investigate the tolerance of five new Achromobacter and Pseudomonas strains to kerosene and to establish if the production of several secondary metabolites increases or not when these bacteria were grown in the presence of kerosene. The biodegradation of kerosene by isolated bacteria was also investigated in this study. Five Proteobacteria were isolated from different samples polluted with petroleum and petroleum products. Based on their morphological, biochemical, and molecular characteristics, isolated bacteria were identified as Achromobacter spanius IBBPo18 and IBBPo21, Pseudomonas putida IBBPo19, and Pseudomonas aeruginosa IBBPo20 and IBBPo22. All these bacteria were able to tolerate and degrade kerosene. Higher tolerance to kerosene and degradation rates were observed for P. aeruginosa IBBPo20 and IBBPo22, compared with that observed for A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19. All these bacteria were able to produce several secondary metabolites, such as surfactants and pigments. Glycolipid surfactants produced by P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 have a very good emulsification activity, and their activity increased when they were grown in the presence of kerosene. The production of rhamnolipid surfactants by P. aeruginosa IBBPo20 and IBBPo22 was confirmed by detection of rhlAB gene involved in their biosynthesis. Pyocyanin and pyoverdin pigments were produced only by P. aeruginosa IBBPo20 and IBBPo22, while carotenoid pigments were produced by all the isolated bacteria. Significant changes in pigments production were observed when P. aeruginosa IBBPo20 and IBBPo22, A. spanius IBBPo18 and IBBPo21, and P. putida IBBPo19 were grown in the presence of kerosene. Due to their ability to tolerate and degrade kerosene, and also to produce several secondary metabolites, the isolated bacteria could be used in the bioremediation of kerosene-polluted environments.     

Imaging and analysis of Pseudomonas aeruginosa swarming and rhamnolipid production

Many bacteria spread over surfaces by "swarming" in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacterium Pseudomonas aeruginosa. First, we quantify the temporal distribution of P. aeruginosa cells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming of P. aeruginosa and Salmonella enterica serovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of several P. aeruginosa strains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.     

Degradation of anthracene by bacteria isolated from oil polluted tropical soils

Four bacteria, identified as Pseudomonas aeruginosa, Alcaligenes eutrophus, Bacillus subtilis and Micrococcus luteus were isolated from crude oil polluted soils using anthracene as the sole carbon and energy source. All the organisms utilized n-hexadecane, n-tetradecane, diesel oil, engine oil and naphthalene as sole carbon sources. None could utilize hexane, cycloheptane, xylene, benzene, toluene, phenol, fluoranthene,and kerosene as carbon sources. Highest cell density obtained with 0.1% (w/v) anthracene were 4.5 x 10(7) (cfu/ml), 8.6 x 10(6) (cfu/ml), 5.4 x 10(6) and 2.4 x 10(6) (cfu/ml) respectively, for P. aeruginosa, A. eutrophus, B. subtilis and M. luteus after 30 days incubation. Growth of the organisms on a Nigerian crude oil resulted in a residual oil concentration of 22.2%, 33.3%, 39.3%, 44% and 91.7% respectively, for P. aeruginosa, A. eutrophus, B. subtilis, M. luteus and the noninoculated control on the 14 th day. Ring fission enzymes of the meta pathway were detected in induced cells of P. aeruginosa and A. eutrophus while ortho pathway enzymes were detected in B. subtilis and M. luteus. P. aeruginosa and A. eutrophus had specific catechol-2,3-dioxygenase activities of 3.8 +/- 0.183 and 0.64 +/- 0.032 micromol/min x mg protein respectively while catechol-1,2-dioxygenase activities of 1.95 +/- 0.029 and 1.89 +/- 0.026 micromol/min x mg protein were detected in B. subtilis and M. luteus respectively. This work, highlights the capability of these unreported tropical strains of A. eutrophus, B. subtilis and M. luteus as anthracene degraders.     

Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils

World production of oils and fats is about 2.5 million tonnes, 75% of which are derived from plants. Most of them are used in the food industry for the manufacture of different products, or directly as salad oil. Great quantities of waste are generated by the oil and fat industries: residual oils, tallow, marine oils, soap stock, frying oils. It is well known that the disposal of wastes is a growing problem and new alternatives for the use of fatty wastes should be studied. Used frying oils, due to their composition, have great potential for microbial growth and transformation. The use of economic substrates such as hydrophobic wastes meets one of the requirements for a competitive process for biosurfactant production. In the Mediterranean countries, the most used vegetable oils are sunflower and olive oil. Here we present a screening process is described for the selection of micro-organism strains with the capacity to grow on these frying oils and accumulate surface-active compounds in the culture media. From the 36 strains screened, nine Pseudomonas strains decreased the surface tension of the medium to 34-36 mN/M; the emulsions with kerosene remained stable for three months. Two Bacillus strains accumulated lipopeptide and decreased the surface tension to 32-34 mN/m. Strain Ps. aeruginosa 47T2 was selected for further studies. The effect of nitrogen and a C/N of 8. 0 gave a final production of rhamnolipid of 2.7 g l-1 as rhamnose, and a production yield of 0.34 g g-1.