Environmental stressor assessment of hydrocarbonoclastic bacteria biofilms from a marine oil spill.

The environmental and economic impact of an oil spill can be significant. Biotechnologies applied during a marine oil spill involve bioaugmentation with immobilised or encapsulated indigenous hydrocarbonoclastic species selected under laboratory conditions to improve degradation rates. The environmental factors that act as stressors and impact the effectiveness of hydrocarbon removal are one of the challenges associated with these applications. Understanding how native microbes react to environmental stresses is necessary for effective bioaugmentation. Herein, Micrococcus luteus and M. yunnanensis isolated from a marine oil spill mooring system showed hydrocarbonoclastic activity on Maya crude oil in a short time by means of total petroleum hydrocarbons (TPH) at 144 h: M. luteus up to 98.79 % and M. yunnanensis 97.77 % removal. The assessment of Micrococcus biofilms at different temperature (30 °C and 50 °C), pH (5, 6, 7, 8, 9), salinity (30, 50, 60, 70, 80 g/L), and crude oil concentration (1, 5, 15, 25, 35 %) showed different response to the stressors depending on the strain. According to response surface analysis, the main effect was temperature > salinity > hydrocarbon concentration. The hydrocarbonoclastic biofilm architecture was characterised using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Subtle but significant differences were observed: pili in M. luteus by SEM and the topographical differences measured by AFM Power Spectral Density (PSD) analysis, roughness was higher in M. luteus than in M. yunnanensis. In all three domains of life, the Universal Stress Protein (Usp) is crucial for stress adaptation. Herein, the uspA gene expression was analysed in Micrococcus biofilm under environmental stressors. The uspA expression increased up to 2.5-fold in M. luteus biofilms at 30 °C, and 1.3-fold at 50 °C. The highest uspA expression was recorded in M. yunnanensis biofilms at 50 °C with 2.5 and 3-fold with salinities of 50, 60, and 80 g/L at hydrocarbon concentrations of 15, 25, and 35 %. M. yunnanensis biofilms showed greater resilience than M. luteus biofilms when exposed to harsh environmental stressors. M. yunnanensis biofilms were thicker than M. luteus biofilms. Both biofilm responses to environmental stressors through uspA gene expression were consistent with the behaviours observed in the response surface analyses. The uspA gene is a suitable biomarker for assessing environmental stressors of potential microorganisms for bioremediation of marine oil spills and for biosensing the ecophysiological status of native microbiota in a marine petroleum environment.

Aerobic biodegradation of sludge with high hydrocarbon content generated by a Mexican natural gas processing facility.

The biodegradation of oil sludge from Mexican sour gas and petrochemical facilities contaminated with a high content of hydrocarbons, 334.7 ± 7.0 g kg(-1) dry matter (dm), was evaluated. Studies in microcosm systems were carried out in order to determine the capacity of the native microbiota in the sludge to reduce hydrocarbon levels under aerobic conditions. Different carbon/nitrogen/phosphorous (C/N/P) nutrient ratios were tested. The systems were incubated at 30 °C and shaken at 100 rpm. Hydrocarbon removals from 32 to 51% were achieved in the assays after 30 days of incubation. The best assay had C/N/P ratio of 100/1.74/0.5. The results of the Microtox(®) and Ames tests indicated that the original sludge was highly toxic and mutagenic, whereas the best assay gave a final product that did not show toxicity or mutagenicity.

Evaluation of haloalkaliphilic sulfur-oxidizing microorganisms with potential application in the effluent treatment of the petroleum industry.

Haloalkaliphilic sulfur-oxidizing mixed cultures for the treatment of alkaline-saline effluents containing sulfide were characterized and evaluated. The mixed cultures (IMP-PB, IMP-XO and IMP-TL) were obtained from Mexican alkaline soils collected in Puebla (PB), Xochimilco (XO) and Tlahuac (TL), respectively. The Ribosomal Intergenic Spacer Analysis (RISA) revealed bacteria related to Thioalkalibacterium and Thioalkalivibrio in IMP-XO and IMP-PB mixed cultures. Halomonas strains were detected in IMP-XO and IMP-TL. In addition, an uncultured Bacteroides bacterium was present in IMP-TL. Mixed cultures were evaluated at different pH and NaCl concentrations at 30°C. IMP-PB and IMP-TL expressed thiosulfate-oxidizing activity in the 7.5-10.5 pH range, whereas IMP-XO presented its maximal activity with 19.0 mg O2 g (protein)⁻¹ min⁻¹, at pH 10.6; it was not affected by NaCl concentrations up to 1.7 M. In continuous culture, IMP-XO showed a growth rate of 15 day⁻¹, productivity of 433.4 mg(protein) l⁻¹ day⁻¹ and haloalkaliphilic sulfur-oxidizing activity was also detected up to 170 mM by means of N-methyl-diethanolamine (MDEA). Saline-alkaline soil samples are potential sources of haloalkaliphilic sulfur-oxidizing bacteria and the mixed cultures could be applied in the treatment of inorganic sulfur compounds in petroleum industry effluents under alkaline-saline conditions.

Evaluation of the effect of nutrient ratios on biosurfactant production by Serratia marcescens using a Box-Behnken design.

The strain SmSA, identified as Serratia marcescens and known as a biosurfactant producer, was isolated from hydrocarbon contaminated soil from Veracruz, México. The interactions among the C/N, C/Mg and C/Fe ratios have not been examined for this microorganism. In this work was evaluated the effect of these nutrients at three levels using a mineral medium with glucose as the carbon source. A Box-Behnken experimental design was utilised to maximise biosurfactant production, which was assessed by oil spreading and surface tension tests. The treatment with C/N=5, C/Fe=26,000 and C/Mg=30 showed the best result since the surface tension was reduced to 30 mN m(-1). The multiple regression and response surface analyses indicated that the interaction between C/N and C/Mg had the utmost effect on the reduction of surface tension and biosurfactant production. The conditions of the best treatment were used to scale up biosurfactant production in a 3L bioreactor giving a yield of 4.1 gL(-1) of pure biosurfactant. It was found that the biosurfactant was mainly produced in the exponential phase and decreased the surface tension to 31 mN m(-1). The contact between the biosurfactant with heavy oil (15° API) increased its displacement from 9.3 to 18 cm.

Microcosm assays and Taguchi experimental design for treatment of oil sludge containing high concentration of hydrocarbons.

Microcosm assays and Taguchi experimental design was used to assess the biodegradation of an oil sludge produced by a gas processing unit. The study showed that the biodegradation of the sludge sample is feasible despite the high level of pollutants and complexity involved in the sludge. The physicochemical and microbiological characterization of the sludge revealed a high concentration of hydrocarbons (334,766+/-7001 mg kg(-1) dry matter, d.m.) containing a variety of compounds between 6 and 73 carbon atoms in their structure, whereas the concentration of Fe was 60,000 mg kg(-1) d.m. and 26,800 mg kg(-1) d.m. of sulfide. A Taguchi L(9) experimental design comprising 4 variables and 3 levels moisture, nitrogen source, surfactant concentration and oxidant agent was performed, proving that moisture and nitrogen source are the major variables that affect CO(2) production and total petroleum hydrocarbons (TPH) degradation. The best experimental treatment yielded a TPH removal of 56,092 mg kg(-1) d.m. The treatment was carried out under the following conditions: 70% moisture, no oxidant agent, 0.5% of surfactant and NH(4)Cl as nitrogen source.