Petroleum contamination and bioaugmentation in bacterial rhizosphere communities from Avicennia schaueriana

ABSTRACT Anthropogenic activity, such as accidental oil spills, are typical sources of urban mangrove pollution that may affect mangrove bacterial communities as well as their mobile genetic elements. To evaluate remediation strategies, we followed over the time the effects of a petroleum hydrocarbon degrading consortium inoculated on mangrove tree Avicennia schaueriana against artificial petroleum contamination in a phytoremediation greenhouse experiment. Interestingly, despite plant protection due to the inoculation, denaturing gradient gel electrophoresis of the bacterial 16S rRNA gene fragments amplified from the total community DNA indicated that the different treatments did not significantly affect the bacterial community composition. However, while the bacterial community was rather stable, pronounced shifts were observed in the abundance of bacteria carrying plasmids. A PCR-Southern blot hybridization analysis indicated an increase in the abundance of IncP-9 catabolic plasmids. Denaturing gradient gel electrophoresis of naphthalene dioxygenase (ndo) genes amplified from cDNA (RNA) indicated the dominance of a specific ndo gene in the inoculated petroleum amendment treatment. The petroleum hydrocarbon degrading consortium characterization indicated the prevalence of bacteria assigned to Pseudomonas spp., Comamonas spp. and Ochrobactrum spp. IncP-9 plasmids were detected for the first time in Comamonas sp. and Ochrobactrum spp., which is a novelty of this study.

Variation analysis of bacterial polyhydroxyalkanoates production using saturated and unsaturated hydrocarbons

ABSTRACT Polyhydroxyalkanoates (PHA) are efficient, renewable and environment friendly polymeric esters. These polymers are synthesized by a variety of microbes under stress conditions. This study was carried out to check the suitability of waste frying oil in comparison to other oils for economical bioplastic production. Six bacterial strains were isolated and identified as Bacillus cereus (KF270349), Klebsiella pneumoniae (KF270350), Bacillus subtilis (KF270351), Brevibacterium halotolerance (KF270352), Pseudomonas aeruginosa (KF270353), and Stenotrophomonas rhizoposid (KF270354) by ribotyping. All strains were PHA producers so were selected for PHA synthesis using four different carbon sources, i.e., waste frying oil, canola oil, diesel and glucose. Extraction of PHA was carried out using sodium hypochlorite method and maximum amount was detected after 72 h in all cases. P. aeruginosa led to maximum PHA production after 72 h at 37 °C and 100 rpm using waste frying oil that was 53.2% PHA in comparison with glucose 37.8% and cooking oil 34.4%. B. cereus produced 40% PHA using glucose as carbon source which was high when compared against other strains. A significantly lesser amount of PHA was recorded with diesel as a carbon source for all strains. Sharp Infrared peaks around 1740-1750 cm-1 were present in Fourier Transform Infrared spectra that correspond to exact position for PHA. The use of waste oils and production of poly-3hydroxybutyrate-co-3hydroxyvalerate (3HB-co-3HV) by strains used in this study is a good aspect to consider for future prospects as this type of polymer has better properties as compared to PHBs.

Draft genome analysis of Dietzia sp. 111N12-1, isolated from the South China Sea with bioremediation activity

Abstract Dietzia sp. 111N12-1, isolated from the seawater of South China Sea, shows strong petroleum hydrocarbons degradation activity. Here, we report the draft sequence of approximately 3.7-Mbp genome of this strain. To the best of our knowledge, this is the first genome sequence of Dietzia strain isolated from the sea. The genome sequence may provide fundamental molecular information on elucidating the metabolic pathway of hydrocarbons degradation in this strain.

Posible transmisión oral de la enfermedad de Chagas en trabajadores del sector de los hidrocarburos en Casanare, Colombia, 2014 / Possible oral transmission of Chagas disease among hydrocarbons sector workers in Casanare, Colombia, 2014

RESUMEN Introducción. Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, puede transmitirse por vía oral tras la ingestión de alimentos o bebidas contaminadas. En la semana epidemiológica 14 del 2014, se notificaron dos casos de enfermedad aguda de Chagas en Paz de Ariporo, Casanare, entre trabajadores del sector de los hidrocarburos, episodio que motivó la investigación epidemiológica en el área. Objetivo. Caracterizar la población afectada, establecer medidas de control y confirmar el brote. Materiales y métodos. Se hizo una investigación de brote con los siguientes componentes: a) búsqueda de personas sintomáticas (cuadro clínico sugerente de enfermedad de Chagas según la definición de caso), para remitirlas a atención médica; b) aplicación de una encuesta entomológica en 192 de 197 viviendas; c) inspección sanitaria y análisis microbiológico de muestras de alimentos, y d) estudio de reservorios. La organización y el análisis de los datos se hicieron mediante estadística descriptiva con el programa Epi-Info 7.1.5. Asimismo, se establecieron los índices de infestación en el domicilio y el peridomicilio. Resultados. Se registraron 552 personas expuestas y se confirmaron por laboratorio 40 casos (7,2 %); siete casos se dieron en mujeres (17,5 %) y 33 en hombres (82,5%), es decir, en una relación de 1:5. La edad promedio fue de 39,1 (± 10,8) años, la tasa de ataque, de 7,2 %, y la letalidad, de 5 % (2/40). Los signos y síntomas incluyeron fiebre en el 100 % de los casos, cefalea en el 80 %, mialgias y artralgias en el 65 %, edema facial en el 55 %, y dolor abdominal en el 37,5 %. El tiempo promedio de incubación fue de 17 (3-21) días. El índice de infestación de Rhodnius prolixus fue de 3,3 % en el domicilio y de 2,2 % en el peridomicilio. En los cinco restaurantes inspeccionados, se encontraron condiciones sanitarias deficientes y alimentos con niveles de contaminación microbiológica inaceptables. Por último, un perro y dos zarigüeyas fueron positivos para los anticuerpos IgG en la prueba ELISA. Conclusiones. Mediante el análisis de las características epidemiológicas, ambientales y sanitarias, se confirmó un brote agudo de enfermedad de Chagas por exposición ocupacional y de posible transmisión oral, que podría ser el de mayor proporción reportado hasta la fecha en Colombia.

ABSTRACT Introduction: Trypanosoma cruzi, the etiological agent for Chagas disease, can be transmitted by oral intake of contaminated food or drinks. During epidemiological week 14 of 2014, two cases of acute Chagas disease were notified among hydrocarbons sector workers in Paz de Ariporo, Casanare. Objective: To characterize the affected population, to establish control and prevention measures and to confirm the outbreak. Materials and methods: We conducted an outbreak investigation that included the following components: a) Search for symptomatic people compatible with Chagas disease according to the case definition for their referral to medical services; b) entomological survey (192/197 houses); c) sanitary inspection and microbiological analysis of food samples; and d) study of reservoirs. Data management and analysis were done with Epi-Info 7.1.5 using descriptive statistics. We also calculated intradomicile and peridomicile triatomine infestation indexes. Results: We detected 552 exposed people; 40 had the disease (7.2%), of whom seven were women (17,5%) and 33, men (82.5%), i.e., a male-female ratio of 5:1. The mean age was 39.1 ± 10.8 years; the attack rate was 7.2% and lethality, 5% (2/40). Symptoms included fever (100% of cases), headache (80%), myalgia and arthralgia (65%), facial edema (55%), and abdominal pain (37.5%). The mean incubation time was 17 days (range: 3-21). Rhodnius prolixus domiciliary infestation index was 3.3 % and 2.2% in the peridomicile. In the five restaurants inspected sanitary conditions were deficient and food samples were microbiologically non-conforming. We found a dog and two opossums positive for IgG antibodies by ELISA. Conclusions: Environmental, sanitary and epidemiological conditions at the place confirmed an outbreak of Chagas diseases related to occupational exposure, possibly by oral transmission, which may be the largest to date in Colombia.

Quick stimulation of Alcanivorax sp. by bioemulsificant EPS2003 on microcosm oil spill simulation

Oil spill microcosms experiments were carried out to evaluate the effect of bioemulsificant exopolysaccharide (EPS2003) on quick stimulation of hydrocarbonoclastic bacteria. Early hours of oil spill, were stimulated using an experimental seawater microcosm, supplemented with crude oil and EPS2003 (SW+OIL+EPS2003); this system was monitored for 2 days and compared to control microcosm (only oil-polluted seawater, SW+OIL). Determination of bacterial abundance, heterotrophic cultivable and hydrocarbon-degrading bacteria were carried out. Community composition of marine bacterioplankton was determined by 16S rRNA gene clone libraries. Data obtained indicated that bioemulsificant addition stimulated an increase of total bacterial abundance and, in particular, selection of bacteria related to Alcanivorax genus; confirming that EPS2003 could be used for the dispersion of oil slicks and could stimulate the selection of marine hydrocarbon degraders thus increasing bioremediation process.

Best conditions for biodegradation of diesel oil by chemometric tools

Diesel oil biodegradation by different bacteria-yeast-rhamnolipids consortia was tested. Chromatographic analysis of post-biodegradation residue was completed with chemometric tools (ANOVA, and a novel ranking procedure based on the sum of ranking differences). These tools were used in the selection of the most effective systems. The best results of aliphatic fractions of diesel oil biodegradation were observed for a yeast consortia with Aeromonas hydrophila KR4. For these systems the positive effect of rhamnolipids on hydrocarbon biodegradation was observed. However, rhamnolipids addition did not always have a positive influence on the biodegradation process (e.g. in case of yeast consortia with Stenotrophomonas maltophila KR7). Moreover, particular differences in the degradation pattern were observed for lower and higher alkanes than in the case with C22. Normally, the best conditions for "lower" alkanes are Aeromonas hydrophila KR4 + emulsifier independently from yeasts and e.g. Pseudomonas stutzeri KR7 for C24 alkane.

In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil

In situ biosurfactant (rhamnolipid) production by Pseudomonas putida CB-100 was achieved during a bioaugmented and biostimulated treatment to remove hydrocarbons from aged contaminated soil from oil well drilling operations. Rhamnolipid production and contaminant removal were determined for several treatments of irradiated and non-irradiated soils: nutrient addition (nitrogen and phosphorus), P. putida addition, and addition of both (P. putida and nutrients). The results were compared against a control treatment that consisted of adding only sterilized water to the soils. In treatment with native microorganisms (non-irradiated soils) supplemented with P. putida, the removal of total petroleum hydrocarbons (TPH) was 40.6%, the rhamnolipid production was 1.54 mg/kg, and a surface tension of 64 mN/m was observed as well as a negative correlation (R = -0.54; p < 0.019) between TPH concentration (mg/kg) and surface tension (mN/m), When both bacteria and nutrients were involved, TPH levels were lowered to 33.7%, and biosurfactant production and surface tension were 2.03 mg/kg and 67.3 mN/m, respectively. In irradiated soil treated with P. putida, TPH removal was 24.5% with rhamnolipid generation of 1.79 mg/kg and 65.6 mN/m of surface tension, and a correlation between bacterial growth and biosurfactant production (R = -0.64; p < 0.009) was observed. When the nutrients and P. putida were added, TPH removal was 61.1%, 1.85 mg/kg of biosurfactants were produced, and the surface tension was 55.6 mN/m. In summary, in irradiated and non-irradiated soils, in situ rhamnolipid production by P. putida enhanced TPH decontamination of the soil.

Transcriptional profiling of genes involved in n-hexadecane compounds assimilation in the hydrocarbon degrading Dietzia cinnamea P4 strain

The petroleum-derived degrading Dietzia cinnamea strain P4 recently had its genome sequenced and annotated. This allowed employing the data on genes that are involved in the degradation of n-alkanes. To examine the physiological behavior of strain P4 in the presence of n-alkanes, the strain was grown under varying conditions of pH and temperature. D. cinnamea P4 was able to grow at pH 7.0-9.0 and at temperatures ranging from 35 ºC to 45 ºC. Experiments of gene expression by real-time quantitative RT-PCR throughout the complete growth cycle clearly indicated the induction of the regulatory gene alkU (TetR family) during early growth. During the logarithmic phase, a large increase in transcriptional levels of a lipid transporter gene was noted. Also, the expression of a gene that encodes the protein fused rubredoxin-alkane monooxygenase was enhanced. Both genes are probably under the influence of the AlkU regulator.

Comparative study of the cuticular hydrocarbon in queens, workers and males of Ectatomma vizottoi (Hymenoptera, Formicidae) by Fourier transform-infrared photoacoustic spectroscopy

Fourier transform-infrared photoacoustic spectroscopy was applied for the first time, to our knowledge, to distinguish different castes of an ant species. The method was applied directly to the abdomen of queens, workers and males of Ectatomma vizottoi ants, without any special sample preparation. The absorption bands of secondary amide and hydrocarbons were identified; using these as variables in a canonical discriminant analysis we found significant differences between the castes. Queens have a greater hydrocarbon content than do workers and males, which is related to their function in the colony. This technique can be used to analyze and distinguish small chemical differences in biological systems, even in opaque samples.

Impact of refined petroleum spills on water quality, macro-invertebrate and microbial communities of a tropical aquatic environment.

Water quality characteristics, benthic macro-invertebrates and microbial communities of three first order streams in South West Nigeria were investigated to assess the effects of refined petroleum five months after spillage. All physical and chemical conditions except temperature and pH were significantly different (P<0.01) at the upstream control stations and impacted stations reflecting the perturbational stress. The benthic macro-invertebrate fauna were dominated by arthropods, but the faunal spectrum was dissimilar at all the stations studied. Sampling stations at the epicentre of the spill showed considerable reduction in faunal compositions and relative abundance. Generally, the microbial density and diversity were highest in both soil and water samples from impacted sites than in control sites. There was a significantly higher proportion (P < 0.05) of hydrocarbon utilizers in soil than in water samples in all stations except in samples from stations (P<0.05).

Production of glycolipids containing biosurfactant by Pseudomonas species.

Microorganisms, that degrade hydrocarbon were isolated and screened for their biosurfactant activity. A total of 68 strains were isolated and tested for their glycolipid activity of which 4 isolates showed good glycolipid activity. Isolate K10 gave the maximum biosurfactant production in medium A (containing kerosene as a sole carbon source) as compared to medium B (containing glucose as a sole carbon source). Characterization of isolate K10 showed that it belongs to Pseudomonas species.

Production biosurfactant by hydrocarbon degrading rhodococcus ruber and rhodococcus erythropolis

There is world wide concern about the liberation of hydrocarbons in the environment, both from industrial activities and from accidental spills of oil-related compounds. Biosurfactants, which are natural emulsifiers of hydrocarbons, are produced by some bacteria, fungi and yeast. They are polymers, totally or partially extracellular, with an amphipathyc structure, which allows them to form micelles that accumulate at the interface between liquids of different polarities such as water and oil. This process is based upon the ability of biosurfactants to reduce surface tension, blocking the formation of hydrogen bridges and certain hydrophilic and hydrophobic interactions. The ability of biosurfactant production by five strains of Rhodococcus isolated from oil prospecting sites was evaluated. Surface tension measurement and emulsifying index were used to quantify biosurfactant production. The influence of environmental conditions was also investigated - pH, temperature, medium composition, and type of carbon source - on cell growth and biosurfactant production. Strain AC 239 was shown to be a potential producer, attaining 63 (per cent) of emulsifying index for a Diesel-water binary system. It could be used, either directly on oil spills in contained environments, or for the biotechnological production of biosurfactant.

Utilización de hidrocarburos por oomycetes II: achlya americana, A. prolifera y dictyuchus monosporus / Hidrocarbons utilization by oomycetes II: achlya americana, A. prolifera and dictyuchus monosporus

Se estudió el crecimiento in vitro de Achlya americana, A prolifera y Dictyuchus monosporus usándose como fuente carbonada heptano, benceno, xileno, tolueno, nafta común, gas oil y kerosene. Estas especies mostraron abundante crecimiento en combustibles (mezclas hidrocarbonadas), como gas oil y kerosene; en cambio,el crecimiento fue limitado en hidrocarburos aromáticos (tolueno, benceno y xileno), en nafia común y en heptano, a excepción de D. monosporus que no creció en estos dos últimos

Deteccíon bacteriológica de contaminacíon por petróleo en el estuario de Bahía Blanca / Bacteriological detection of crude oil contamination in the Bahía Blanca estuary

Para conocer la deriva y determinar el área afectada por las pérdidas de petróleo producidas durante las tareas de transvasamiento mediante Boyas Suecas, se cuantificaron bacterias degradadoras de petróleo por el método del Número Más Probable en sedimentos superficiales del estuario de Bahía Blanca. Se establecieron diez estaciones intermareales de nuestreo, localizadas entre las boyas 21 y 16. Se realizó el recuento de bacterias heterótrofas aerobias marinas sobre Agar Marine 2216, y la determinación de materia orgánica. De acuerdo a la abundancia de bacterias degradadoras, el análisis estadístico diferenció dos zonas con distinto grado de contaminación. Una comprendida por las estaciones ubicadas en el Canal Principal de navegación, con recuentos medios, expresados en log10, de 2,98 a 3,65 bactérias/g de sedimento seco, y la otra con valores de 4,16 a 4,37, que se interna en canales y arroyos del veril sur. Estos resultados indican una deriva del contaminante en sentido perpendicular al Canal Principal, debido a la influencia de los vientos predominantes con dirección N y NNO. Mediante el análisis de regresión lineal simple se estableció una relación altamente significativa de la materia orgánica con los dos grupos bacterianos. Este estudio señala que el petróleo proveniente de los volcados está afectando zonas productivas del estuario

Hydrocarbon emulsification by Candida tropicalis and Debaryomyces polymorphus.

Potentiality of C. tropicalis and D. polymorphus, to produce surface active compounds (bioemulsifiers/biosurfactants) during shake cultivation on hexadecane and oily waste was studied. Better emulsification activity, specific towards aromatic hydrocarbons, was observed with C. tropicalis culture broth. Emulsification activity of culture broth was quite stable and was unaffected by change in pH and by increasing the concentration of NaCl up to 5%. The activity was marginally affected by heating in boiling water bath for 15 min, but inhibited to the tune of 90% by 0.3% CaCl2. The isolated bioemulsifying factor contained 40, 22 and 17.5% lipid, protein and carbohydrate, respectively.