Conversion of renewable substrates for biosurfactant production by Rhizopus arrhizus UCP 1607 and enhancing the removal of diesel oil from marine soil

BACKGROUND: The use of agro-industrial wastes to produce high value-added biomolecules such as biosurfactants is a promising approach for lowering the total costs of production. This study aimed to produce biosurfactants using Rhizopus arrhizus UCP 1607, with crude glycerol (CG) and corn steep liquor (CSL) as substrates. In addition, the biomolecule was characterized, and its efficiency in removing petroderivatives from marine soil was investigated. RESULTS: A 22 factorial design was applied, and the best condition for producing the biosurfactant was determined in assay 4 (3% CG and 5% CSL). The biosurfactant reduced the surface tension of water from 72 to 28.8 mN/m and produced a yield of 1.74 g/L. The preliminary biochemical characterization showed that the biosurfactant consisted of proteins (38.0%), carbohydrates (35.4%), and lipids (5.5%). The compounds presented an anionic character, nontoxicity, and great stability for all conditions tested. The biomolecule displayed great ability in dispersing hydrophobic substrates in water, thereby resulting in 53.4 cm2 ODA. The best efficiency of the biosurfactant in removing the pollutant diesel oil from marine soil was 79.4%. CONCLUSIONS: This study demonstrated the ability of R. arrhizus UCP1607 to produce a low-cost biosurfactant characterized as a glycoprotein and its potential use in the bioremediation of the hydrophobic diesel oil pollutant in marine soil

Biosurfactants produced by Microbacterium sp, isolated from aquatic macrophytes in hydrocarbon-contaminated area in the Rio Negro, Manaus, Amazonas / Biossurfactantes produzidos por Microbacterium sp, isolada de macrófita aquática em área impactada por hidrocarbonetos no rio Negro, Manaus, Amazonas

Endophytic bacteria isolated from Eichhornia crassipes (Mart) Solms., collected in oil contaminated wastewater of effluent generated by Petrobras refinery in Manaus were investigated to determine their potential for producing biosurfactants. Assay with 2.6-dichlorophenol indophenol (DCPIP) indicator to verify hydrocarbon biodegradation activity; oil emulsification test; drop-collapse method; surface tension and growth curve of biosurfactant production. The M87 Microbacterium sp. strain chosen for this work was identified by the sequencing of the rDNA region and the chemical characterization was performed by FTIR, UFLC/MS and 1H RMN techniques. The selected bacterial isolate provided 3g L-1 of biosurfactant, using diesel oil as sole carbon source, being efficient in biodegrading oil as demonstrated by the DCPIP test. Fractions obtained by column chromatography were efficient in reducing water surface tension around 40 mN m-1, especially fraction 1, which reduced it to 34.17 mN m-1. The different techniques of chemical analysis used for the identification of the biosurfactant isolate indicated that this is probably a long - chain fatty acid lipid type, which may be used in the future as both biosurfactant in decontamination processes of hydrocarbon-polluted areas or as bioemulsifier in countless processes, since it exhibited no toxicity as determined by Alamar Blue assay.

Foram investigadas bactérias endofíticas isoladas de Eichhornia crassipes (Mart.) Solms., coletadas em águas contaminadas com resíduos de petróleo em um afluente da refinaria da Petrobrás/Manaus, para avaliação da produção de biossurfactantes. Para selecionar o micro-organismos à produção e caracterização de biossurfactantes, foram realizados os seguintes testes: a descoloração do indicador 2,6 indofenol (DCPIP), emulsificação do diesel, colapso da gota, tensão superficial e curva de produção. A caracterização química foi realizada por meio das técnicas de FT-IR, UFLC/MS e RMN1H. A bactéria M87 Microbacterium neste estudo, foi identificada pelo sequenciamento da região rDNA e produziu 3g L-1 de biossurfactantes utilizando o diesel como fonte de carbono, mostrando-se eficiente na ação biodegradadora do petróleo, por meio do teste de Indofenol (DCPIP). As frações obtidas, mostraram-se eficazes na redução da tensão superficial da água abaixo de 40 mN m-1, com destaque para a fração 1 que reduziu a tensão superficial para 34,17 mN m-1. Pelas análises química utilizadas, pode-se inferir que, provavelmente, se trata de um ácido graxo de cadeia longa, que pode ser utilizado futuramente tanto como biossurfactante em processos de descontaminação de ambientes impactados por hidrocarbonetos, assim como bioemulsificante em inúmeros processos uma vez que não apresentou toxicidade por meio do teste realizado.

GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium.

Hepatotoxicity and Nephrotoxicity of Automobile Gas Oil (Diesel Oil) and Its Biochemical Effect on Hematological and Oxidative Stress Parameters in Wistar Male Albino Rats.

Aims: The study was designed to investigate the hepatotoxicity and nephrotoxicity of automobile gas oil (Diesel oil) and its biochemical effect on hematological and oxidative stress parameters in Wistar male albino rats. Methodology: Preliminary toxicity study to determine the volume of diesel oil that could cause toxicity was carried out using 56 healthy albino rats. Another set of 20 albino rats were grouped into two groups and used for the biochemical analysis. Group I animals were the control group and Group II animals were administered with 1 ml of diesel oil per kg body weight. The hematological parameters were determined using BC-3200 Auto Hematology Analyzer. Liver injuries were measured in the plasma using AST, ALT, ALP, GGT, TP and TB Randox kits respectively. Liver histopathological examination was also determined. The oxidative stress parameters assayed in the liver homogenate were TBARS, SOD, CAT and GSH. Kidney injuries were assayed using urea and creatinine Randox kits. Results: Preliminary toxicity study shows that group II albino rats exhibited changes in behavioural pattern such as salivation, respiratory distress, coma, sedation and death. The LD50 was calculated to be 2.5ml/Kg body weight of diesel oil. The results of this study show that WBC, lymphocyte and granulocyte values were significantly increased (P<0.05) in group II animals compared to group I animals. HGB, RBC and HCT values were significantly reduced (P<0.05) in the group administered with diesel oil compared to the healthy group. All the liver biomarker enzymes: AST, ALT, ALP and GGT were significantly increased (P<0.05) in group II rats compared to group I rats. The plasma TB values increased in group II animals while their plasma TP values significantly reduced (P<0.05) when compared to group I animals. Liver histopathological examination also confirmed that administration of diesel oil caused liver lesions in group II animals. Administration of diesel oil to group II rats caused significant reduction (P<0.05) in the activities of the enzymic antioxidants (SOD and CAT) and non enzymic antioxidant (GSH) values of the liver homogenate. The TBARS values were significantly high in group II rats compared to group I rats. These biochemical parameters (SOD, CAT, GSH and TBARS) indicate oxidative stress in animals administered with diesel oil. Conclusion: The results of this study show that administration of diesel oil is hematotoxic, its affects the liver, kidney and oxidative stress parameters.

Laboratory and field evaluation of Metarhizium anisopliae var. anisopliae for controlling subterranean termites

The efficacy of the Metarhizium anisopliae strain ARSEF 6911 was determined in the laboratory and field against two sugarcane pests, Microtermes obesi Holmgren and Odontotermes obesus Rambur (Termitidae: Isoptera). The susceptibility of both termite species to different conidial suspensions (1 × 10(10), 1 × 10(8), 1 × 10(6) and 1 × 10(4) conidia/ml) was determined in laboratory. All conidial suspensions were able to induce mortality. Termite mortality caused by the fungal suspensions was dose dependent. There were no significant differences in the LT50 values between species. Field evaluation of M. anisopliae alone or in combination with diesel oil and thiamethoxam was carried out in two growing seasons (autumn 2005 and spring 2006) at two sites located in Punjab, Pakistan. Dipping the sugarcane setts in these suspensions was tried to determine their effects on germination and percentage of bud damage to sugarcane setts. All treatments significantly reduced termite infestation compared to the untreated control. The combined treatment of M. anisopliae and diesel oil significantly reduced insect damage by attaining higher germination > 55 percent and lower bud damage < 5.50 percent at both sites in both seasons. The results suggest that the application of M. anisopliae and diesel oil in combination might be a useful treatment option for the management of termites in sugarcane.

Investigation about the efficiency of the bioaugmentation technique when applied to diesel oil contaminated soils

This work investigated the efficiency of the bioaugmentation technique when applied to diesel oil contaminated soils collected at three service stations. Batch biodegradation experiments were carried out in Bartha biometer flasks (250 mL) used to measure the microbial CO2 production. Biodegradation efficiency was also measured by quantifying the concentration of hydrocarbons. In addition to the biodegradation experiments, the capability of the studied cultures and the native microorganisms to biodegrade the diesel oil purchased from a local service station, was verified using a technique based on the redox indicator 2,6 -dichlorophenol indophenol (DCPIP). Results obtained with this test showed that the inocula used in the biodegradation experiments were able to degrade the diesel oil and the tests carried out with the native microorganisms indicated that these soils had a microbiota adapted to degrade the hydrocarbons. In general, no gain was obtained with the addition of microorganisms or even negative effects were observed in the biodegradation experiments.

Este trabalho investigou a eficiência da técnica do bioaumento quando aplicada a solos contaminados com óleo diesel coletados em três postos de combustíveis. Experimentos de biodegradação foram realizados em frascos de Bartha (250 mL), usados para medir a produção microbiana de CO2. A eficiência de biodegradação também foi quantificada pela concentração de hidrocarbonetos. Conjuntamente aos experimentos de biodegradação, a capacidade das culturas estudadas e dos microrganismos nativos em biodegradar óleo diesel comprado de um posto de combustíveis local, foi verificada utilizando-se a técnica baseada no indicador redox 2,6 - diclorofenol indofenol (DCPIP). Resultados obtidos com esse teste mostraram que os inóculos empregados nos experimentos de biodegradação foram capazes de biodegradar óleo diesel e os testes com os microrganismos nativos indicaram que estes solos previamente apresentavam uma microbiota adaptada para degradar hidrocarbonetos. Em suma, nenhum ganho foi obtido com a adição dos microrganismos ou mesmo efeitos negativos foram observados nos experimentos de biodegradação.

The use of vinasse as an amendment to ex-situ bioremediation of soil and groundwater contaminated with diesel oil

This work investigated the possibility of using vinasse as an amendment in ex-situ bioremediation processes. Groundwater and soil samples were collected at petrol stations. The soil bioremediation was simulated in Bartha biometer flasks, used to measure the microbial CO2 production, during 48 days, where vinasse was added at a concentration of 33 mL.Kg-1of soil. Biodegradation efficiency was also measured by quantifying the total petroleum hydrocarbons (TPH) by gas chromatography. The groundwater bioremediation was carried out in laboratory experiments simulating aerated (bioreactors) and not aerated (BOD flasks) conditions. In both the cases, the concentration of vinasse was 5 percent (v/v) and different physicochemical parameters were evaluated during 20 days. Although an increase in the soil fertility and microbial population were obtained with the vinasse, it demonstrated not to be adequate to enhance the bioremediation efficiency of diesel oil contaminated soils. The addition of the vinasse in the contaminated groundwaters had negative effects on the biodegradation of the hydrocarbons, since vinasse, as a labile carbon source, was preferentially consumed.

Este trabalho investigou a possibilidade de se usar a vinhaça como um agente estimulador de processos de biorremediação ex-situ. Amostras de água subterrânea e solo foram coletadas em três postos de combustíveis. A biorremediação do solo foi simulada em frascos de Bartha, usados para medir a produção de CO2, durante 48 dias, onde a vinhaça foi adicionada a uma concentração de 33 mL.Kg-1 de solo. A eficiência de biodegradação também foi medida pela quantificação de hidrocarbonetos totais de petróleo (TPH) por cromatografia gasosa. A biorremediação da água subterrânea foi realizada em experimentos laboratoriais simulando condições aeradas (bioreatores) e não aeradas (frascos de DBO). Em ambos os casos, a concentração de vinhaça foi de 5 por cento (v/v) e diferentes parâmetros físico-químicos foram avaliados durante 20 dias. Embora um aumento da fertilização e da população microbiana do solo foram obtidos com a vinhaça, esta estratégia não se mostrou adequada em aumentar a eficiência da biorremediação dos solos contaminados com óleo diesel. A adição de vinhaça às águas subterrâneas contaminadas teve efeitos negativos na biodegradação dos hidrocarbonetos, uma vez que a vinhaça, como uma fonte de carbono facilmente assimilável, foi preferencialmente consumida.

Evaluation of emulsifier stability of biosurfactant produced by Saccharomyces lipolytica CCT-0913

Surface-active compounds of biological origin are widely used for many industries (cosmetic, food, petrochemical). The Saccharomyces lipolytica CCT-0913 was able to grow and produce a biosurfactant on 5 percent (v/v) diesel-oil at pH 5.0 and 32ºC. The cell-free broth emulsified and stabilized the oil-in-water emulsion through a first order kinetics. The results showed that the initial pH value and temperature influenced the emulsifier stability (ES), which was the time when oil was separated. The biosurfactant presented different stabilization properties for vegetable and mineral oil in water solution, despite the highest values of the ES occurring with vegetable oil. The biosurfactant presented smallest ES when compared to commercial surfactants; however, this biosurfactant was not purified.

Os tensoativos de origem biológica são amplamente utilizados em diversas aplicações. O microrganismo Saccharomyces lipolytica CCT-0913 possui a habilidade de crescer em 5 por cento (v/v) óleo diesel a pH 5,0 e 32ºC e produzir biosurfactante. O caldo fermentado livre de células e produzido por S. lipolytica emulsiona e estabiliza emulsões óleo em água de acordo com uma cinética de primeira ordem. Os resultados mostram que o valor do pH inicial e a temperatura influenciam a estabilidade emulsificante (ES), que é medido pelo tempo que a quantidade de óleo. O biosurfactante apresenta diferentes valores de estabilidade emulsificante para óleos vegetais e minerais em emulsões óleo-água, os maiores valores de ES ocorrem nas emulsões utilizando óleo vegetal. O biosurfactante apresenta valores baixos de ES quando comparado com emulsificantes comerciais, entretanto sem sofrer nenhum processo de purificação.

Caracterização físico-química das misturas binárias de biodiesel e diesel comercializados no Amazonas / Physical chemical characterization of binary mixtures of biodiesel and diesel commercialized in Amazonas

O uso de energias renováveis aliadas à preservação do meio ambiente tem despertado interesse cada vez maior em nível nacional e internacional principalmente a utilização do biodiesel em substituição ao diesel de petróleo. Sua utilização traz uma série de vantagens sociais, econômicas e ambientais. Nos últimos anos, houve no Brasil um aumento nas pesquisas sobre o biodiesel. A lei brasileira nº 11.097 de 2005 dispõe sobre a introdução do biodiesel na matriz energética brasileira, misturado ao óleo diesel em um percentual mínimo obrigatório de 2 por cento, com aumento na adição para 5 por cento até 2012. Contudo, essa adição só é possível se o biodiesel estiver dentro dos padrões de qualidade exigidos. Diante disto, neste trabalho foram preparadas misturas de biodiesel adicionado ao diesel interior, nas proporções 2, 5, 10 e 20 por cento (v/v), sendo posteriormente caracterizados a partir da determinação dos parâmetros físico-químicos citados na Resolução da Agência Nacional do Petróleo, Gás Natural e Biocombustíveis (ANP), visando avaliar se as misturas estão em conformidade com a norma. De acordo com os resultados concluiu-se que, tanto o biodiesel quanto suas misturas binárias (B2 a B20) encontram-se dentro dos padrões de qualidade estabelecido pela ANP.

The use of renewable energies allied to the preservation of the environment has awakening more and more interest on the national and international level, especially the utilization of biodiesel in substitution of petroleum diesel. Its use brings a series of social, economical and environmental advantages. In the last few years, there has been an increase in biodiesel research in Brazil. The Brasilian law no. 11.097 of 2005 proposes the introduction of biodiesel in the Brazilian energetic matrix, mixing it with diesel oil at an obligatory percentile minimum of 2 percent, with an increase of 5 percent in the addition, until 2012. However, the addition is only permitted if the biodiesel is within the quality patterns demanded. Before this, mixtures of biodiesel added to interior diesel were prepared, in proportions of 2, 5, 10 and 20 percent (v/v), which were later characterized, as of the physical chemical parameters determined in the Agência Nacional do Petróleo, Gás Natural e Biocombustíveis (ANP) Resolution for evaluating mixtures in accordance with the norm. The results show that, biodiesel as well as their binary mixtures (B2 to B20) are within the quality patterns established by the ANP.

Growth and biosurfactant synthesis by Nigerian hydrocarbon-degrading estuarine bacteria

The ability of microorganisms to degrade petroleum hydrocarbons is important for finding an environmentally-friendly method to restoring contaminated environmental matrices. Screening of hydrocarbon-utilizing and biosurfactant-producing abilities of organisms from an estuarine ecosystem in Nigeria, Africa, resulted in the isolation of five microbial strains identified as Corynebacterium sp. DDv1, Flavobacterium sp. DDv2, Micrococcus roseus DDv3, Pseudomonas aeruginosa DDv4 and Saccharomyces cerevisae DDv5. These isolates grew readily on several hydrocarbons including hexadecane, dodecane, crude oil and petroleum fractions. Axenic cultures of the organisms utilized diesel oil (1.0 % v/v) with generation times that ranged significantly (t-test, P < 0.05) between 3.25 and 3.88 day, with concomitant production of biosurfactants. Kinetics of growth indicates that biosurfactant synthesis occurred predominantly during exponential growth phase, suggesting that the bioactive molecules are primary metabolites. Strains DDv1 and DDv4 were evidently the most metabolically active in terms of substrate utilization and biosurfactant synthesis compared to other strains with respective emulsification index of 63 and 78 %. Preliminary biochemical characterization indicates that the biosurfactants are heteropolymers consisting of lipid, protein and carbohydrate moieties. The hydrocarbon catabolic properties coupled with biosurfactant-producing capabilities is an asset that could be exploited for cleanup of oil-contaminated matrices and also in food and cosmetic industries. Rev. Biol. Trop. 56 (4): 16031611. Epub 2008 December 30.

La capacidad de los microorganismos para degradar hidrocarburos del petróleo es de gran importancia para hallar un método aceptable y ambientalmente amigable para la restauración de terrenos ambientalmente contaminados. Al investigar las capacidades de los organismos de un ecosistema de estuario que utilizan hidrocarburos y producen biosurfactantes, se produjo como resultado el aislamiento de cinco cepas microbianas identificadas como Corynebacterium sp. DDv1, Flavobacterium sp. DDv2, Micrococcus roseus DDv3, Pseudomonas aeruginosa y DDv4 Saccharomyces cerevisiae DDv5. Estas cepas crecieron fácilmente en varios hidrocarburos incluyendo hexadecanos, dodecanos, petróleo crudo y fracciones de petróleo. Los cultivos axénicos de organismos utilizaron diesel (1.0% v/v) con períodos por generación con ámbitos significativos (t-test, P <0.05) de entre 3.25 y 3.88 días, con la consiguiente producción de bio-surfactantes. La cinética del crecimiento indica que la síntesis de bio-surfactante se produjo principalmente durante la fase de crecimiento exponencial, lo que sugiere que las moléculas bioactivas son metabolitos primarios. Las cepas DDv1 y DDv4 fueron evidentemente las más metabólicamente activas en términos de utilización del sustrato y la síntesis de bio-surfactantes en comparación con otras cepas con índices respectivos de emulsificación de 63 y 78%. La caracterización bioquímica preliminar indica que los bio-surfactantes son heteropolímeros constituidos de fracciones de lípidos, proteínas y carbohidratos. Las propiedades catabólicas de los hidrocarburos, junto con las capacidades de producción de bio-surfactantes, es una ventaja que puede ser aprovechada para la limpieza de terrenos contaminados con petróleo y también en la industria alimentaria y cosmética.

Biodegradability of commercial and weathered diesel oils

This work aimed to evaluate the capability of different microorganisms to degrade commercial diesel oil in comparison to a weathered diesel oil collected from the groundwater at a petrol station. Two microbiological methods were used for the biodegradability assessment: the technique based on the redox indicator 2,6 - dichlorophenol indophenol (DCPIP) and soil respirometric experiments using biometer flasks. In the former we tested the bacterial cultures Staphylococcus hominis, Kocuria palustris, Pseudomonas aeruginosa LBI, Ochrobactrum anthropi and Bacillus cereus, a commercial inoculum, consortia obtained from soil and groundwater contaminated with hydrocarbons and a consortium from an uncontaminated area. In the respirometric experiments it was evaluated the capability of the native microorganisms present in the soil from a petrol station to biodegrade the diesel oils. The redox indicator experiments showed that only the consortia, even that from an uncontaminated area, were able to biodegrade the weathered diesel. In 48 days, the removal of the total petroleum hydrocarbons (TPH) in the respirometric experiments was approximately 2.5 times greater when the commercial diesel oil was used. This difference was caused by the consumption of labile hydrocarbons, present in greater quantities in the commercial diesel oil, as demonstrated by gas chromatographic analyses. Thus, results indicate that biodegradability studies that do not consider the weathering effect of the pollutants may over estimate biodegradation rates and when the bioaugmentation is necessary, the best strategy would be that one based on injection of consortia, because even cultures with recognised capability of biodegrading hydrocarbons may fail when applied isolated.

Este trabalho objetivou avaliar a capacidade de diferentes microrganismos em degradar óleo diesel comercial em comparação com um óleo diesel intemperizado coletado da água subterrânea em um posto de combustíveis. Dois métodos microbiológicos foram usados para a avaliação da biodegradabilidade: a técnica baseada no indicador redox 2,6-diclorofenol indofenol (DCPIP) e os experimentos respirométricos usando os respirômetros de Bartha. No primeiro, testamos as culturas bacterianas Staphylococcus hominis, Kocuria palustris, Pseudomonas aeruginosa LBI, Ochrobactrum anthropi e Bacillus cereus, um inóculo comercial, consórcios obtidos do solo e da água subterrânea contaminados com hidrocarbonetos e um consórcio de uma área não contaminada. Nos experimentos respirométricos, foi avaliada a capacidade dos microrganismos nativos do solo de um posto de combustíveis em biodegradar os óleos diesel. Os experimentos com o indicador redox mostraram que apenas os consórcios, mesmo aquele de uma área não contaminada, foram capazes de biodegradar o diesel intemperizado. Em 48 dias, a remoção de hidrocarbonetos totais de petróleo (HTP) nos experimentos respirométricos foi aproximadamente 2,5 vezes maior quando o óleo diesel comercial foi usado. Esta diferença foi causada pelo consumo de hidrocarbonetos facilmente biodegradáveis, presentes em maior quantidade no óleo diesel comercial, como demonstrado pelas análises cromatográficas. Assim, resultados indicam que estudos de biodegradabilidade que não consideram o efeito de intemperização dos poluentes pode sobre estimar as taxas de biodegradação e quando o bioaumento é necessário, a melhor estratégia seria aquela baseada na injeção de consórcios, pois mesmo culturas com reconhecida capacidade de biodegradar hidrocarbonetos podem falhar quando aplicadas isoladamente.