Search

As lipases (E.C. 3.1.1.3) são uma classe de enzimas que catalisam a hidrólise dos triglicerídeos de ácidos gordos livres e glicerol. Essas enzimas encontram uma grande aplicação nas indústrias de alimentos, detergentes, cosméticos, síntese orgânica e farmacêutica. Neste trabalho, as leveduras Candida tropicalis e Meyerozyma caribbica isoladas do bagaço de caju foram testadas quanto à sua capacidade de produção de lipase. As fermentações foram conduzidas em agitador rotatório a 30 °C e 170 rpm. A medida da atividade foi feita através da hidrólise com o pNFL (p-nitrofenil laurato). Para a produção da enzima foi utilizado um meio de cultura contendo resíduos agroindustriais, nomeadamente melaço 10 g/L, milhocina 4 g/L, águas russas 1,0 % (v/v). A atividade do sobrenadante da C. tropicalis foi 233 ± 10 U/L e da M. caribbica foi 146 ± 7,30 U/L em 24 h de fermentação. Esses resultados mostram o potencial dessas estirpes para a produção da enzima lipase a partir de resíduos agroalimentares.

info:eu-repo/semantics/publishedVersion

Subsurface oil reservoirs are complex ecosystems comprising physiological diverse indigenous microbial communities. Those populations have been the subject of intense investigation in recent years. Their study and characterization is of great relevance to understanding the activities and roles of indigenous microorganisms in oil reservoirs, as well as the genesis and transformations of petroleum hydrocarbons. Furthermore, their study can contribute to predict or avoid detrimental effects caused by deleterious microorganisms as well as to promote the development of new and innovative technologies to enhance oil productivity and recovery efficiency from poorly performing and depleted oil fields. As microorganisms inhabiting oil reservoirs are adapted to extreme environmental conditions, they usually harbor enzymes or metabolic functions that might be of great interest for scientific and industrial applications. The current knowledge of the microbial ecology of oil fields, however, is still largely insufficient and further studies are required. This review provides a microbiological perspective of oil reservoirs, focused on the application of microorganisms to increase oil recovery from mature reservoirs as an alternative to the traditional chemical-enhanced oil-recovery methods.

Surface active compounds (SACs) produced by microorganisms are attracting a pronounced interest due to their potential advantages over synthetic counterparts, and to the fact that they could replace some of the synthetics in many environmental and industrial applications. Bioemulsifier production by a Paenibacillus strain isolated from crude oil was studied. The bioemulsifier was produced using sucrose with and without adding hydrocarbons (paraffin or crude oil) under aerobic and anaerobic conditions at 40ºC. It formed stable emulsions with several hydrocarbons, exhibiting similar or better emulsifying activity when compared with chemical SACs, and its emulsifying ability was not affected by exposure to high salinities (up to 300 g/l), high temperatures (100-121ºC) or a wide range of pH values (2-13). In addition, it presented low toxicity and high biodegradability when compared with chemical surfactants, implying a greater environmental compatibility. A preliminary chemical characterization by Fourier Transform Infrared Spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance (1H NMR and 13C CP-MAS NMR) and size exclusion chromatography indicated that the bioemulsifier is a low molecular weight oligosaccharide-lipid complex. To the best of our knowledge, bioemulsifier production by a Paenibacillus strain has not been previously reported. This is also the first description of a low molecular weight bioemulsifier. The features of this novel bioemulsifier make it an interesting biotechnological product for many environmental and industrial applications., Financial support from the projects BIOCLEAN-Desenvolvimento de produtos contendo surfactantes microbianos para limpeza e desinfeção de superfícies industriais e domésticas. QREN-n.º 2013/030215, and NCMICROBIOS -Desenvolvimento de bioprocessos usando microrganismos não convencionais para a produção de biosurfactantes - Convénio FCT-CNPq Nº 17/2013 – Ref.: Projecto nº 6818.

In this work, biosurfactant production by Pseudomonas aeruginosa and Bacillus subtillis strains was optimized using low-cost substrates. The highest biosurfactant production (3.2 g/L) by the P. aeruginosa strain was obtained using a culture medium containing corn steep liquor (CSL) (10% (v/v)) and molasses (10% (w/v)), whereas the best biosurfactant production by the B. subtillis isolate (1.3 g/L) was obtained using a culture medium consisting of 10% (v/v) of CSL. Subsequently, for the B. subtillis strain, the effect of different metals (iron, manganese and magnesium) on biosurfactant production was evaluated. When the culture medium CSL 10% was supplemented with the optimum concentration of those metals simultaneously, the biosurfactant production was increased up to 4.8 g/L. The biosurfactant produced by the P. aeruginosa strain was characterized as a mixture of eight different rhamnolipid congeners, being the most abundant the mono-rhamnolipid Rha-C10-C10, and the biosurfactant produced by the B. subtillis isolate consisted of a mixture of C13-, C14- and C15-surfactin. Both biosurfactants exhibited a good performance in oil recovery assays when compared with chemical surfactants, suggesting their potential use as an alternative to traditional chemical surfactants in enhanced oil recovery or bioremediation.

Oil is a crucial source of energy and one of the main factors driving the world’s economy. Currently, oil production technologies recover only about 40% of the oil originally present in the reservoirs. As the price of petroleum increases and petroleum reserves shrink, exploitation of oil resources in mature reservoirs is essential for meeting future energy demands. Consequently, in the last few years more attention has been focused on enhanced oil recovery (EOR) techniques to mobilize entrapped oil from mature reservoirs. EOR generally involves the use of chemical agents that interfere with the reservoir wettability, pressure and permeability. However, these processes are expensive and often environmentally hazardous. Microbial enhanced oil recovery (MEOR) represents a cost-effective and eco-friendly alternative in which selected microbial strains are used to synthesize compounds analogous to the EOR chemicals to increase the recovery of oil from depleted and marginal reservoirs, thereby extending their life., info:eu-repo/semantics/publishedVersion

Microbial Enhanced Oil Recovery (MEOR) is potentially useful to increment oil recovery from reservoirs beyond primary and secondary recovery operations using microorganisms and their metabolites. In situ stimulation of microorganisms that produce biosurfactants and degrade heavy oil fractions reduces the capillary forces that retain the oil inside the reservoir and decreases oil viscosity, thus promoting its flow and increasing oil production. Bacillus subtilis #573, isolated from crude oil samples obtained from a Brazilian oil field with a moderate temperature (40ºC), was selected for further use in MEOR. This isolate can grow at temperatures up to 55ºC and salinities up to 100 g/l, and produces extracellular biosurfactants under both aerobic and anaerobic conditions in the presence of hydrocarbons. The biosurfactants produced reduced the surface tension to 30 mN/m, decrease the interfacial tension oil-water and exhibited a high emulsifying activity, as well as thermo- and salt-tolerance. The microbial isolate also showed the ability of degrading long-chain n-alkanes under aerobic and anaerobic conditions. Mobilization of heavy crude oil by this isolate was evaluated using sand-pack columns at 40ºC. Growing in situ B. subtilis #573 for 14 days allowed a 17% recovery of the entrapped crude oil. The recovered crude oil showed a decrease in the percentage of n-alkanes higher than C25 and its viscosity was reduced about 32%, which contributed to enhance its mobility. A core flooding equipment was used for a better simulation of the oil reservoir conditions (40ºC and 32.4 bars). Preliminary results showed an additional oil recovery of 4%. The results obtained demonstrated that the selected isolate can be useful to recover residual oil from mature reservoirs.

Surfactants are amphiphilic molecules that comprise both hydrophilic and hydrophobic moieties, allowing the reduction the surface and interfacial tensions, as well as the formation of oil in water or water in oil emulsions. These surface-active compounds are extensively used by petroleum industries in order to reduce the capillary forces that entrapped the oil inside the reservoir. The compounds synthetized chemically, chemical surfactants, have some applicability limitations according some environmental restrictions. Contrarily, sustainable surfactants compounds can be produced naturally by microorganisms, designed by biosurfactants. The biosurfactants are a reliable alternative, since they exhibit lower toxicity, higher biodegradability, and effectiveness at extreme temperature, salinity and pH conditions. This work studies the production of different biosurfactant produced by microorganisms isolated from Brazilian oils. It was evaluated their abilities by measuring surface tensions, interfacial oil-water tensions and emulsification activities. Two Pseudomonas and three Bacillus strains demonstrated capability to grow and produce extracellular biosurfactants at 40ºC. Additionally, the biosurfactants produced were characterized using different spectroscopic techniques, namely FTIR, 1H NMR, ESI/MS and MS/MS, being the biosurfactants produced by Pseudomonas and Bacillus strains characterized as rhamnolipids and surfactins, respectively. The results obtained show that it is important to characterize the biosurfactants in order to understand their surface-active properties, as well as their formation of molecular aggregates: The biosurfactants chemical characterization allows the optimization of their application in bioremediation with crude oil, or in microbial enhanced oil recovery processes.

Surface-active agents or surfactants are amphiphilic molecules that comprise both hydrophilic and hydrophobic moieties, allowing the reduction of the surface and interfacial tensions, as well as the formation of oil in water or water in oil emulsions. Due to their interesting properties, surfactants are widely used by petroleum industries to reduce the capillary forces that retain the oil inside the reservoir. However, since chemical surfactants present some limitations related to environmental issues and restrictive laws, the biosurfactants constitute a reliable alternative due to their lower toxicity, higher biodegradability and effectiveness at extreme temperature, salinity and pH conditions. In this work, different biosurfactant-producing microorganisms under reservoir conditions were isolated from Brazilian oils. Biosurfactant production was evaluated by measuring surface tensions, interfacial oil-water tensions and emulsification activities. Among the isolated microorganisms, two Pseudomonas and three Bacillus strains were able to grow and produce extracellular biosurfactants at 40ºC. Furthermore, the biosurfactants were characterized using different spectroscopic techniques, namely FTIR, 1H NMR, ESI/MS and MS/MS. Structural characterization of these molecules is important to understand their surface-active properties, as well as the formation of molecular aggregates. Biosurfactants produced by Pseudomonas and Bacillus strains were found to be rhamnolipids and surfactins, respectively. The results obtained show that it is important to characterize the biosurfactants in order to optimize their application in bioremediation with crude oil, or in microbial enhanced oil recovery processes.

Microbial Enhanced Oil Recovery (MEOR) is potentially useful to increment oil recovery from reservoirs beyond primary and secondary recovery operations using microorganisms and their metabolites. In situ stimulation of microorganisms that produce biosurfactants and degrade heavy oil fractions reduces the capillary forces that retain the oil inside the reservoir and decreases oil viscosity, thus promoting its flow and increasing oil production. Bacillus subtilis #573, isolated from crude oil samples obtained from a Brazilian oil field with a moderate temperature (40ºC), was selected for further use in MEOR. This isolate can grow at temperatures up to 55ºC and salinities up to 100 g/l, and produces extracellular biosurfactants under both aerobic and anaerobic conditions in the presence of hydrocarbons. The biosurfactants produced reduced the surface tension to 30 mN/m, decrease the interfacial tension oil-water and exhibited a high emulsifying activity, as well as thermo- and salt-tolerance. The microbial isolate also showed the ability of degrading long-chain n-alkanes under aerobic and anaerobic conditions. Mobilization of heavy crude oil by this isolate was evaluated using sand-pack columns at 40ºC. Growing in situ B. subtilis #573 for 14 days allowed a 17% recovery of the entrapped crude oil. The recovered crude oil showed a decrease in the percentage of n-alkanes higher than C25 and its viscosity was reduced about 32%, which contributed to enhance its mobility. A core flooding equipment was used for a better simulation of the oil reservoir conditions (40ºC and 32.4 bars). Preliminary results showed an additional oil recovery of 4%. The results obtained demonstrated that the selected isolate can be useful to recover residual oil from mature reservoirs.

Microbial surfactants are amphipathic molecules produced by a variety of microorganisms that exhibit pronounced surface and emulsifying activities. Biosurfactants can replace synthetic surfactants in environmental and industrial applications, such as bioremediation and microbial enhanced oil recovery. Furthermore, some biosurfactants have been reported as suitable alternatives to synthetic medicines and antimicrobial agents and may be used as effective therapeutic agents, due to their antibacterial, antifungal, antiviral and anti-adhesive activities. The main advantages of biosurfactants when compared with synthetic surfactants include their diversity, specificity, environmentally friendly nature, non-toxicity and high biodegradability, effectiveness at extreme temperatures or pH values, as well as their suitability for scale-up production. Many of the potential applications that have been considered for biosurfactants depend on whether they can be produced economically at large-scale. Several efforts have been conducted to reduce production costs, including the use of agro-industrial wastes as substrates, optimization of medium and culture conditions and efficient recovery processes. In this work, biosurfactant production by a Pseudomonas aeruginosa strain isolated from a crude oil sample was optimized using agro-industrial wastes. A culture medium containing corn step liquor (10% v/v) and molasses (10% w/v) led to the production of 5 g biosurfactant/l, which is about ten times the amount of biosurfactant produced when using LB medium. The crude biosurfactant reduced the surface tension of water to 31 mN/m and exhibited high emulsifying activity (60%), with a critical micelle concentration of 200 mg/l. Moreover, it showed antimicrobial activity against a broad range of Gram-positive and Gram-negative bacteria, as well as a high efficiency in removing oil from contaminated sand, when compared with chemical surfactants. The results obtained suggest the possibility of using this biosurfactant as an alternative to traditional chemical surfactants.

Microbial Enhanced Oil Recovery (MEOR) is a tertiary oil recovery process in which microorganisms and their metabolites are used to retrieve unrecoverable oil from mature reservoirs. Stimulation of microorganisms that produce biosurfactants and degrade heavy oil fractions in situ reduces the capillary forces that retain the oil into the reservoir and decreases oil viscosity, thus promoting its flow. As a result, oil production can be increased. In previous work, Bacillus subtilis strains that are useful for application in MEOR were isolated from crude oil samples. Those isolates were able to grow and produce extracellular biosurfactants at 40ºC under anaerobic conditions in mineral medium supplemented with hydrocarbons. In addition, some isolates degraded the long-chain n-alkanes of paraffinic mixtures. Mobilization of residual oil by those isolates was evaluated using sand-pack columns at 40ºC. Additional oil recoveries obtained with the different isolates using paraffin ranged from 16 to 31%. The recovered paraffin showed a decrease in the percentage of large alkyl chains and its viscosity was reduced. In the present work, the degradation of long-chain n-alkanes and the mobilization of residual oil by the same isolates were studied using heavy oil. The results obtained confirmed that the isolates were able of degrading the long chain n-alkanes of crude oil, and also additional oil recoveries between 8 and 10% were obtained. Furthermore, for a better simulation of the oil reservoir conditions, a core flooding equipment that can work at the oil reservoir pressure was designed and will be used to confirm the applicability of selected isolates to increase oil recovery. The sampled reservoir consists of sands with an average porosity of 25% and a permeability of 50mD. The oil is paraffinic, with low viscosity, high pour point and a gravity of 25º API, with very low solution gas. The reservoir pressure and temperature are respectively 398-440psi and 40ºC., (undefined)

Microbial Enhanced Oil Recovery (MEOR) is a tertiary oil recovery process where microorganisms and their metabolites are used to retrieve unrecoverable oil from mature reservoirs. Stimulation of biosurfactant production by indigenous microorganisms can reduce the capillary forces that retain the oil into the reservoir. The studied reservoir is characterized by alternated oil and water sand layers, with an average porosity of 25% and a permeability of 50 mD. It’s a flat structure at 450 m depth, with an initial pressure of 32.4 bars and a temperature of 42.5ºC. The oil is paraffinic, with low viscosity, high pour point and a gravity of 25º API, with no gas dissolved. Due to these properties, the wells can’t flow naturally and the production has to be lifted, making this reservoir a good candidate for MEOR application. This work addresses the isolation and identification of microorganisms capable of producing biosurfactants and degrading heavy oil fractions at the oil reservoir conditions. Five Bacillus subtilis strains isolated from oil samples were able to grow and produce extracellular biosurfactants at 40ºC under anaerobic conditions in medium supplemented with hydrocarbons. In addition, some of the isolates displayed a capacity to degrade, both in aerobic and anaerobic conditions, the large alkyl chains, and reduce the viscosity of hydrocarbon mixtures. A sand-pack column model was designed to simulate the oil recovery operations and evaluate the mobilization of residual oil by microorganisms. Additional oil recovery using B. subtilis isolates ranged from 19.8 to 35.0%, suggesting that stimulation of biosurfactant production by these strains in situ can contribute to mobilize entrapped oil. The novelty of this technique, compared to the conventional EOR methods, is the application of indigenous microorganisms to increase the oil recovery., (undefined)

Biosurfactant-producing Bacillus subtilis strains isolated from crude oil samples enhance oil recovery at lab scale Eduardo J Gudiña, Lígia R. Rodrigues, José A. Teixeira IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal Microbial Enhanced Oil Recovery (MEOR) is potentially useful to increment oil recovery from reservoirs beyond primary and secondary recovery operations using microorganisms and their metabolites. Stimulation of bacterial growth and biosurfactant production by indigenous microorganisms can reduce the capillary forces that retain the oil into the reservoir. MEOR offers major advantages over conventional EOR, namely low amounts of energy consumption and independence of the price of crude oil [1]. In this work, a sand pack column model was designed to simulate the oil recovery operations in oil reservoirs and evaluate the mobilization of residual oil. Three Bacillus subtilis strains, previously isolated from crude oil samples [2], were used. Those strains grow and produce extracellular biosurfactants at 40ºC under anaerobic conditions in medium supplemented with hydrocarbons. Biosurfactants produced reduce the surface tension of water from 72 to 30 mN/m, exhibit emulsifying activity and are not affected by exposure to high temperatures (121ºC) which makes them good candidates for application in biosurfactant mediated MEOR. Sand pack column assays were performed using paraffin and crude oil. Additional oil recovery using paraffin ranged from 19 to 35% with the different isolates. When crude oil was used as hydrocarbon, the isolates recovered between 19 and 21% of the entrapped oil. The results obtained suggest that stimulation of biosurfactant production by these strains in situ can contribute to mobilize entrapped oil and improve the oil fluidity. [1] Sen R, “Biotechnology in petroleum recovery: The microbial EOR”, Progress in Energy and Combustion Science (2008) 34: 714-724. [2] Gudiña EJ, Pereira JFB, Rodrigues LR, Coutinho JAP, Teixeira JA, “Isolation and study of microorganisms from oil samples for application in Microbial Enhanced Oil Recovery”, International Biodeterioration and Biodegradation (2012) 68: 56-64.

Biosurfactant production was screened in four lactobacilli strains. The highest biosurfactant production (excreted and cellbound biosurfactants) was achieved with Lactobacillus paracasei ssp. paracasei A20, a strain isolated from a Portuguese dairy plant, with a decrease in the surface tension of 6.4mNm−1 and 22.0mNm−1, respectively. Biosurfactant production by this strain was evaluated under different culture broth compositions. The use of different nitrogen sources revealed that yeast extract is essential for bacterial growth, while peptone is crucial for biosurfactant synthesis. For biosurfactant production, the use of peptone and meat extract yielded a higher production when compared to the standard medium, with a surface tension reduction of 24.5mNm−1. Furthermore, experiments were also conducted in a reactor with pH and temperature control. Biomass and biosurfactant production in bioreactor was higher comparing with the experiments conducted in shake flaks. The optimization procedure adopted in the current work was found to improve the biosurfactant production and opened new perspectives for the use of L. paracasei ssp. paracasei A20 as a promising biosurfactant-producer.

Oil recovery comprises a primary phase, which produces oil using the natural pressure drive of the reservoir, and a secondary phase, which includes the injection of water to improve the flow of oil to the wellhead [1,2]. While primary recovery produces 5-10% of the original oil in place, recovery efficiencies in the secondary stage vary from 10% to 40% [1]. Most of the unrecovered oil (up to two-thirds of the total oil reserves) is trapped in the reservoir pores by high capillary forces [2]. Microbial Enhanced Oil Recovery (MEOR) is a tertiary oil recovery process where microorganisms and their metabolites are used to retrieve unrecoverable oil from mature reservoirs. Stimulation of biosurfactant production by indigenous or injected microorganisms can reduce the capillary forces that retain the oil into the reservoir. In this work, a sand pack column model was designed to simulate the oil recovery operations in oil reservoirs and evaluate the mobilization of residual oil by microorganisms. Three Bacillus subtilis strains (309, 311 and 573), previously isolated from crude oil samples, were used in this study. They grow and produce extracellular biosurfactants at 40ºC under anaerobic conditions in medium supplemented with hydrocarbons. Biosurfactants produced by those isolates reduce the surface tension of water from 72 to 30 mN/m, exhibit emulsifying activity and are not affected by exposure to high temperatures (121ºC) which makes them good candidates for application in biosurfactant mediated MEOR. Acrylic columns (250 ml) packed with acid washed sand were first flooded with water, after that saturated with paraffin, and then washed with water to remove the excess of paraffin. Afterwards, the isolates were injected into the columns with the optimized medium and incubated at 40ºC. After 14 days, the columns were flooded with water and the additional oil recovery (AOR) was calculated as the percentage of paraffin recovered. AOR using B. subtilis 309, 311 and 573 was 35.0 ± 1.0 %, 23.5 ± 1.2 % and 19.8 ± 1.9 %, respectively. The results obtained suggest that stimulation of biosurfactant production by these strains in the oil reservoir can contribute to mobilize entrapped oil

The renewed interest in Enhanced Oil Recovery (EOR) techniques as a consequence of the current oil prices is boosting the development of the Microbial Enhanced Oil Recovery (MEOR). This technique is useful to recover incremental oil from a reservoir beyond primary and secondary recovery operations and can be carried by the injection of exogenous or stimulation of indigenous microorganisms. This last approach is here investigated. In this work we address the isolation and identification of microorganisms from Brazilian heavy oil samples capable of producing biosurfactants and to promote the degradation of heavy oil fractions, in particular long-chain hydrocarbons.Different crude oil samples obtained from an oil reservoir were used to isolate microorganisms for application in MEOR. Most of isolates were Pseudomonas and Bacillus strains. The growth of different microbial isolates was studied under both aerobic and anaerobic conditions at 40ºC. These isolates were found to produce extracellular biosurfactants, reduce surface tension and showed a high emulsifying activity. In addition to these capabilities, we studied the ability of these microorganisms to degrade the heavy oil fraction, in particular long-chain hydrocarbons. Several parameters were studied, such as nutritional conditions, incubation time and paraffinic composition of the mixture. Our results show that some of the isolates displayed a capacity to degrade, under aerobic and anaerobic conditions, the large alkyl chains (18+ carbons in alkyl chains) and to reduce the viscosity of hydrocarbon mixtures. Our results, show the ability of the Bacillus subtilis strains to enhance the recovery of paraffinic oil on sand pack columns. These results suggest that the microorganisms’ here isolated have interesting characteristics to be applied for MEOR.

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/10968

Fundação para a Ciência e a Tecnologia (FCT)