Emulsan Production Research Articles

Synergistic Effect of Biosurfactant and Nanoparticle Mixture on Microbial Enhanced Oil Recovery

AbstractRecently, nanoparticles have become an attractive agent for enhanced oil recovery (EOR). Because much of the work on nanoparticles for enhanced oil recovery is still at the laboratory stage and to gain a better understanding of this technique, it is essential to study the effect of nanoparticles on EOR. In addition, the world is now more environmentally aware, presenting the opportunity to use biosurfactants for EOR. In this paper, the synergistic effect of biosurfactant and nanoparticles on the removal of oil in a glass micromodel was evaluated. In this study, an aqueous solution of emulsan biosurfactant with addition of SiO2 nanoparticles was used as a nanofluid. The emulsan biosurfactant was produced by Acinetobacter calcoaceticus PTCC1318. The production of emulsan was confirmed by FTIR and 1H NMR analysis. According to our results, the use of the mixture of biosurfactant and nanoparticle (nanofluid) permitted a 90% reduction of interfacial tension in comparison with biosurfactant solution alone. Micromodel oil displacement experiments with kerosene showed around 10 and 20% recovery of residual oil after water flooding when the emulsan and nanofluid were injected, respectively. These results are useful in extending the application of nanostructures in ex situ microbial enhanced oil recovery.

Optimizing emulsan production of A. venetianus RAG-1 using response surface methodology

Statistical experimental design was used to optimize medium constituents for emulsan production by Acinetobacter venetianus RAG-1 in batch cultivation. The factors affecting emulsan production were screened by a two-level factorial design, and the optimal concentration of medium constituents for emulsan production were determined by the method of steepest path ascent and central composite experimental design. Experimental results showed that the optimal medium constituents were 9.16 g/L ethanol, 8.2 g/L KH(2)PO(4), 23.32 g/L K(2)HPO(4), 5.77 g/L (NH(4))(2)SO(4) and 0.354 g/L MgSO(4)*7H(2)O. Under this optimal composition, the predicted emulsan production was 72.198 mg/L, and experimental value was 73.312 mg/L for 80 h culture in the shake flasks, and the emulsan yield by A. venetianus RAG-1 was enhanced nearly 1.48-fold (from 49.5 to 73.312 mg/L). Based on the results, we identify the optimal medium constituents for emulsan production and could take advantage of strategy for scale up the fermentation of emulsan production.

Emulsan quantitation by Nile red quenching fluorescence assay

A Nile red fluorescent technique to quantify 20-200 microg ml(-1) of emulsan was developed. Nile red dissolved in DMSO showed an adsorption peak at 552 nm, and emission peak at 636 nm, with molar extinction coefficient of 19,600 cm(-1) M(-1). Nile red fluorescence in DMSO was proportionally quenched by emulsan and the quenching was time-dependent. The assay was used to follow the production of emulsan by cultures of Acinetobacter venetianus RAG-1.

Involvement of a protein tyrosine kinase in production of the polymeric bioemulsifier emulsan from the oil-degrading strain Acinetobacter lwoffii RAG-1.

The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V(max) of 12 micromol of PNPP min(-1) mg(-1) and a K(m) of 8 mM PNPP at 30 degrees C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.

Analysis of the wee gene cluster responsible for the biosynthesis of the polymeric bioemulsifier from the oil-degrading strain Acinetobacter lwoffii RAG-1.

A cluster (27 kbp) of genes responsible for the biosynthesis of the amphipathic, polysaccharide bioemulsifier emulsan from the oil-degrading Acinetobacter lwoffii RAG-1 was isolated and characterized. The complete sequence of this cluster, termed wee, consisted of 20 ORFs. One set of 17 ORFs was transcribed in one direction, while a second set of three ORFs, 607 bp upstream of the first, was transcribed in the opposite direction. Mutations in either of the two regions caused defects in emulsan production, yielding specific activities of 5-14% of parental emulsifying activity. Putative functions could be assigned to proteins involved in production of nucleotide amino sugar precursors, transglycosylation, transacetylation, polymerization and transport. However, no JUMPstart or ops sequences, normally found associated with some polysaccharide biosynthetic gene clusters, were identified. Evidence is presented suggesting that the bioemulsifier may be a member of the group 1 or group 4 polysaccharides.

Fuzzy control of ethanol concentration for emulsan production in a fed-batch cultivation ofAcinetobacter Calcoaceticus RAG-1

A fuzzy control system was organized and applied to the control of ethanol concentration in a fed-batch cultivation process for emulsan production byAcinetobacter calcoaceticus RAG-1. The membership functions and fuzzy rules were determined by sets of data and experiences obtained from the preliminary culture experiments. The input variables, error (the difference between the set point value and the process variable) and the change of the error, were fuzzified by using the membership functions and the output variable, change of the ethanol feed rate, was inferred based on the membership functions and the given fuzzy rules. To obtain the numerical value for the output variable, the center-of-gravity method was used in the defuzzification procedure. The results showed that the ethanol concentration was well regulated around optimal level and the emulsan yield was increased compared with that of the cultivation controlled by the conventional feedback control loop.

Control of ethanol concentration in a fed-batch cultivation of Acinetobacter calcoaceticus RAG-1 using a feedback-assisted iterative learning algorithm

A control scheme using a feedback-assisted iterative learning control algorithm to maintain the optimal ethanol concentration for the enhancement of cell growth and emulsan production in fed-batch cultivation of Acinetobacter calcoaceticus RAG-1 is presented. The optimal concentration of ethanol for cell growth and emulsan production was determined by considering the inhibitory effect of ethanol. To maintain the optimal concentration of ethanol, continuous feeding of ethanol was adopted using the feedback-assisted iterative learning control algorithm. A mathematical kinetic model is used to simulate the objective process. The transfer function of the objective process was approximated to the FOPDT (First Order Plus Dead Time) model and model parameters were determined by the LSE (Least Square Estimation) method. In the transfer function modeling, to evaluate the process model parameters which can describe the objective process, the disturbance included in the process was eliminated by linearization of two data sets of preliminary fed-batch runs. Fed-batch cultivation experiments using a feedback-assisted learning control algorithm were performed. The results showed that the convergence performance was improved as the run was iterated and the emulsan yield was increased compared with that of the runs controlled by only the feedback loop.

Kinetic model for effects of ethanol and phosphate on cell growth and emulsan production inAcinetobacter calcoaceticus RAG-1

A mathematical kinetic model was proposed to describe the cell growth and the emulsan production in batch cultivations ofAcinetobacter calcoaceticus RAG-1. Ethanol and phosphate concentrations were chosen as the key variables, which affected the cell growth and emulsan production in the batch cultivations. The cell growth was inhibited by high concentrations of ethanol and was slightly affected by intracellular phosphate level. And the emulsan production was related to the intracellular phosphate level dependent upon the extracellular phosphate concentration. Kinetic model for the cell growth was formulated using the ethanol inhibition term and the intracellular phosphate level. The relationship between extracellular and intracelluiar phosphate level was expressed by the concept of active transport. Kinetic model for the emulsan production was represented using growth-associated term and intracellular phosphate inhibition term. Release of emulsan was presumed as the primary release from the surface of viable cell and the secondary release by cell lysis. The model predicted the experimental results with good agreement.

Effects of ethanol and phosphate on emulsan production by Acinetobacter calcoaceticus RAG-1

To enhance emulsan production, the effects of ethanol and phosphate on cell growth and emulsan synthesis by Acinetobacter calcoaceticus RAG-1 were investigated in batch cultivations. It was observed that an optimal concentration of ethanol and phosphate existed for the maximization of emulsan production in batch cultivations. High concentrations of ethanol (above 10 g l −1) inhibited cell growth, which resulted in decreased emulsan synthesis rates. Optimum level of ethanol for emulsan production (about 6.5 g l −1) was decided to maximize the specific growth rate and specific production rate of emulsan. High concentrations of phosphate (above 18.15 g l −1) inhibited cell growth and emulsan production. The intracellular phosphate level affected the specific growth rate. The optimum level of phosphate for emulsan production (about 12.1 g l −1) was identified in order to maximize the specific growth rate as well as the specific production rate. In a fed-batch cultivation, high volumetric production of emulsan was achieved by continuous feeding of ethanol and phosphate to maintain ethanol and phosphate concentrations at the optimal level.

Production of emulsan in a fermentation process using soybean oil (SBO) in a carbon-nitrogen coordinated feed

A fermentation process for the microbial production of an amphipatic lipopolysaccharide, emulsan, has been established using a triglyceride carbon source in a coordinated carbon–nitrogen feed strategy. The polysaccharide was produced by the Acinetobacter strain at a productivity of about 0.5 g emulsan/L h while utilizing only the fatty acids (FA) portion of the triglycerides or free fatty acids that were fed into the medium. A useful correlation between the utilization of the fatty acids and the consumption of the nitrogen was found and employed for the practical feed strategy using an appropriate CN ratio (7.7 g C/g N) of the soybeanoil (SBO) (carbon source) to the ammonium hydroxide base (nitrogen source). Either the pH control or the supervising computer system could accomplish the adequate balanced feed in to the fermentor. Lipolysis slowdown was overcome by switching from a triglyceride carbon source to a free fatty acids one. A yield of about 0.2 g emulsan/g fatty acids was obtained and a final concentration of over 20 g/L was reached. The polymeric product was found to be partially associated with the cell-oil complexes in the fermentation broth unless the oily carbon source was efficiently exhausted. A fedbatch fermentation that employed a shift of the carbon source feed from triglycerides to free fatty acids appeared to be an appropriate and feasible way of producing the polymer.

Self-Cycling Fermentation Applied to Acinetobacter calcoaceticus RAG-1

The self-cycling fermentation (SCF) technique was applied to a culture of Acinetobacter calcoaceticus RAG-1. This method was shown to result in synchronization of the cells, achieving a 77% improvement in cell synchrony over that of the batch case. Cellular occurrences, averaged out by asynchronous batch cultures, were magnified by the temporal alignment of metabolic events brought about by the synchronization associated with SCFs. The cell population doubled only once per cycle, thus establishing an equality between cycle time and doubling time. Parameters of interest were biomass concentration, total bioemulsifier (emulsan) production, cycle time, and residual carbon concentration. Cycle-to-cycle variation of these parameters was, in most cases, insignificant. Repeatability of doubling time estimates (based on 95% confidence intervals) was roughly 7 to 10 times better between cycles in an SCF than between batch replicates. The carbon substrate was completely utilized in all cases in which it was measured, giving this technique an advantage over chemostat-type fermentations. The dissolved-oxygen profiles monitored throughout a cycle were found to be repeatable. A characteristic shape, which can be related to the growth of the organism, was associated with each carbon source. The specific emulsan productivity of SCFs was found to be approximately 50 times greater than that of the batch process and 2 to 9 times greater than that of the chemostat, depending on the dilution rate considered. With respect to specific emulsan production, a 25-fold improvement over that in an immobilized cell system recently introduced was obtained. Thus, SCFs are a viable alternative to established fermentation techniques.

Production of emulsan in a fermentation process using soybean oil (SBO) in a carbon–nitrogen coordinated feed

A fermentation process for the microbial production of an amphipatic lipopolysaccharide, emulsan, has been established using a triglyceride carbon source in a coordinated carbon-nitrogen feed strategy. The polysaccharide was produced by the Acinetobacter strain at a productivity of about 0.5 g emulsan/L h while utilizing only the fatty acids (FA) portion of the triglycerides or free fatty acids that were fed into the medium.A useful correlation between the utilization of the fatty acids and the consumption of the nitrogen was found and employed for the practical feed strategy using an appropriate C--N ratio (7.7 g C/g N) of the soybeanoil (SBO) (carbon source) to the ammonium hydroxide base (nitrogen source). Either the pH control or the supervising computer system could accomplish the adequate balanced feed in to the fermentor. Lipolysis slowdown was overcome by switching from a triglyceride carbon source to a free fatty acids one. A yield of about 0.2 g emulsan/g fatty acids was obtained and a final concentration of over 20 g/L was reached. The polymeric product was found to be partially associated with the cell-oil complexes in the fermentation broth unless the oily carbon source was efficiently exhausted. A fedbatch fermentation that employed a shift of the carbon source feed from triglycerides to free fatty acids appeared to be an appropriate and feasible way of producing the polymer.

Adherence of emulsan-producing Acinetobacter calcoaceticus to hydrophobic liquids

The adherence of Acinetobacter calcoaceticus ATCC 31012 cells to hexadecane and perfluorocarbon FC-43 was measured using the Bacterial Adherence To Hydrocarbon (BATH) assay. In batch culture the adherence of cells to both hydrophobic liquids increased sharply during the exponential growth phase and remained high for the remainder of the culture period. No correlation was found between the surface emulsan concentration and the adherence to perfluorocarbon FC-43 and hexadecane. In continuous cultures, the production of cell-free emulsan was found to be growth-associated. The adherence to both hydrophobic liquids decreased with increasing dilution rate while the amount of surface emulsan increased. Furthermore, exogenously added emulsan decreased the adherence to hydrophobic liquids. Thus, the accumulation of surface emulsan does not appear to have a beneficial effect for cell adherence to hydrophobic liquids.

Enhanced emulsan production in mutants of Acinetobacter calcoaceticus RAG-1 selected for resistance to cetyltrimethylammonium bromide.

Mutants of Acinetobacter calcoaceticus RAG-1 that produced elevated levels of the polymeric bioemulsifier emulsan were isolated on the basis of their resistance to the cationic surfactant cetyltrimethylammonium bromide (CTAB). Such mutants showed maximum enhancement in both overall yield and specific productivity of some two- to threefold over that of the wild type. In addition, the effect was also observed in a resting cell system in the presence of chloramphenicol, indicating that the mutation is not simply the result of faster growth. When CTAB-tolerant mutants were subjected together with the sensitive parent to the detergent under growing conditions, only the mutants were found to grow. The results suggest that the mutation for CTAB resistance leads to enhanced capsule production. This was confirmed quantitatively by a specific enzyme-linked immunosorbent assay for the cell-bound emulsan minicapsule.

Localization of emulsan-like polymers associated with the cell surface of acinetobacter calcoaceticus.

Various immunochemical techniques were employed to probe the relationship between the extracellular emulsifying agent (emulsan) and the cell-associated form of the polymer in Acinetobacter calcoaceticus RAG-1. Using an emulsan-specific antibody preparation, immunocytochemical labeling revealed that an emulsan-like antigen is a major component of the 125-nm minicapsule which envelopes the exponential-phase cell of the parent strain. The marked reduction of this capsule in stationary-phase cells was correlated with the production of extracellular emulsifying activity. Crossed immunoelectrophoresis techniques demonstrated that the major antigenic component (S1) of the culture supernatant fluid is immunochemically identical to purified emulsan, yet electrophoretically distinct. The characteristics of the parent strain were compared with those of two phage-resistant mutant strains which are defective in extracellular emulsan production. One of these mutants, termed TR3, lacked both the emulsan-like capsule on the cell surface and the extracellular S1 component. A second phage-resistant emulsan-defective mutant (TL4) was characterized by an antigenically altered and inactive form of extracellular emulsan. A relatively small amount of emulsan-like capsular material was consistently demonstrated on the cell surface of this mutant. The correlation between phage sensitivity and extracellular emulsan production was strengthened by the fact that emulsan-specific antibodies inhibited both emulsification activity and phage adsortion onto cells of the parent strain.

Immunochemical identification of the major cell surface agglutinogen of Acinetobacter calcoaceticus RAG-92.

The immunochemical and immunocytochemical characteristics of three Acinetobacter calcoaceticus RAG strains were compared in order to clarify the relationship between antibody-induced agglutination and the production of polyanionic extracellular emulsifier (termed emulsan). In addition to the parent, RAG-92, two mutant strains were examined: (1) a non-agglutinating emulsan-producer (AB15), and (2) an agglutinating mutant (16TLU) defective in the production of emulsan. A combined genetic-immunochemical approach was employed. This included the comparison of crossed immunoelectrophoresis patterns of parent and mutant supernates and the effect of absorption of anti-whole cell antiserum with mutant cells. In addition, agglutinability and competition studies were performed as well as electron microscopic cytochemistry. The results demonstrated that three major antigenic components were associated with the cell surface and the supernate. Mutant cells were altered both in their cell surface properties and in their extracellular products. One antigenic component, termed component C3, was the major cell surface agglutinogen; this component was absent in non-agglutinating mutants. Component C3 may be identical with or attached to the 300 nm projections on the parent cell surface, but it is not directly related to the presence of emulsan. It appears that emulsan plays little or no role in the phenomenon of antibody-induced agglutination of this organism.

Emulsan production by Acinetobacter calcoaceticus in the presence of chloramphenicol.

When exponentially growing cultures of Acinetobacter calcoaceticus RAG-1 or RAG-92 were either treated with inhibitors of protein synthesis or starved for a required amino acid, there was a stimulation in the production of emulsan, an extracellular polyanionic emulsifier. Emulsan synthesis in the presence of chloramphenicol was dependent on utilizable sources of carbon and nitrogen and was inhibited by cyanide or azide or anaerobic conditions. Radioactive tracer experiments indicated that the enhanced production of emulsan after the addition of chloramphenicol was due to both the release of material synthesized before the addition of the antibiotic (40%) and de novo synthesis of the polymer (60%). Chemical analysis of RAG-1 cells demonstrated large amounts of polymeric amino sugars; it was estimated that cell-associated emulsan comprised about 15% of the dry weight of growing cells. The data are consistent with the hypothesis that a polymeric precursor of emulsan accumulates on the cell surface during the exponential growth phase; in the stationary phase or during inhibition of protein synthesis, the polymer is released as a potent emulsifier.

Emulsan in Acinetobacter calcoaceticus RAG-1: Distribution of Cell-Free and Cell-Associated Cross-Reacting Material

Emulsan is an extracellular polymeric bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. Antibodies prepared against purified emulsan inhibited the activity of the polymer in a standard emulsification test. These antibodies were used to develop a sensitive enzyme-linked immunosorbent assay to monitor changes in cell-free emulsan throughout the growth cycle. This assay was also used to detect emulsan associated with the cell surface and to monitor changes in the distribution of cell-free and cell-associated emulsan throughout the growth cycle. Cells in the early exponential phase exhibited relatively large amounts of cell-associated emulsan which decreased rapidly between the midexponential and early stationary phases. This drop in cell-associated material was accompanied by a rise in the concentration of extracellular polymer. Moreover, in agreement with previous results (C. Rubinovitz, D. L. Gutnick, and E. Rosenberg, manuscript in press), production of cell-free emulsan was enhanced in the presence of chloramphenicol. The release of this material from the cell surface in the presence of chloramphenicol apparently involved the synthesis of cell-associated cross-reacting material since the relative amount of such cell-bound polymer remained constant during the treatment with the drug.

Relationship between phage resistance and emulsan production, interaction of phages with the cell-surface of Acinetobacter calcoaceticus RAG-1

The hydrocarbon-degrading strain Acinetobacter calcoaceticus RAG-1 produces an extracellular emulsifying agent capable of forming stable oil-in-water emulsions. The bioemulsifier, termed emulsan, is a polyanionic heteropolysaccharide (M.W. 106) composed mainly of N-acyl D-galactosamine and an N-acyl hexosamine uronic acid. In order to probe the interaction of emulsan with the cell surface prior to its release into the growth medium, two new virulent bacteriophages for A. calcoaceticus RAG-1 were isolated from sewage and the properties of phage resistant mutants were studied. The two phages, ap-2 and ap-3, were differentiated on the basis of plaque morphology, electron microscopy and buoyant density. Isolated mutants of A. calcoaceticus RAG-1 which were resistant to one of the two phages retained sensitivity to the other phage. Resistance to phage ap-3 was accompanied by a severe drop in emulsan production. Independently isolated derivatives of A. calcoaceticus RAG-1 with a defect in emulsan production also turned out to be resistant towards phage ap-3. Antibodies prepared against purified emulsan specifically inhibited phage ap-3 adsorption to the cell surface of the parental strain.