Tetradecane 1,14-Dicarboxylic Acid Production from n-Hexadecane by Candida cloacae

The better condition of cultivation for tetradecane 1,14-dicarboxylic acid (DC-16) production from n-hexadecane (n-C₁₆) by Candida cloacae MR-12 was investigated by using acetic acid as carbon source for the growth. In general, the condition suitable for the growth was also favorable for the production of DC-16. The change of pH during cultivation, the use of NaOH solution as pH controlling agent after pH-change and the addition of antifoam stimulated the production of DC-16.

Under the optimum conditions where the culture medium contained 15% (v/v) n-C₁₆, 1.4% (w/v) acetic acid, inorganic salts and growth factors, and pH was changed from 6.5 to 7.75 at 16 hr after the inoculation, the highest level of DC-16 production was attained after about 72 hr cultivation and the amount of the product accumulated was 61.5 g per liter of the medium.

When a mixture of various n-alkanes was used as starting material, DCs corresponding to the respective n-alkanes were produced as mixture.     

Utilization of tannery solid waste for protease production by Synergistes sp. in solid‐state fermentation and partial protease characterization

Synergistes sp. DQ560074 produced a protease in submerged fermentation (SmF) at 400–420 U/mL and in solid‐state fermentation (SSF) at 745–755 U/g. The protease, which belongs to the aspartic protease class, was active over a wide range of pH (5–7) and at high temperatures (25–45°C). The protease is stable and active in various polar protic solvents (50% v/v) like ethanol, isopropanol, n–butanol, in polar aprotic solvents (50% v/v) like acetonitrile, and in non‐polar solvents (50% v/v) such as ethylacetate and toluene, but not in hydrophilic organic solvents (methyl alcohol and acetone). As far as we know, this is the first contribution to the production of a mesophilic protease with solvent stability in SSF using a proteinaceous solid waste.     

Microbial Oxidation of Tetradecanols and Related Substances in Organic Solvents

Microbial oxidations of n-tetradecane, tetradecanols and tetradecanoic acid were investigated by using intact cells of Corynebacterium equi, a hydrocarbon-assimilating bacterium, in an aqueous phase and organic solvents. The bacterial cells were hydrophobic and could be well dispersed in all organic solvents employed to give homogeneous reaction mixtures, and among them, isooctane was found to be the best for the reaction. n-Tetradecane and tetradecanoic acid were completely oxidized in the aqueous phase, but not in isooctane. In contrast, 1-tetradecanol was oxidized much more readily in isooctane than in the aqueous phase, and an oxidation product identified as myristyl myristate was accumulated in isooctane at the conversion rate of 80%. 2-Tetradecanol was also readily oxidized in isooctane, and 2-tetradecanone was obtained at the conversion rate of nearly 100%. Similar results were obtained when toluene and n-hexane were used as the solvent in place of isooctane, while no reaction was observed when chloroform was employed.     

Isolation of new toluene-tolerant marine strains of bacteria and characterization of their solvent-tolerance properties

Aims:  To isolate and characterize new marine bacteria capable of tolerating high concentrations of organic solvents, and to understand the toxic effects of these chemicals on marine bacteria. Methods and Results:  Five marine bacteria able to tolerate 0·1% (v/v) toluene were isolated and characterized on the basis of their growth and survival rates in the presence of different organic solvents. The toluene-tolerant marine bacteria identified in this study could not grow in the presence of 0·1% (v/v) of several organic solvents with a log P_ow higher than that of the toluene (which in theory should be less toxic than toluene). The mechanisms underlying solvent tolerance were explored. Conclusions:  Isolates of four different genera were identified as toluene-tolerant. Toxicity of a second phase of an organic solvent toward these isolates could not be predicted on the basis of the solvents’ log P_ow. Significance and Impact of the Study:  To improve the biodegradation rate of some water-insoluble compounds, double-phase bioreactors can be used. This type of bioreactor will require strains able to grow in a salt-containing environment and able to tolerate a second phase of an organic solvent.     

Role of the Multidrug Efflux Systems of Pseudomonas aeruginosa in Organic Solvent Tolerance

Multidrug efflux pumps with a broad substrate specificity make a major contribution to intrinsic and acquired multiple antibiotic resistance in Pseudomonas aeruginosa. Using genetically defined efflux pump mutants, we investigated the involvement of the three known efflux systems, MexA-MexB-OprM, MexC-MexD-OprJ, and MexE-MexF-OprN, in organic solvent tolerance in this organism. Our results showed that all three systems are capable of providing some level of tolerance to organic solvents such as n-hexane and p-xylene. Expression of MexAB-OprM was correlated with the highest levels of tolerance, and indeed, this efflux system was a major contributor to the intrinsic solvent tolerance of P. aeruginosa. Intrinsic organic solvent tolerance was compromised by a protonophore, indicating that it is substantially energy dependent. These data suggest that the efflux of organic solvents is a factor in the tolerance of P. aeruginosa to these compounds and that the multidrug efflux systems of this organism can accommodate organic solvents, as well as antibiotics.     

Effects of water miscible organic solvents on the activity and conformation of the Baeyer-Villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus: a comparative study

A broader exploitation of enzymes in organic synthesis can be achieved by increasing their tolerance toward organic solvents. In this study, the stability and activity of Baeyer–Villiger monooxygenases from Thermobifida fusca (PAMO) and Acinetobacter sp. (CHMO) in the presence of water miscible organic solvents were compared. PAMO was more stable than CHMO. The concentration of solvent (v/v) at which it halved its activity (C₅₀) was 4‐ to 16‐fold higher than that observed for CHMO. For PAMO, the C₅₀ varied from 16% to 55% of solvent and followed the destabilizing order methanol < ethanol < 1,4‐dioxane < acetonitrile < trifluoroethanol. In the case of CHMO, the maximal C₅₀ was 7% with methanol and even lower with the other solvents. Therefore, methanol was the most tolerated solvent. In the case of PAMO, methanol induced a significant increase of enzyme activity (up to fivefold), which was optimal at 20% (v/v) solvent. Only minor spectral variations were observed with PAMO in 20% methanol, suggesting that the increase of activity observed in this condition is not due to marked conformational changes. Fluorescence and circular dichroism analyses showed that the lower stability of CHMO toward organic solvent correlates with a more pronounced destructive effect on its secondary and tertiary structure. A possible rationale for the higher stability of PAMO could be inferred from inspection of the PAMO and CHMO (two enzymes of similar size) structure, which revealed a higher (up to twofold) number of ionic bridges in PAMO with respect to CHMO. Biotechnol. Bioeng. 2011; 108:491–499. © 2010 Wiley Periodicals, Inc.     

Bioproduction of vanillin using an organic solvent-tolerant <Emphasis Type="Italic">Brevibacillus agri</Emphasis> 13

Nowadays, majority of vanillin supplied to the world market is chemically synthesized from a petroleum-based raw material, raising a concern among the consumers regarding the product safety. In this study, an organic solvent-tolerant Brevibacillus agri 13 previously reported for a strong predilectic property was utilized as a whole-cell biocatalyst for bioproduction of vanillin from isoeugenol (IG). B. agri 13 is the first biocatalyst reported for bioproduction of vanillin at a temperature as high as 45°C. Both pH and temperature were found to affect vanillin production significantly. An extreme level of organic solvent tolerance of B. agri 13 allowed us to utilize it in a biphasic system using organic solvents generally considered as highly toxic to most bacteria. With an addition of butyl acetate at 30% (v/v) as an organic second phase, toxicity of IG exerted onto the biocatalyst was reduced dramatically while faster and more efficient vanillin production was obtained (1.7 g/L after 48 h with 27.8% molar conversion).     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.     

Improvement in lipase-catalyzed methanolysis of triacylglycerols for biodiesel production using a solvent engineering method

A solvent engineering strategy was applied to the lipase-catalyzed methanolysis of triacylglycerols for biodiesel production. The effect of different pure organic solvents and co-solvent mixtures on the methanolysis was compared. The substrate conversions in the co-solvent mixtures were all higher than those of the corresponding pure organic solvents. Further study showed that addition of co-solvent decreased the values of |log P_interface − log P_substrate| and thus led to a faster reaction. The more the values of |log P_interface − log P_substrate| decreased, the faster the reaction proceeded and the higher the conversion attained. Different co-solvent ratio was further investigated. The co-solvent mixture of 25% t-pentanol:75% isooctane (v/v) was optimal, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. There was no obvious loss in lipase activity even after being repeatedly used for 60 cycles (720 h) with this co-solvent mixture as reaction medium. Other lipases and lipase combinations can also catalyze methanolysis in this co-solvent mixture. Furthermore, other vegetable oils were also explored for biodiesel production in this co-solvent mixture and it had been found that this co-solvent mixture media has extensive applicability.     

Removal of dibenzothiophene and its oxidized product in anhydrous water-immiscible organic solvents by immobilized cytochrome c

In various anhydrous water-immiscible organic solvents, dibenzothiophene (DBT) was removed by more than 80% by cumene hydroperoxide (,-dimethylbenzyl hydroperoxide) catalyzed by the immobilized cytochrome c on Celite. DBT-sulfone, the oxidation product of DBT, strongly adsorbed on Celite in highly hydrophobic organic solvents such as hexadecane, decane and n-octane. The adsorbed DBT-sulfone was desorbed from Celite by a simple washing of the Celite with a less hydrophobic solvent such as octanol.     

Investigation of saturated and aromatic hydrocarbon resistance mechanisms in Pseudomonas aeruginosa IBBML1

Pseudomonas aeruginosa IBB_ML1, isolated from Poeni petroleum sludge, was able to tolerate and degrade both saturated (n-hexane, n-heptane, n-hexadecane, cyclohexane) and aromatic (benzene, ethylbenzene, propylbenzene, xylene isomers, styrene) hydrocarbons. Molecular studies have revealed that the high hydrocarbon resistance of Pseudomonas aeruginosa IBB_ML1 could be due to the action of members of the HAE1 (hydrophobe/amphiphile efflux 1) family of transporters. It is further possible that additional mechanisms may account for the tolerance of Pseudomonas aeruginosa IBB_ML1 to hydrocarbons, and a combination of short-term and long-term mechanisms may act together in the adaptation of Pseudomonas aeruginosa IBB_ML1 cells to saturated and aromatic hydrocarbons. β-galactosidase activity measurements revealed that there was significant induction of the lacZ gene in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of either 5% and 10% (v/v) saturated or aromatic hydrocarbons, compared with control (cells incubated without hydrocarbons). Rhodamine 6G accumulation in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of 5% and 10% (v/v) saturated hydrocarbons was higher than rhodamine 6G accumulation in cells grown in the presence of 5% and 10% (v/v) aromatic hydrocarbons. The study of cellular and molecular modifications to Pseudomonas aeruginosa IBB_ML1 induced by 5% and 10% (v/v) saturated and aromatic hydrocarbons revealed a complex response of bacterial cells to the presence of different hydrophobic substrates in the culture medium.     

Screening of biocompatible organic solvents for enhancement of lipid milking from Nannochloropsis sp.

To extract the microalgal lipid in situ, biocompatible solvents were screened for lipid milking of Nannochloropsis sp. in an aqueous–organic system. The effects of organic solvents on the microalgal growth, the lipid extractability, the dehydrogenases activity and the cell membrane integrity were investigated by UV–visible spectrophotometer, FT-IR spectroscopy, 2,3,5-triphenyltetrazolium chloride (TTC) and Evans Blue stain method, respectively. The results showed that alkane solvents with log P > 5.5 were biocompatible while the hydrophilic solvents with log P < 5.5 were toxic to Nannochloropsis sp. due to the deactivated dehydrogenase and increased cell membrane permeability. As 10% (v/v) hexadecane was used to establish biphasic system, the total lipid production of Nannochloropsis sp. was increased by 28.9% compared to the control. The screened biocompatible solvent hexadecane enhanced not only the algal growth but also the lipid accumulation, showing an effective way to facilitate the process for in situ lipid milking from Nannochloropsis sp.     

Immobilization of Thermoanaerobium brockii alcohol dehydrogenase on SBA-15

The activity of Thermoanaerobium brockii alcohol dehydrogenase (TBADH) adsorbed on mesoporous silica SBA-15 was compared with that of the free enzyme in water and in biphasic system (water phase up to 50% v/v water). TBADH was active at a water concentration ≥10% v/v. In the reduction reaction of sulcatone to sulcatol carried out in biphasic systems, the yield obtained with SBA-15-adsorbed TBADH was up to 5.5-fold higher than that with the free enzyme, which suggests a higher stability of the immobilized enzyme toward the organic solvent. The nature of the organic solvent substantially influenced the degree of conversion that, for example, was 7.4% in toluene and 31.6% in petroleum ether.     

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

Abstract

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55?°C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72?h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.     

Synthesis of propyl gallate by mycelium-bound tannase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in organic solvent

Aspergillus niger with mycelium-bound tannase activity was employed to investigate the synthesis of propyl gallate from gallic acid and 1-propanol in organic solvents. The effects of various organic solvents (log P: −1.0 to 6.6) on the enzymatic reactions showed that benzene (log P: 2.0) was the most suitable solvent. The water content and protonation state of mycelium-bound enzyme both had significant effects on the activity of tannase. The maximum molar conversion (65%) was achieved with 7.3% (v/v) 1-propanol and 5.56 mM gallic acid at stirring speeds of 200 rev/min, 40 °C in presence of anhydrous sodium sulfate and PEG-10,000. Enzyme specificity for the alcohol portion (C₁–C₈) of the ester showed that the optimum synthesis was observed with alcohols ranging from C₃ to C₅.     

Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium

Aims: To isolate and characterize new bacteria capable of tolerating high concentrations of organic solvents at high temperature. Methods and Results: A solvent‐tolerant, thermophilic bacterium was isolated from hot spring samples at 55°C. The strain PGDY12 was characterized as a Gram‐positive bacterium. It was able to tolerate 100% solvents, such as toluene, benzene and p‐xylene on plate overlay and high concentrations of these solvents in liquid cultures. A comparison of growth showed that 0·2% (v/v) benzene and 0·15% (v/v) p‐xylene were capable of enhancing the final cell yields. Transmission electron micrographs showed the incrassation of electron‐transparent intracellular material and the distorted cytoplasm in case of the cells grown in toluene. A phylogenetic analysis based on 16S rRNA sequence data indicated that the strain PGDY12 was member of the genus Anoxybacillus. Conclusions: The thermophilic, Gram‐positive Anoxybacillus sp. PGDY12 exhibited a unique and remarkable ability to tolerate solvents at 55°C. Significance and Impact of the Study: The solvent tolerance properties are less known in thermophilic bacteria. The Anoxybacillus sp. PGDY12 is the first strictly thermophilic bacterium able to tolerate a broad range of solvents. This strain is a promising candidate for use as a high temperature biocatalyst in the biotechnological applications.     

Formation of Glutaric and Adipic Acids from n-Alkanes with Odd and Even Numbers of Carbons by Candida tropicalis OH23

Many strains of yeast which can utilize n-alkanes as the sole source of carbon were isolated from flowers and fruits. Among them, a strain, OH23, identified as Candida tropicalis, formed acidic substances from n-alkanes. The principal products from n-alkanes with odd and even numbers of carbons were identified as glutaric and adipic acids, respectively. The culture conditions for their formation were investigated. n-Pentadecane and n-hexadecane were the best substrates for the formation of glutaric and adipic acids, respectively. Yields of 170 mg of glutaric and 64 mg of adipic acid were obtained from 100 ml of media containing 4% (v/v) n-pentadecane and n-hexadecane, respectively, and 0.5% casamino acids.     

In situ recovery of 2,3-butanediol from fermentation by liquid–liquid extraction

End-product conversion, low product concentration and large volumes of fermentation broth, the requirements for large bioreactors, in addition to the high cost involved in generating the steam required to distil fermentation products from the broth largely contributed to the decline in fermentative products. These considerations have motivated the study of organic extractants as a means to remove the product during fermentation and minimize downstream recovery. The aim of this study is to assess the practical applicability of liquid–liquid extraction in 2,3-butanediol fermentations. Eighteen organic solvents were screened to determine their biocompatibility, and bioavailability for their effects on Klebsiella pneumoniae growth. Candidate solvents at first were screened in shake flasks for toxicity to K. pneumoniae. Cell density and substrate consumption were used as measures of cell toxicity. The possibility of employing oleyl alcohol as an extraction solvent to enhance end product in 2,3-butanediol fermentation was evaluated. Fermentation was carried out at an initial glucose concentration of 80 g/l. Oleyl alcohol did not inhibit the growth of the fermentative organism. 2,3-Butanediol production increased from 17.9 g/l (in conventional fermentation) to 23.01 g/l (in extractive fermentation). Applying oleyl alcohol as the extraction solvent, about 68% of the total 2,3-butanediol produced was extracted. An erratum to this article can be found at     

Effects of oxygen and nitrate on growth of Escherichia coli and Pseudomonas aeruginosa in the presence of organic solvents

Escherichia coli and Pseudomonas aeruginosa grown in the presence of certain harmful organic solvents become susceptible to these solvents during the cultivation. This susceptibility is conspicuous in the stationary phase of growth. The organic solvent tolerance levels of these microorganisms were maintained when the oxygen concentration was kept high. The tolerance levels were maintained also when these organisms were grown with nitrate present under anaerobic respiratory conditions. Received: 21 March, 1997 / Accepted: July 20, 1997     

Characteristics of butanol fermentation by a low-acid-producing Clostridium acetobutylicum B18

Fermentation characteristics of Clostridium acetobutylicum B18 were studied in batch experiments with and without pH control. This strain is shown to be potentially useful in simultaneous acetone-butanol-ethanol fermentation-separation systems because of its low acid production. In a pH-uncontrolled batch culture this strain produced mostly solvents, including 15 g/l of butanol. Ethanol production was low. Strain B18 recycled organic acids more efficiently than other strains. In particular, butyric acid was completely recycled when glucose was not limiting. Yield of liquid products (solvents plus organic acids) and carbon recovery in total products (gas plus liquid) were 33.1–36.4 wt% and 90–91 mol%, respectively, for 20–80 g/l of initial glucose. Glucose consumption and the percentage of butanol among solvents were higher at 32°C than at 37°C. Strain B18 required approximately 0.4 g/l of undissociated butyric acid at the onset of solvent production in pH-uncontrolled batch culture. The low undissociated butyric acid requirement enabled this strain to produce 13.8 g/l of butanol at a controlled pH of 6.0.Contribution no. 19998 of the Minnesota Agricultural Experiment Station Correspondence to: C.-H. Park