Increased secretion of lignolytic enzymes by the Lentinus tigrinus fungus after addition of butanol and toluene in submerged cultivation

We studied the effects of butanol and toluene on secretion of lignolytic enzymes by the Lentinus tigrinus fungus during submerged cultivation. Addition of butanol and toluene during the trophophase was followed by an increase in laccase and peroxidase activity of the culture and change in the composition of phospholipids and fatty acids. The ratio of phosphatidylcholine and phosphatidic acid decreased, while the amount of lysophosphatidylcholine, phosphatidylethanolamine, phosphoinositides, phosphatidylserine, and unsaturated fatty acids decreased. These changes resulted in an increase in the unsaturation index.     

Isolation and phenotypic characteristics of the <Emphasis Type="Italic">Escherichia coli</Emphasis> butanol-tolerant mutants

Resistance to butanol is a key factor affecting microbial ability to produce economically profitable amounts of butanol. In this study, an Escherichia coli strain capable of growth in the presence of 1.5% butanol was isolated. The mutant MG1655 ButR was characterized by increased resistance to ethanol, isopropanol, and bivalent ions but exhibited supersensitivity to osmotic shock. Compared to the wild type strain, the butanol-tolerant mutant was more sensitive to antibiotics inhibiting protein synthesis but was more resistant to membrane-penetrating antibiotics, such as surfactin. Increased content of unsaturated fatty acids was found in the membranes of butanol-tolerant mutants. It was revealed that overexpression of the genes encoding cold-shock proteins decreased butanol tolerance of both mutant and the wild-type strain. It was concluded that butanol tolerance was associated with multiple rearrangements of the cell genetic system, rather than with single mutations.     

Changes in Membrane Plasmalogens of Clostridium pasteurianum during Butanol Fermentation as Determined by Lipidomic Analysis

Changes in membrane lipid composition of Clostridium pasteurianum NRRL B-598 were studied during butanol fermentation by lipidomic analysis, performed by high resolution electrospray ionization tandem mass spectrometry. The highest content of plasmalogen phospholipids correlated with the highest butanol productivity, which indicated a probable role of these compounds in the complex responses of cells toward butanol stress. A difference in the ratio of saturated to unsaturated fatty acids was found between the effect of butanol produced by the cells and butanol added to the medium. A decrease in the proportion of saturated fatty acids during conventional butanol production was observed while a rise in the content of these acids appeared when butanol was added to the culture. The largest change in total plasmalogen content was observed one hour after butanol addition i.e. at the 7th hour of cultivation. When butanol is produced by bacterial cells, then the cells are not subjected to severe stress and responded to it by relatively slowly changing the content of fatty acids and plasmalogens, while after a pulse addition of external butanol (to a final non-lethal concentration of 0.5 % v/v) the cells reacted relatively quickly (within a time span of tens of minutes) by increasing the total plasmalogen content.     

Effect of Butanol Challenge and Temperature on Lipid Composition and Membrane Fluidity of Butanol-Tolerant Clostridium acetobutylicum

The effect of butanol challenge (0, 1.0, 1.5% [vol/vol]) and growth temperature (22, 37, 42°C) on the membrane composition and fluidity of Clostridium acetobutylicum ATCC 824 and a butanol-tolerant mutant, SA-2, was examined in chemically defined medium. Growth of strain ATCC 824 into the stationary phase coincided with a gradual increase in the percent saturated to percent unsaturated (SU) fatty acid ratio. When challenged with butanol at 22 and 37°C, ATCC 824 demonstrated an immediate (within 30 min) dose-response increase in the SU ratio. This strain showed little additional change over a 48-h fermentation. Compared with ATCC 824, growth of SA-2 into the late stationary phase at 22 or 37°C resulted in an overall greater increase in the SU ratio for both unchallenged and challenged cells. This effect was minimized when SA-2 was challenged at 42°C, probably due to the combination of the membrane fluidizing effect of butanol and the elevated temperature. Growth at 42°C resulted in an increase in longer acyl chain fatty acids at the expense of shorter acyl chains for both strains. The membrane fluidity exhibited by SA-2 remained essentially constant at various butanol challenge and temperature combinations, while that for the ATCC 824 strain increased with increasing butanol challenge. By synthesizing an increased amount of saturated fatty acids, the butanol-tolerant SA-2 strain has apparently developed a mechanism for maintaining a more stable membrane environment. Growth of the microorganism is necessary for butanol to fluidize the membrane. Incorporation of exogenous fatty acids (18:1) did not significantly improve the butanol tolerance of either strain. Since SA-2 was able to produce only trace amounts of either butanol or acetone, increased tolerance to butanol does not necessarily coincide with greater solvent yields in this strain.     

Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes.

Pseudomonas putida S12 was more tolerant to ethanol when preadapted to supersaturating concentrations of toluene. Cellular reactions at the membrane level to the toxicities of both compounds were different. In growing cells of P. putida S12, sublethal concentrations of toluene resulted in an increase in the degree of saturation of the membrane fatty acids, whereas toxically equivalent concentrations of ethanol led to a decrease in this value. Contrary to this, cells also reacted to both substances with a strong increase of the trans unsaturated fatty acids and a corresponding decrease of the cis unsaturated fatty acids under conditions where growth and other cellular membrane reactions were totally inhibited. While the isomerization of cis to trans unsaturated fatty acids compensates for the fluidizing effect caused by ethanol, a decrease in the degree of saturation is antagonistic with respect to the chemo-physical properties of the membrane. Consequently, the results support the hypothesis that the decrease in the degree of saturation induced by ethanol is not an adaptation mechanism but is caused by an inhibitory effect of the compound on the biosynthesis of saturated fatty acids.     

Membrane fatty acids adaptive profile in the simultaneous presence of arsenic and toluene in <Emphasis Type="Italic">Bacillus</Emphasis> sp. ORAs2 and <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. ORAs5 strains

Bacillus sp. ORAs2 and Pseudomonas sp. ORAs5, two arsenic-resistant bacterial strains previously isolated from sediments of the Orbetello Lagoon, Italy, were tested for their adaptation to mixed contaminants on the level of membrane fatty acid composition. The two bacterial strains were characterized by high levels of arsenic resistance, and Pseudomonas sp. ORAs5 was also shown to be solvent-tolerant. The bacterial strains were exposed to mixtures of two toxic compounds: arsenic at fixed concentrations and toluene in variable amounts or, alternatively, toluene at constant values along with arsenic added at variable concentrations. Both strains react to the contaminants by changing the composition of their membrane fatty acids. Bacillus sp. strain ORAs2 showed a correlation between growth rate decreases and fatty acids degree of saturation increases in both cases, although pointedly in the presence of 1, 2, and 3 mM of toluene and different additions of arsenic, counteracting membranes fluidity induced by toxic compounds. In Pseudomonas sp. ORAs5, adaptive changes in membrane composition was observed both in terms of increases in the degree of saturation and in the trans/cis ratio of unsaturated fatty acids in the presence of varying toluene and constant arsenic concentrations, whereas only minor changes occurred with increasing arsenic and constant toluene concentrations. Thus, on the level of membrane composition, Bacillus sp. ORAs2 showed a higher potential for adaptation to the presence of mixed pollutants, suggesting its probable suitability for bioremediation purposes.     

Interesterification of phosphatidylcholine with lipases in organic media

Summary Lipases were investigated with respect to their ability to catalyse the incorporation of fatty acids into phosphatidylcholine (PC) by interesterification reactions. The enzymes were dried onto solid support materials and the conversions were carried out in water-saturated toluene. Three lipases (two fungal and one plant enzyme) had the desired activity; immobilized lipase from Mucor miehei (Lipozyme) was the most active enzyme. The Lipozyme-catalysed interesterification was selective for the sn-1 position of PC and during 48 h of reaction around 50% of the fatty acids in this position were replaced with heptadecanoic acid, a fatty acid which was practically absent in the original phospholipid. Due to adsorption on the support material and the competing hydrolysis reaction the total amount of PC in the reaction solution decreased to about 40% of the original amount. Higher interesterification rates were obtained with free fatty acids as acyl donors than with fatty acid esters. Offprint requests to: I. Svensson     

Intracellular metabolic changes of Clostridium acetobutylicum and promotion to butanol tolerance during biobutanol fermentation

During the fermentation process, Clostridium acetobutylicum cells are often inhibited by the accumulated butanol. However, the mechanism underlying response of C. acetobutylicum to butanol stress remains poorly understood. This study was performed to clarify such mechanism through investigating the butanol stress-associated intracellular biochemical changes at acidogenesis phase (i.e., middle exponential phase) and solventogenesis phase (i.e., early stationary phase) by a gas chromatography-mass spectrometry-based metabolomics strategy. With the aid of partial least-squares-discriminant analysis, a pairwise discrimination between control group and butanol-treated groups was revealed, and 27 metabolites with variable importance in the projection value greater than 1 were identified. Under butanol stress, the glycolysis might be inhibited while TCA cycle might be promoted. Moreover, changes of lipids and fatty acids compositions, amino acid metabolism and osmoregulator concentrations might be the key factors involved in C. acetobutylicum metabolic response to butanol stress. It was suggested that C. acetobutylicum cells might change the levels of long acyl chain saturated fatty acids and branched-chain amino acids to maintain the integrity of cell membrane through adjusting membrane fluidity under butanol stress. The increased level of glycerol was considered to be correlated with osmoregulation and regulating redox balance. In addition, increased levels of some amino acids (i.e., threonine, glycine, alanine, phenylalanine, tyrosine, tryptophan, aspartate and glutamate) might also confer butanol tolerance to C. acetobutylicum. These results highlighted our knowledge about the response or adaptation of C. acetobutylicum to butanol stress, and would contribute to the construction of feasible butanologenic strains with higher butanol tolerance.     

The nature of fatty acid modifies the equilibrium position in the esterification catalyzed by lipase

The equilibrium position in lipase mediated esterification of various fatty acids and butanol was studied. The influence of the chain length and the presence of unsaturations in the fatty acids on the equilibrium position was measured and predicted. To predict equilibrium position the program TREP extended (TREPEX) based on the UNIFAC group contribution method was used. Using an equilibrium constant of 35, calculated on the basis of thermodynamic activities, the equilibrium position between butanol and saturated and/or unsaturated fatty acids with different chain lengths can be predicted. The ester mole fraction at equilibrium increases with the fatty acid chain length, and for fatty acids with the same carbon number, the highest values are found for unsaturated fatty acids. For reaction systems containing two saturated fatty acids, a slightly higher mole fraction is obtained for the fatty acid with the higher chain length, while for mixtures consisting of saturated and unsaturated fatty acids, the mole fractions of the unsaturated esters are lower than those of the saturated ones, regardless the chain length of the fatty acid. These experimental results are in good agreement with the calculations with TREPEX.     

Human fetal liver fatty acid binding proteins. Role on glucose-6-phosphate dehydrogenase activity

Fatty acid binding proteins (FABPs) may play an important role in the transport and metabolism of fatty acids during human embryogenesis. Three fractions of FABP, namely, DE-I, DE-II and DE-III, having Mr 14,200 Da each and pI values 7.8, 6.9 and 5.4, respectively, have been detected in human fetal liver. These proteins were purified by heat and butanol precipitation of fetal liver supernatant as well as by gel filtration and ion-exchange chromatography. Fetal liver FABPs are immunochemically identical to each other. Concentrations of DE-I, DE-II and DE-III increase gradually from early gestation to term. DE-I is almost lipid-free, DE-II binds long-chain fatty acids nonspecifically and DE-III transports mainly arachidonic acid. DE-II and DE-III protect glucose-6-phosphate dehydrogenase, which furnishes NADPH for fatty acid synthesis, from the feed-back inhibition exerted by added palmitoyl-CoA and oleate. In the absence of exogenous inhibitors, this enzyme is stimulated by FABPs. DE-I has no effect on such inhibition. Thus, FABPs play a regulatory role in critical aspects of cellular physiology during human embryogenesis.     

The whey proteins of the milk of red deer (Cervus elaphus L.). A homologue of bovine beta-lactoglobulin.

Increasing plasma free fatty acids decreased the degree of glycogen depletion, and increased the citrate concentration, in slow-red (soleus) and fast-red (deep portion of vastus lateralis) muscle during exercise (approx. 50% depletion of glycogen, as against 75% in control animals). There was no effect in fast-white muscle (superficial portion of vastus lateralis). Glycogen concentration in the liver decreased by 83% in controls, but only by 23% in animals with increased free fatty acids during exercise. The decreased glycogen depletion may be partly explained by the findings that (a) plasma-insulin concentration was two- to three-fold higher in animals with increased plasma free fatty acids and (b) the exercise-induced increase in plasma glucagon was lessened by increased free fatty acids. Blood glucose was higher in the animals with increased free fatty acids after the exercise. The rats with increased plasma free fatty acids utilized approx. 50% as much carbohydrate as did the controls during the exercise.     

Changes in cellular composition during magnesium deficiency.

1. Concentrations and compositions of liver, serum and milk lipids of cows were measured during 6 days' starvation and serum lipids during 60 days' re-feeding. 2. The concentration of free fatty acid in serum increased fivefold during starvation. 3. The content of total lipid in liver (g/100g of liver dry matter) doubled owing to a 20-fold increase in triglyceride, an eightfold increase in cholesterol ester, a three fold increase in free fatty acid and a 20% increase in cholesterol. There were no changes in the content or composition of liver phospholipids. 4. Starvation lowered the concentrations of total lipid, phospholipid and cholesterol ester of dextran sulphate-precipitable serum lipoproteins. Total lipid and cholesterol ester concentrations in lipoproteins of d greater than 1.055 and in lipoproteins not precipitable by dextran sulphate decreased from day 4 of the starvation period and during the first 20 days' re-feeding. 5. During starvation there were decreases in percentages of stearic acid and increases in oleic acid in serum free fatty acids and triglycerides and in liver neutral lipid. 6. Throughout starvation total milk lipid yield decreased, yields and percentages of C4-14 fatty acids decreased and percentages of C18 fatty acids increased. 7. It is suggested that accumulation of triglyceride in liver may be caused by increased uptake of plasma free fatty acids without corresponding increase in lipoprotein secretion.     

Seasonal Changes of Fatty Acid Compositions in Overwintering Larvae of Rice Stem Borer,Chilo suppressalis (Lepidoptera: Pyralidae)

Cold acclimation and overwintering state can affect fatty acid compositions of insects. To determine compositional change of fatty acids during nondiapause and diapause stages, an experiment was conducted to investigate fatty acid constituents from whole body of C. suppressalis larvae. Five most abundant fatty acids were found to be palmitoleic (35–58%), palmitic (18–44%), oleic (14–23%), stearic (0.5–2.5%) and linoleic acid (0.4–2%). However, linolenic, erucic, lauric and myristic acid were found at lower level. Saturated fatty acids significantly decreased and conversely unsaturated fatty acids increased from August (pre-diapause) to October (initiation of diapause). The increase in seasonal cold hardiness during cold acclimation, exposed at −15°C for 24 h, was related to degree of fatty acid unsaturation. The elevation of palmitoleic acid content at low temperature resulted in an increase in the overall degree of unsaturation in the whole body. These results indicated the importance of unsaturated fatty acids composition to prepare larvae entering diapause phase.     

Survey of Extreme Solvent Tolerance in Gram-Positive Cocci: Membrane Fatty Acid Changes in Staphylococcus haemolyticus Grown in Toluene

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.     

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.     

Impact of impurities in biodiesel-derived crude glycerol on the fermentation by <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis> ATCC 6013

During the production of biodiesel, crude glycerol is produced as a byproduct at 10% (w/w). Clostridium pasteurianum has the inherent potential to grow on glycerol and produce 1,3-propanediol and butanol as the major products. Growth and product yields on crude glycerol were reported to be slower and lower, respectively, in comparison to the results obtained from pure glycerol. In this study, we analyzed the effect of each impurity present in the biodiesel-derived crude glycerol on the growth and metabolism of glycerol by C. pasteurianum. The crude glycerol contains methanol, salts (in the form of potassium chloride or sulfate), and fatty acids that were not transesterified. Salt and methanol were found to have no negative effects on the growth and metabolism of the bacteria on glycerol. The fatty acid with a higher degree of unsaturation, linoleic acid, was found to have strong inhibitory effect on the utilization of glycerol by the bacteria. The fatty acid with lower or no degrees of unsaturation such as stearic and oleic acid were found to be less detrimental to substrate utilization. The removal of fatty acids from crude glycerol by acid precipitation resulted in a fermentation behavior that is comparable to the one on pure glycerol. These results show that the fatty acids in the crude glycerol have a negative effect by directly affecting the utilization of glycerol as the carbon source, and hence their removal from crude glycerol is an essential step towards the utilization of crude glycerol.     

Improvements of metabolites tolerance in <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> by micronutrient zinc supplementation

Micronutrient zinc is of great importance for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. The effect of zinc supplementation on toxic metabolites (formic, acetic, butyric acid and butanol) tolerance during ABE fermentation was investigated under various stress-shock conditions without pH control. Great improvements on cell growth, glucose utilization and butanol production were achieved. In the presence of 0.45 g/L formic acid, zinc contributed to 11.28 g/L butanol produced from 55.24 g/L glucose compared to only 5.27 g/L butanol from 29.49 g/L glucose in the control without zinc supplementation. More importantly, relatively higher levels of 7.5 g/L acetic acid, 5.5 g/L butyric acid and 18 g/L butanol could be tolerated by C. acetobutylicum with zinc supplementation while no fermentation was observed under the same stress-shock condition respectively, suggesting that the acids and butanol tolerance in C. acetobutylicum could be significantly facilitated by pleiotropic regulation of micronutrient zinc. Thus, this paper provides an efficient bioprocess engineering strategy for improving stress tolerance in Clostridium species.     

Wirkung von Ölen und Fettsäuren auf Wachstum und Enzymbildung bei Thermoactinomyees vulgaris. V. Untersuchungen zur Metabolisierung des Öles

The splitting of vegetable and animal oils used as antifoam agents and the metabolization of fatty acids released were analysed during fermentation of T. vulgaris in a 30 l jar fermenter. During cultivation time of 22 hours the oil amount decreased by 60 to 70%. The metabolization of fatty acids is 50 to 90% of the starting level depending on the kind of acid. The eventual effect of the fatty acids released from antifoam oils on the protease production is discussed.     

Effects of boiling,deep‐frying,and microwave treatment on the proximate composition of rainbow trout fillets: changes in fatty acids,total protein,and minerals

This study was conducted to investigate the differences in the proximate composition (moisture, fat, protein and ash), cholesterol content, energy, mineral composition (Na, Ca, Mg, K, P, Fe and Zn) and fatty acid profile of rainbow trout, Oncorhynchus mykiss, after frying, boiling, or microwaving. Results showed that all cooking methods reduced moisture and increased total protein, fat and ash contents. Also all minerals increased significantly during the microwave and frying methods. Statistical results showed that the major fatty acids among the saturated and monounsaturated fatty acids in each fish were palmitic (C16:0) and oleic (C18:1) acids, respectively. In addition, linoleic acid (C18:2) was predominant in the n‐6 polyunsaturated fatty acids (PUFA) in both cooked and raw trout. The EPA (eicosapentaenoic acid; C20:5 ω3) and DHA (docosahexaenoic acid; C22:6 ω6) acids were the major fatty acids among total n‐3 acids in fish samples. The fatty acids profile of the cooked fish showed a saturated (SFA) and monounsaturated (MUFA) decrease and an increase in PUFA contents. However, the ω‐3 fatty acids content increased in microwaved samples but decreased in the fried samples. Moreover, the PUFA/SFA and Hypocholesterolaemic/Hypercholesterolaemic (HH) ratios increased in both fried and microwaved trout, whereas significant increases in ω3/ω6 as well as EPA + DHA/C16 content were observed only in microwaved samples. A significant increase in energy content was observed in all cooked samples, whereas the cholesterol decreased. Research results show that microwaving is recommended as the best cooking method for a healthy consumption of rainbow trout.     

Effect of Environmental Factors on the trans/cis Ratio of Unsaturated Fatty Acids in Pseudomonas putida S12

The membrane reactions of Pseudomonas putida S12 to environmental stress were investigated. Cells reacted to the addition of six different heavy metals with an increase in the ratio of trans to cis unsaturated fatty acids. A correlation among the increase in the trans/cis ratio, the toxic effects of the heavy metals, and nonspecific permeabilization of the cytoplasmic membrane, as indicated by an efflux of potassium ions, was measured. Cells previously adapted to toxic concentrations of toluene exhibited increased tolerance to all applied concentrations of zinc compared with nonadapted cells. Cells exposed to different temperatures grew optimally at 30(deg)C. The degree of saturation of the membrane fatty acids of these cells decreased with decreasing temperature. An increase in the trans/cis ratio of unsaturated fatty acids took place only at higher temperatures. Osmotic stress, expressed as reduced water activity, was obtained by using different types of solutes. Only in the presence of toxic concentrations of sodium chloride or sucrose did the trans/cis ratio increase. At no applied water activity a significant effect of glycerol on the trans/cis ratio was measured. When cells were exposed to different pHs, a distinct optimum cis/trans isomerase activity was measured at pHs between 4.0 and 5.0, whereas at higher or lower pHs no reaction occurred. This optimum coincided with a loss of viability between pH 4 and 5.