Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

Biosynthesis of cannabinoid acids

Malonic acid, mevalonic acid, geraniol and nerol were incorporated into tetrahydrocannabinolic acid and cannabichromenic acid in Cannabis sativa. The pathway from cannabigerolic acid to tetrahydrocannabinolic acid via cannabidiolic acid was established by feeding labelled cannabinoid acids. Cannabichromenic acid was shown to be formed on a side pathway from cannabigerolic acid.     

Aminolysis of thiazolinones in manual amino acid sequence analysis : Direct detection of carboxyl, amide, and tryptophan residues in conjunction with the dansyl-Edman method

Aminolysis of thiazolinones with methylamine and identification of the resulting phenylthiocarbamyl-amino acid methylamides were shown to be highly useful steps in manual sequence degradations. The effects of different conditions in the degradative steps on the detectability of phenylthiocarbamyl-amino acid methylamides were studied, and the influence of aminolysis on subsequent dansylations was examined. Phenylthiocarbamyl-amino acid methylamides may be identified by thin-layer chromatography or high-performance liquid chromatography without interference from thioureas or other background material. Aminolysis is especially useful when used in conjunction with the dansyl-Edman method. It provides a way to distinguish aspartic acid from asparagine, and glutamic acid from glutamine as well as a means of identification of tryptophan residues.     

Identification of the prosthetic group of urocanase. The mode of its reaction with sodium borohydride and of its photochemical reactivation.

Urocanase from Pseudomonas putida and from beef liver were isolated by modifying described procedures. Both enzymes were inactivated and labeled on treatment with tritiated sodium borohydride and gave, upon subsequent hydrolysis, a radioactive acid. The previously reported identity of this acid as 2-hydroxybutanoic acid was disproved by several criteria. Other hydroxy acids were also proved to be different from the radioactive acid derived from urocanase. A large portion of the radioactive material from P. putida was found to be nicotinic acid by 1H NMR spectroscopy, gas-liquid chromatography of its methyl ester, and co-crystallization with authentic reference compounds both as the acid and as the hydrazide. A significant portion of the radioactive material derived from beef liver urocanase also co-crystallized with nicotinic acid. Sodium borohydride-treated inactive urocanase was partially reactivated by light. The action spectrum of the photoreactivation showed a maximum at 330 nm. Treatment of urocanase with sodium borodeuteride followed by hydrolysis afforded a sample of nicotinic acid which carried deuterium mainly in position 6. Both the reversible reducibility of urocanase and its action spectrum of photoreactivation suggest that urocanase contains an enzyme-bound nicotinamide nucleotide molecule which is essential for enzymic activity.     

Green biorefinery: separation of lactic acid from grass silage juice by chromatography using neutral polymeric resin

The aim of this work was to recover lactic acid in undissociated form from grass silage juice. For this aim, chromatographic separation using neutral polymeric resin Amberlite XAD1600 was investigated. Up to now, there is no hint in the literatures about using neutral polymeric resin for lactic acid separation from a mixture. Important factors (flow-rate, concentration of feed and loaded volume) that affect separation performance were firstly investigated with model solutions. The obtained results showed that lactic acid solutions with the purity varying from 93.2% to 99.9% could be obtained at the recovery yields over 99.4%. After that, trials with silage juice were carried out. Due to the complex composition of the feed, the purity of products decreased to 94% at a recovery yield of 97%. Although 99% of inorganic salts and sugars were separated from lactic acid organic acids in general and acetic acid in particular caused a purity problem. It seems that organic acids could not be separated from lactic acid by neutral resin Amberlite XAD1600. Besides the organic acid problem, some amino acids were remained in the products as impurities.     

Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

Haemoglobin adducts and specific immunoglobulin G in humans as biomarkers of exposure to hexahydrophthalic anhydride

The aim of this study was to determine whether haemoglobin adducts Hb of hexahydrophthalic anhydride HHPA and HHPA specific immunoglobulin G IgG can be used as biomarkers of exposure to HHPA. The exposures of HHPA in 10 workers were determined from the mean urinary hexahydrophthalic acid HHP acid levels range 76-3300 nmol HHP acid mmol-1 creatinine during a period of 4 weeks. Blood was collected at the end of the period and Hb-HHPA adducts were analysed by gas chromatography mass spectrometry. The Hb-HHPA adduct levels ranged from 0.45 to 24.7 pmol g-1 Hb. There was a close correlation between the urinary HHP acid levels and the amount of Hb-HHPA adducts r = 0.87 . One day exposures to HHPA and methylhexahydrophthalic anhydride MHHPA in 142 workers were determined from analysis of urinary HHP acid range 0-3300 nmol HHP acid mmol-1 creatinine and methylhexahydrophthalic acid MHHP acid; range 0-1700 nmol MHHP acid mmol-1 creatinine . HHPA specific IgG were analysed in the 142 workers with an ELISA method. The optical density for HHPA specific IgG varied between 0 and 1.25. There was no statistically significant correlation between the sum of the urinary HHP acid and MHHP acid and the HHPA specific IgG r = 0.12; p = 0.14 . Thus, Hb-HHPA adducts seem to be applicable as biomarkers of exposure to HHPA while the possible role of HHPA specific IgG as an indicator of exposure has to be further evaluated.     

Kinetics and mass transfer for lactic acid recovered with anion exchange method in fermentation solution

An anion exchange method for lactic acid recovered from lactic acid-glucose solution in an ion-exchange membrane-based extractive fermentation system was examined. The exchange isotherms of anion exchange resins for lactic acid recovered were measured batchwise, and the exchange-desorption kinetics of lactic acid passing through the exchange column was investigated. The determined typical breakthrough and elution curves were measured and simulated by conventional mode. The mass transfer coefficients were identified by numberical method. The effects of the velocity of the fluid on the dynamics were studied. Aqueous NaOH solution was found to be the best solvent for elution. An experiment on anioun exchange from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the ion exchange system from a borth. The ion-exchange mass-transfer coefficient and efficiency from the fermentation broth is found to be lower when compared with aqueous solutions of pure lactic acid. The results show that the separation method with anion exchange resins may be used in the production of lactic acid by fermentation.(c) 1995 John Wiley & Sons, Inc.     

Aromatic and fatty acids of triterpene esters and rubber content of Hoya latices and their taxonomic significance

The acid composition and the rubber content of the latices of 20 Hoya species were determined. All acids isolated from the latices were esterified with triterpenols and particle-bound. Cinnamic acid was the main acid in most latices. Acetic acid occurred in all latices, and predominated in three of the investigated species. Isovaleric acid and benzoic acid were present in some species; in one latex (H. bella) isovaleric acid was the predominant acid. Almost all species contained small amounts of phenylpropionic acid, and in two species this acid was a major compound. Longchain fatty acids were present as minor compounds. Rubber occurred in all particle-containing latices; its contribution to the total lipid fraction ranged from 1.7–24.0%. The acid profiles proved to be species-specific in Hoya and may be useful in species identification. Patterns of latex particle synthesis are proposed.     

Bile acid profiles in bile, urine, and feces of a patient with cerebrotendinous xanthomatosis

Bile acid profiles of bile, urine, and feces obtained from a patient with cerebrotendinous xanthomatosis on the same day have been analyzed by gas-liquid chromatography-mass spectrometry after fractionation into groups by mode of conjugation by an ion-exchange chromatography. The predominant biliary bile acid was cholic acid conjugated with glycine and taurine. Lesser amounts of the amino acid conjugates of chenodeoxycholic acid, ursodeoxycholic acid, 7-ketodeoxycholic acid, allocholic acid, and deoxycholic acid, and of unconjugated norcholic acid and allonorcholic acid were also present in the bile. The major fecal bile acid was 7-epicholic acid. Relatively large amounts of bile acids were excreted in the urine. Unconjugated 7-epicholic acid, norcholic acid, allonorcholic acid, and cholic acid predominated. The bile acid profiles of the patient were different from those of normal subjects and should be useful for the diagnosis.     

Haemoglobin adducts and specific immunoglobulin G in humans as biomarkers of exposure to hexahydrophthalic anhydride

The aim of this study was to determine whether haemoglobin adducts Hb of hexahydrophthalic anhydride HHPA and HHPA specific immunoglobulin G IgG can be used as biomarkers of exposure to HHPA. The exposures of HHPA in 10 workers were determined from the mean urinary hexahydrophthalic acid HHP acid levels range 76-3300 nmol HHP acid mmol-1 creatinine during a period of 4 weeks. Blood was collected at the end of the period and Hb-HHPA adducts were analysed by gas chromatography mass spectrometry. The Hb-HHPA adduct levels ranged from 0.45 to 24.7 pmol g-1 Hb. There was a close correlation between the urinary HHP acid levels and the amount of Hb-HHPA adducts r = 0.87 . One day exposures to HHPA and methylhexahydrophthalic anhydride MHHPA in 142 workers were determined from analysis of urinary HHP acid range 0-3300 nmol HHP acid mmol-1 creatinine and methylhexahydrophthalic acid MHHP acid; range 0-1700 nmol MHHP acid mmol-1 creatinine. HHPA specific IgG were analysed in the 142 workers with an ELISA method. The optical density for HHPA specific IgG varied between 0 and 1.25. There was no statistically significant correlation between the sum of the urinary HHP acid and MHHP acid and the HHPA specific IgG r = 0.12; p = 0.14 . Thus, Hb-HHPA adducts seem to be applicable as biomarkers of exposure to HHPA while the possible role of HHPA specific IgG as an indicator of exposure has to be further evaluated.     

Isolation and identification of lactic acid bacteria from soil using an enrichment procedure

AIMS: To survey, and identify and classify the ecological distribution of lactic acid bacteria from soil in Japan and Taiwan. METHODS AND RESULTS: Acid-producing bacteria were isolated from 68 soil samples, collected from Japan and Taiwan, in the rhizospheres of fruit trees, from the floor of a henhouse and around a horse farm. All isolates were identified by physiological and genetic tests. Thirty-two of the 54 isolates were identified as lactic acid bacteria (LAB), 16 as spore-forming lactic acid bacteria, five as Clostridium and one as Bacillus. These lactic acid bacteria represent five genera: Lactobacillus, Lactococcus, Enterococcus, Leuconostoc and Weissella. CONCLUSIONS: A high rate of isolating lactic acid bacteria was obtained from soil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that soil may be a common source for the isolation of lactic acid bacteria.     

Production of lactic acid by Lactobacillus rhamnosus with vitamin-supplemented soybean hydrolysate

Batch fermentation studies were performed to evaluate the potentials of a complex nitrogen source, soybean, as an alternative to yeast extract for the economical production of lactic acid by Lactobacillus rhamnosus. An enzyme-hydrolysate of soybean meal, Soytone, with an adequate supplementation of vitamins was found to be highly effective in supporting lactic acid production from glucose and lactose. The effects of seven selected vitamins: d-biotin, pyridoxine, p-aminobenzoic acid, nicotinic acid, thiamine, pantothenic acid, and riboflavin, on cell growth and lactic acid production were investigated to provide the basis for the optimization of vitamin supplementation to minimize the cost. Pantothenic acid was the most required compound while the other six vitamins were also essential for high lactic acid productivity. As a result of the optimization, 15 g/l yeast extract could be successfully replaced with 19.3 g/l Soytone supplemented with the vitamins, resulting in a production of 125 g/l lactic acid from 150 g/l glucose. The volumetric productivity and lactate yield were 2.27 g/l/h and 92%, respectively, which were higher than those with 15 g/l yeast extract. The raw material cost was estimated to be 21.4 cent/kg lactic acid, which was only approximately 41% of that with yeast extract.     

The ferulic acid esterases of Chrysosporium lucknowense C1: purification, characterization and their potential application in biorefinery

Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45 °C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6.     

Isolation of 2-Phenylacetamide as a Plant Growth Regulator Produced by Actinomyces

Isolation and screening tests were carried out in order to find microorganisms which were able to produce citric acid directly from blackstrap molasses. Some strains were obtained which accumulate considerable quantities of citric acid. Certain temperature changes during the course of incubation were found to increase the yield of citric acid.

The present investigation was undertaken to see if a simple method could be found to improve the yield of citric acid from blackstrap molasses, and we could obtain the yield of more than 70% from the untreated molasses using a newly isolated strain of Asp. niger.     

Biosynthetic incorporation of fluorescently labelled fatty acids in Escherichia coli

Escherichia coli cells were cultivated in a medium containing 1-pyrene butanoic acid, a fluorescent probe. Total lipids were extracted from the cells, and the extract was separated by thin-layer chromatography. The fluorescent fractions were examined using spectrofluorimetry. The starting 1-pyrene butanoic acid was shown to be biosynthetically incorporated into the bacterial lipid. Four fluorescent fractions appeared as a result; the fractions were derivatives of this compound modified in the chromophore and the fatty acid chain. The results indicate that the formation of 1-pyrene butanoic acid fluorescent metabolites can be used for studying the oxidation-reduction systems of the bacterium.     

Aqueous solubility and acidity constants of cholic, deoxycholic, chenodeoxycholic, and ursodeoxycholic acids.

Cholic acid, deoxycholic acid, chenodeoxycholic acid, and ursodeoxycholic acid were purified by a foam fractionation method. Using thermogravimetric analysis, the attached water molecule was found to be completely removed from solids of the latter three at 100 degrees C, while cholic acid still had one water molecule of crystallization per two cholic acid molecules at that temperature. The acidity constants of the acids were accurately determined from aqueous solubility measurements at different pH values and at 15, 25, 35, and 45 degrees C. The enthalpy change of dissolution from temperature dependence of solubility as undissociated acid monomer was much less than those of common ionic surfactants. This results from a smaller entropy increase on dissolution due to the hardly flexible hydrophobic structure of these bile acids.     

HepG2. A human hepatoblastoma cell line exhibiting defects in bile acid synthesis and conjugation

We used capillary gas chromatography/mass spectrometry to demonstrate that a cell line derived from a well differentiated human hepatoblastoma, HepG2, synthesized and secreted the following bile acids (ng/10(7) cells/h): chenodeoxycholic acid (131.4), cholic acid (3.3), 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid (DHCA; 4.5), and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA; 32.0). Deuterium from [7 beta-2H]7 alpha-hydroxycholesterol, which was added to the media, was incorporated into newly synthesized chenodeoxycholic acid, DHCA, and THCA, but not into cholic acid. Since THCA is a known precursor of cholic acid, these data suggest that HepG2 is specifically deficient in the side chain cleavage that transforms THCA into cholic acid. Greater than 90% of the bile acids synthesized and secreted by HepG2 were unconjugated. Conjugation could not be stimulated by the addition of glycine or taurine to the media. Approximately 30% of newly synthesized DHCA and THCA were sulfated. Chenodeoxycholic acid and cholic acid were not appreciably sulfated. In summary, cultured HepG2 cells synthesize bile acid, but in a pattern distinct from that of adult human liver. This cell line may be a model for studying pathways of human bile acid synthesis, conjugation, and sulfation.     

Properties of lipid bilayer membranes made from lipids containing phytanic acid

Besides the preparation of phytanic acid (3,7,11,15-tetramethylhexadecylic acid) according to the Dumas-Stass reaction, the synthesis of four different lipids containing phytanic acid residues is described. Diphytanoyl phophatidylcholine was systhesized beginning from glycerylphosphorylcholine, whereas the other lipids, diphytanoyl phosphatidylethanolamine, diphytanoyl phosphatidylserine and monophytanoyl glyceride were prepared by total synthesis.Some properties of lipid bilayer membranes made from the lipids containing phytanic acid were investigated. The specific capacity of these membranes was measured. Its value of approximately 400 nF cm^?2 was found to be similar to the value of membranes from lipids with unbranched fatty acid residues. Charge pulse experiments were performed using dipicrylamine as a molecular probe of membrane structure. The results were discussed on the basis of a higher viscosity of the membranes from lipids containing phytanic acid residues compared with unbranched fatty acid residues.     

N-malonyltransferases from peanut

Three distinct N-malonyltransferases were purified from peanut seedlings, accepting either anthranilic acid, D-tryptophan, or 3,4-dichloroaniline, respectively, as a substrate. Partially purified malonyl-CoA:D-tryptophan malonyltransferase also catalyzed the formation of the corresponding malonic acid conjugate when 1-aminocyclopropane-1-carboxylic acid was employed as a substrate. These N-malonyltransferases were clearly distinguished from several O-malonyltransferase activities also present in the same seedlings. N-Malonic acid conjugates have been previously isolated from peanut either as a natural constituent or after feeding with xenobiotics. By analogy to the results reported with cultured parsley cells, multiple malonyltransferases in peanut may have a role in vacuolar transport. Crude extracts of young peanut seedlings were incapable of hydrolyzing the respective N-malonic acid conjugates. However, dialyzed extracts of older plants released malonic acid from malonyl-1-aminocyclopropane-1-carboxylic acid but not from malonyl-3,4-dichloroaniline, suggesting that some N-malonic acid conjugates may be metabolized in plants which are approaching senescence.