Comparison of Growth and Primary Shunt Product Formation by Claviceps purpurea Cultured on Succinic Acid and Glucose as Carbon Sources

Growth on a medium containing succinic acid as the sole carbon source produced 1 g (dry weight) of mycelium per liter of medium by 50 days of incubation, whereas 25 g of mycelium was produced in 10 days when glucose was also present in the medium. Primary shunt metabolism took place during growth on succinic acid in spite of the extremely slow growth. Mycelia grown on succinic acid contained a higher percentage of residual mycelium and phosphate, but a lower percentage of mannitol, carbohydrate, lipid, and water-soluble nitrogen, than mycelia grown on a mixture of glucose and succinic acid. Thus, although primary shunt metabolism is favored by rapid growth on a rich, balanced sugar medium, it can also take place during extremely restricted growth in a medium containing succinic acid as the sole carbon source.     

Studies on the Enzymatic Production of l-Aspartic Acid from Maleic Acid

The enzymatic production of l-aspartic acid from maleic acid with cell suspensions of Alcaligenes faecalis 5-24, isolated from solid by the authors, was investigated.

The optimum conditions of this reaction and some cultural conditions which influenced on the ability of the cells to catalyze the above reaction were mainly studied.

The cells grown on maleic acid as a sole source of carbon showed exclusively the strong ability. The cells grown on a carbon source other than maleic acid showed no activity of this reaction.

It was concluded that an inducibles enzyme whose formation was stimulated by the presence of maleic acid might be involved in the reaction for the production of l-aspartic acid from maleic acid.

It was found that malonic acid was replaceable for maleic acid which played an inductive role for the formation of the enzyme system concerned with the reaction of l-aspartic acid production from maleic acid.

The cells grown in the medium containing malonic acid showed a stronger activity of the above reaction than the cells grown on maleic acid. The induction effect of malonic acid was remarkable when the organism was cultured in an acid medium. Whereas, consumption of C¹⁴-malonic acid in the medium by the organism was not observed at all in any pH milieu even where the formation of the enzyme system essential for the reaction was fully conducted. It indicated that malonic acid penetrated preferentially in acid milieu into the cells was a non-metabolic inducer like thiomethyl-β-d-galactoside in β-galactosidase system and that permeability barrier might exist in the organism.

The formation of cis-trans isomerase which catalyzed the conversion of maleic acid to fumaric acid was much stimulated by the addition of either malonic acid or maleic acid. From these results, it was concluded that l-aspartic acid was produced from maleic acid and ammonium ion by both actions of the inducible cis-trans isomerase and the constitutive aspartase.     

Requirement of heme for growth of Bacteroides fragilis.

Heme or protoporphyrin IX was required for growth of Bacteroides fragilis in a defined medium. The amount of heme necessary for half-maximal growth was 2 to 10 ng/ml (3.8 to 15 pmol/ml) among the Bacteroides species and strains tested. The growth rate, metabolic products from glucose fermentation, and cell yields were affected by the concentration of heme in the medium and by the length of time the culture was incubated. When heme was growth limiting (4 ng/ml), growth rates decreased by 50%, cultures started producing lactic and fumaric acids, and the cell yields declined. The cell yield for B. fragilis (ATCC 25285) at 24 h in medium containing 6.5 microgram of heme per ml was 69 g (dry weight) of cells per mol of glucose compared to 16 g (dry weight) of cells per mol of glucose with 4 ng of heme per ml. B. fragilis was unable to grow in defined medium when a porphyrin precursor, delta-aminolevulenic acid or porphobilinogen, was added in place of heme.     

Influence of Nutritional Conditions on Production of l-Glutamine by Flavobacterium rigense

The nutritional conditions for the production of l-glutamine by Flavobacterium rigense strain 703 were investigated. The optimum concentration of ammonia for achieving the highest yield of l-glutamine (25 mg/ml of broth) was relatively broad, from 0.9 to 1.6%, whereas fumaric acid had a narrow optimum range, near 5.5%. High concentration of inorganic ions such as chloride or sulfate ion clearly inhibited cell growth. Therefore, ammonium salts other than (NH(4))(2)-fumarate were unsuitable for the highest production. The optimum concentration of (NH(4))(2)-fumarate was 7%. To reduce the concentration of fumaric acid in the medium, many substances were evaluated as substitutes. The fumaric acid concentration required for highest l-glutamine yield could not be replaced by any one of the compounds tested. However, part of fumaric acid could be replaced with succinic acid and cupric ion; 4% (NH(4))(2)-fumarate plus 2.5% succinic acid or 5% (NH(4))(2)-fumarate plus 1 mM cupric ion produced results similar to 7% (NH(4))(2)-fumarate in the fermentation medium.     

Detection of Glycollic Acid in Etiolated Barley Shoots

A modified method for carboxylic acid analysis has been developedin order to study the metabolism of glycollic acid in barleysap. Glycollic, malic, citric, malonic, succinic, and fumaric acidshave been detected in alcoholic extracts from etiolated barleyshoots, and the amounts present roughly estimated.     

Immobilization of Brevibacterium flavum with carrageenan and its application for continuous production of l-malic acid

Extensive experiments were carried out to improve the productivity ofl-malic acid from fumaric acid using Brevibacterium flavum immobilized with carrageenan. The most favourable preparation for the continuous production ofl-malic acid was obtained when 16 g of B. flavum cells was entrapped in 100 ml 3.4% carrageenan gel. However, the immobilized cells produced an unwanted by-product, succinic acid. Treatment of the immobilized cells with 0.6% bile extract suppressed the side reaction and gave the highest operational stability of fumarase activity. By the immobilization of intact cells, the optimal temperature of the enzyme reaction shifted to 10°C higher, the optimal pH became broader, and the operational stability of fumarase activity increased. The effect of temperature on the stability of fumarase activity in the immobilized cell column was investigated under conditions of continuous enzyme reaction. The decay of fumarase activity during continuous enzyme reaction was expressed by an exponential relationship. The productivity of the immobilized B. flavum using carrageenan was as high as 5.2 times that of the conventional immobilized B. ammoniagenes using polyacrylamide.     

Biochemical differences between serotypes of Cryptococcus neoformans.

Prior studies have shown that C. neoformans isolates belonging to serotypes B and C differed from serotype A and D isolates in respect to the morphology of the perfect state, geographic distribution within the U.S.A. and frequency of isolation from avian droppings. The current study found that uptake of radiolabeled l-malic acid was approximately tenfold greater in serotype B and C than in A and D isolates. Assimilation of l-malic, fumaric and succinic acids also distinguished serotypes B and C from A and D. Greening of Guizotia seed agar occurred with 18 of 32 serotype B and C but in none of 91 serotype A or D isolates. The one property not following the same line of division was relatively slow creatinine assimilation, which distinguished type A alone.     

Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.     

Simultaneous Production and Recovery of Fumaric Acid from Immobilized Rhizopus oryzae with a Rotary Biofilm Contactor and an Adsorption Column

An integrated system of simultaneous fermentation-adsorption for the production and recovery of fumaric acid from glucose by Rhizopus oryzae was investigated. The system was constructed such that growing Rhizopus mycelia were self-immobilized on the plastic discs of a rotary biofilm contactor during the nitrogen-rich growth phase. During the nongrowth, production phase, the biofilm was alternately exposed to liquid medium and air upon rotation of the discs in the horizontal fermentation vessel. The product of fermentation, fumaric acid, was removed simultaneously and continuously by a coupled adsorption column, thereby moderating inhibition, enhancing the fermentation rate, and sustaining cell viability. Another beneficial effect of the removal of fumaric acid is release of hydroxyl ions from a polyvinyl pyridine adsorbent into the circulating fermentation broth. This moderates the decrease in pH that would otherwise occur. Polyvinyl pyridine and IRA-900 gave the highest loading for this type of fermentation. This fermentation system is capable of producing fumaric acid with an average yield of 85 g/liter from 100 g of glucose per liter within 20 h under repetitive fed-batch cycles. On a weight yield basis, 91% of the theoretical maximum was obtained with a productivity of 4.25 g/liter/h. This is in contrast to stirred-tank fermentation supplemented with calcium carbonate, whose average weight yield was 65% after 72 h with a productivity of 0.9 g/liter/h. The immobilized reactor was operated repetitively for 2 weeks without loss of biological activity.     

Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

尖顶羊肚菌液体培养基质与条件的研究

通过对尖顶羊肚菌液体培养基质与条件的研究,明确其菌丝生长的最适pH值、最适温度、适宜光照条件、适宜葡萄糖和蛋白胨浓度、适宜培养基,以便应用于尖顶羊肚菌液体菌种的生产和工业发酵。结果表明:菌丝的最适生长温度为2 5℃;最适生长pH值为6 ;葡萄糖和蛋白胨最适浓度分别为2 0 0g/L和10g/L ;菌丝在黑暗环境下生长良好,光照对菌丝生长具有抑制作用;用胡萝卜酵母膏培养基振荡培养形成的菌丝球多,菌丝生长量大;菌丝球在不同培养基中生长,可引起培养液pH值的上升或者下降;菌丝球可利用培养基内的氨基酸,使氨基酸降解,在胡萝卜酵母膏培养基中振荡培养8d的菌液总氨基酸含量较原液减少了36 71% ,亮氨酸、异亮氨酸和甲硫氨酸含量的下降幅度最大     

Thermal decomposition of fungal poly(beta,L-malic acid) and Poly(beta,L-malate)s

The thermal decomposition of poly(beta,l-malic acid), poly(alpha-methyl beta,l-malate), and ionic complexes of the polyacid with alkyltrimethylammonium salts was studied by TGA, GPC, and FTIR and NMR spectroscopy. It was found that poly(beta,l-malic acid) depolymerized above 200 degrees C by an unzipping mechanism with generation of fumaric acid which is then partially converted in a mixture of maleic acid and anhydride. On the contrary, random scission of the main chain was found to happen in the thermal decomposition of poly(alpha-methyl beta,l-malate). On the other hand, ionic poly(beta,l-malate)s degraded through a well defined three-stage process, the first one being depolymerization of the poly(malate) main chain along with decomposition of the ionic complex. Decomposition of the previously generated alkyltrimethylammonium salts followed by unspecific cracking of the resulting nitrogenated compounds happened at higher temperatures. Mechanisms partially explaining the decomposition processes of the three studied systems were proposed according to collected data.     

Continuous production of L-malic acid by immobilized cells

l-Malic acid is used extensively in the pharmaceutical industry and as a food additive. It is now produced on an industrial scale by the enzymatic conversion of fumaric acid using immobilized cells of Brevibacterium flavum. Recent improvements to this system, especially the use of x-carrageenan supports, have resulted in a continuous process capable of yielding 30 tonnes of l-malic acid per month.     

Regulation of formation of aerial mycelia and spores of Streptomyces viridochromogenes.

Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.     

The ability of filamentous fungi to produce acids on indoor building materials

Sixty two filamentous fungi isolated from paint coatings, wallpaper, carton-gypsum board, and indoor air in buildings were screened for acid activity. It was found that 64.5% of strains produce acids on medium with bromo-cresol purple, where 18% of the strains were distinguished by very high acid activity (acid activity coefficient Q = 1.32–2.83), including the species:Aspergillus niger, Aspergillus versicolor, Penicillium expansum, Penicillium brevicom pactum, Penicillium chrysogenum, Cladosporium cladosporioides, Stachybotrys chartarum, Mucor globosus, Ulocladium chartarum andAlternaria alternata. Research indicated that filamentous fungi considerably decrease the pH of the medium when that medium containing building material. The greatest acid production and pH decrease of the medium was observed during the growth of filamentous fungi in a medium with mortar, while the production of acids was less in a medium with cartongypsum board, gypsum, and wallpaper. Filamentous fungi produced succinic, oxalic, malic and fumaric acids in the medium with indoor building materials. It was stated that the type of building material affects the spectrum and quantity of organic acids produced by filamentous fungi.     

紫色小白菜有机酸的提取优化及UPLC定量分析

为建立一种紫色小白菜中精准的有机酸提取及定性定量分析方法。本研究在单因素试验的基础上,采用Box-Behnken的中心组合试验设计原理,设计响应面试验优化紫色小白菜有机酸的提取工艺。结果表明,紫色小白菜有机酸提取的最优工艺参数为:乙醇浓度73%,料液比1∶21,超声时间11 min,超声温度70℃。通过超高效液相色谱法(ultra performance liquid chromatography,UPLC)对紫色小白菜叶片和叶柄部位有机酸进行定性定量分析,检测出苹果酸、柠檬酸、丙二酸、琥珀酸和酒石酸,其中苹果酸含量最高,分别为15.968、5.019 mg/g;其次为柠檬酸,分别为9.293、1.385 mg/g。定性定量结果表明,紫色小白菜叶片及叶柄部位含有丰富有机酸类物质,叶片部位苹果酸、柠檬酸、丙二酸、酒石酸含量均高于叶柄部位,而琥珀酸含量较低。     

Carbon sources,growth, sclerotium formation and carbohydrate composition of Sclerotinia sclerotiorum

Summary Sclerotinia sclerotiorum (Lib.) D By. was grown in stationary liquid mineral-salts medium, pH 4.3, containing various carbon sources and the weight of mycelia and sclerotia was determined at regular intervals. When grown on various glucose concentrations (0–24 g of C/l), more sclerotia were produced at 8–12 g of C/l. Sclerotia were not usually formed in shake cultures. The ability of the fungus to use other carbon sources for growth and sclerotium formation was tested at 12 g of C/l in the stationary mineral-salts medium. The highest weights of mycelia and sclerotia occurred with raffinose, sucrose, maltose, lactose, d-mannose, d-glucose, d-fructose or l-arabinose. Good growth but decreased sclerotium production were found on cellobiose and d-xylose. Reduced or poor growth, a long lag period and few or no sclerotia occurred on trehalose, melibiose, l-sorbose, l-rhamnose, d-ribose, d-arabinose, l-xylose or 8 polyols. No growth was observed with erythritol or i-inositol. A combination of glucose plus trehalose or polyols resulted in increased growth and the formation of sclerotia. Organic acids supported little or no growth and no sclerotia were produced. Generally culture filtrates which supported growth and formation of sclerotia became acid (about pH 3.5). The pH of the culture filtrate usually increased slowly during the growth period when the fungus grew poorly and no sclerotia were formed. The alcoholsoluble sugars and polyols present in culture filtrates, mycelia and sclerotia were determined by paper and thin-layer chromatography. Regardless of the carbon source, mannitol was usually present in culture filtrates. The occurrence of other compounds in the filtrates depended on the carbon source. Trehalose, mannitol and usually small quantities of glucose or fructose were present in mycelia and sclerotia from all carbon sources. Galactitol or pentitols occurred in mycelia and sclerotia when the fungus grew on galactose and oligosaccharides containing galactose or the corresponding pentose, sugars. Acid hydrolyzates of the alcohol-insoluble fraction of mycelia or sclerotia contained glucose, smaller amounts of galactose and mannose and traces of ribose and rhamnose.     

Inhibition by glucose of the synthesis of proline from glutamic acid

While in the absence of glucose, proline is not a required amino acid, in the presence of glucose the growth of Micrococcus pyogenes var. aureus in amino acid medium is proportional to the concentration of proline when all other amino acids and growth factors are present in amounts adequate for optimal growth. The data presented here and the ideas prevailing in the literature indicate that glutamic acid is a precursor of proline. Glucose inhibits the conversion of glutamic acid into proline, which in turn causes failure of growth. Thus, 1 μg. and 10 μg. glucose/ ml. cause 50% and 100% inhibition, respectively, of the growth dependent on the synthesis of proline. One μg. proline antagonizes completely the inhibition in the presence of 5,000 μg. glucose/ml.One μg. glycerol, 100 μg. pyruvate, 250 μg. lactate, or 100 μg. α-glycerophosphate/ml., individually, cause from 25 to 50% inhibition of the growth dependent on the synthesis of proline from glutamic acid. Five thousand μg./ml. either of malic, succinic, fumaric, α-keto-glutaric, cis-aconitic acid, or dihydroxyacetone, or 500 μg. citric acid/ml. fails to cause inhibition.Pyrrolidone carboxylic acid was found to substitute for glutamic acid but not for proline in tests with M. pyogenes var. aureus. Also, seven proline-less mutant strains of Escherichia coli were unable to utilize pyrrolidone carboxylic acid in place of proline. No evidence was obtained to indicate that pyrrolidone carboxylic acid could serve as a direct precursor of proline.     

Effects of Additives on Alum Hematoxylin Staining Solutions

All additives tested (ethyl alcohol, glycerine, chloral hydrate, ethylene and propylene glycol, and citric, malonic and maleic acids) in varying degrees limited the conversion of hematein to insoluble compounds. Peak absorbance increased slightly in hematoxylin solutions containing citric, malonic and maleic acids, but decreased with other additives, and in controls. After four months storage the absorbance in all solutions increased about 50%, acidity increased and staining effetiveness increased.     

Effects of additives on alum hematoxylin staining solutions.

All additives tested (ethyl alcohol, glycerine, chloral hydrate, ethylene and propylene glycol, and citric, malonic and maleic acids) in varying degrees limited the conversion of hematein to insoluble compounds. Peak absorbances increased slightly in hematoxylin solutions containing citric, malonic and maleic acids, but decreased with other additives, and in controls. After four months storage the absorbance in all solutions increased about 50%, acidity increased and staining effectiveness increased.