Influence of carbohydrates and polyols on l-lactic acid production and fatty acid formation by Rhizopus arrhizus

Growth and l-lactic acid production on 24 different carbohydrates and polyols (glycerol, mannitol and sorbitol) by Rhizopus arrhizus CCM 8109 were determined. The d- but not the l-forms of xylose, fructose, galactose, mannose, glucose, cellobiose, maltose and sucrose and partially hydrolysed starch were converted to l-lactic acid. Changes in lipid formation and fatty acid composition were detected in biomass grown on the different sugars. In the presence of polyols, growth and considerable production of lipids were observed with little or no lactate production. Invertase was mainly associated with the mycelium during growth on sucrose, whereas glucoamylase and -amylase were produced extracellularly during growth on starch.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Polytechnical University, Radlinského 9, SK-812 37 Bratislava, Slovak Republic     

Production of 5′-dRiboflavin-α-d-glucopyranoside in Growing Cultures by the Mold Mucor

During the investigations on riboflavin glycoside formation by Aspergillus, Mucor, Penicillium and Rhizopus, a remarkable production of 5′-d-riboflavin-α-d-glucopyranoside was observed in several strains belonging to the genus Mucor when grown on a, medium containing maltose and riboflavin. Several conditions on 5′-d-riboflavin-α-d-glucopyranoside formation were also investigated with washed mycellium of M. javanicus. Maltosyl compounds such as maltose, dextrin, amylose and soluble starch were the effective glucosyl donor, whereas glucose, fructose, sucrose, lactose and dextran were inactive.     

Effect of cultural conditions on production of eicosapentaenoic acid byPythium irregulare

Summary The effect of culture conditions upon lipid content and fatty acid composition of mycelia ofPythium irregulare was investigated with particular attention to increasing the yield of 5,8,11,14,17-eicosapentaenoic acid (205; –3) (EPA). All experiments were done by shake flask culture using a yeast extract + malt extract medium. The maximum growth rate was obtained at 25°C, but maximum EPA production was obtained at 12°C. The highest EPA production was 76.5 g EPA/ml 13 days fermentation at 12°C. Addition of glucose during fermentation increased the yield considerably. The highest yield was 112 g/ml, obtained at 13 days fermentation with spiking on day 11. Fermentation time could be shortened by initial incubation at 25°C for 2 days, followed by incubation at 12°C for 6 days. The culture also produced arachidonic acid and other -6 polyunsaturated fatty acids. EPA production was also obtained with lactose or sweet whey permeate, a by-product of cheese manufacture that contains lactose as the main carbohydrate.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions

We attempted to enhance the growth and total lipid production of three microalgal species, Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1, and Dunaliella salina, which are capable of accumulating high content of lipid in cells. Low nitrogen concentration under photoautotrophic conditions stimulated total lipid production, but a decreasing total lipid content and an increasing biomass were observed with increasing nitrogen concentration. Among the different carbon sources tested for heterotrophic cultivation, glucose improved the growth of all three strains. The optimal glucose concentration for growth of I. galbana LB987 and N. oculata CCAP849/1 was 0.02 M, and that of D. salina was 0.05 M. Enhanced growth occurred when they were cultivated under heterotrophic or mixotrophic conditions compared with photoautotrophic conditions. Meanwhile, high total lipid accumulation in cells occurred when they were cultivated under photoautotrophic or mixotrophic conditions. During mixotrophic cultivation, biomass production was not affected significantly by light intensity; however, both chlorophyll concentration and total lipid content increased dramatically with increasing light intensity up to 150 µmol/m²/s. The amount and composition ratio of saturated and unsaturated fatty acids in cells were different from each other depending on both species and light intensity. The highest accumulation of total fatty acid (C16–C18) among the three strains was found from cells of N. oculata CCAP849/1, which indicates that this species can be used as a source for production of biodiesel.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

Kinetic study on starch hydrolysis using a glucose/maltose sensing system

Summary A dual-enzyme electrode flow injection system that can simultaneously determine glucose and maltose is used for an on-line study of starch hydrolyses catalysed by amylases. With the working system, determinations can be made every 2 minutes. A 10 L sample size with recycled back-flow minimises any loss of the reaction medium. The production, growth and decay of glucose and maltose concentrations during starch hydrolysis under various enzymatic conditions can thus be closely monitored, making it useful for the study of the catalytic kinetics of amylases and in screening and analysing enzyme systems.     

Formation of a raw starch-hydrolyzing alpha-amylase by Clostridium 2021: Effect of carbon sources

Summary Clostridium 2021 was found to produce -amylase effective at hydrolyzing raw starch. Of the carbohydrates examined, starch at 3 % concentration was found to be the best carbon source for enzyme production. The products of -amylase action on starch were: maltose. glucose and higher dextrins.     

Thermoanaerobium brockii gen. nov. and sp. nov., a new chemoorganotrophic,caldoactive, anaerobic bacterium

The isolation of a new anaerobic thermophilic bacterium, Thermoanaerobium brockii, from volcanic features is described. Successful enrichment required a complex medium containing glucose or other fermentable sugars and incubation temperatures of 55–80° C. Strains of T. brockii were gram positive, rods of uneven length that existed singly, in pairs, chains or filaments. Electron micrographs of thin sections of cell revealed a monolayered cell wall and a constrictive or pinching off cell division process. The organism was nonsporeforming, obligately anaerobic and chemoorganotrophic. The optimal temperature for growth was 65–70° C, the maxium was below 85° C and the minimum above 35° C. The doubling time at the optimal temperature for growth was about 1 h. The DNA base composition of three strains of T. brockii varied from 30.0–31.4 mol % guanosine plus cytosine. Fermentable carbohydrates included glucose, sucrose, maltose, lactose, cellobiose and insoluble starch. The fermentation products of cells grown on glucose were ethanol, lactic acid, acetic acid, hydrogen and carbon dioxide. Growth of all strains tested was inhibited by fairly low concentrations of cycloserine, penicillin, streptomycin, tetracycline and chloramphenicol. The possible ecological, evolutionary, and industrial significance, and taxonomic relationships of Thermoanaerobium are discussed.Abbreviations TYEG complex medium containing mineral salts, 0.3% yeast extract, 1.0% tryptone and 0.5% glucose - O.D. optical density - G+C guanosine plus cytosine     

Metabolic characteristics and lipid composition of yeastlike cells and mycelium of <Emphasis Type="Italic">Mucor circinelloides</Emphasis> var. <Emphasis Type="Italic">lusitanicus</Emphasis> INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of γ-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

Production of ψ-linolenic acid by the fungus Mucor rouxii on cheap nitrogen and carbon sources

Summary The production of -linolenic acid (GLA) by the fungus Mucor rouxii CBS 416.77 was studied on low budget nitrogen and carbon sources, i.e. rape meal, cocos expeller and two types of yeast extract (nitrogen sources), and starch, starch hydrolysate, beet molasses and cocos expeller (carbon sources). As references, Difco yeast extract and glucose were used. In flask cultivations the three yeast extracts were fully interchangeable, while the Difco yeast extract (the most expensive of those tested) gave a higher productivity of GLA in fermentor cultures (14 mg·l^–1·h^–1). The yield of lipids and GLA were increased in the order yeast extract < rape meal < cocos expeller. Thus the amount of lipid increased from 0.56 to 2.8 g·l^–1, and that of GLA from 0.15 to 0.33 g·l^–1. Use of beet molasses or cocos expeller as carbon sources gave poor growth. Starch and starch hydrolysate resulted in better productivity of GLA than glucose (4.7 and 4.9 compared to 3.4 mg·l^–1·h^–1). Offsprint requests to: A.-M. Lindberg     

Transient responses of <Emphasis Type="Italic">Wickerhamia</Emphasis> sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis

The purpose of this study was to evaluate the inductive effect of starch and maltose, and the repressive/inhibitory effect of glucose, on amy-1 gene expression and α-amylase production by Wickerhamia sp., using continuous culture under transient-state conditions at a dilution rate (D) of 0.083 h^?1. Induction and repression kinetics of α-amylase were studied by changing the medium feed from glucose to maltose or starch in the induction experiments and vice versa in the repression experiments. Expression levels of amy-1 gene were measured by RT-qPCR. Results showed that starch was a more efficient inducer of α-amylase synthesis compared to maltose, with maximum accumulation rate constants of 0.424 and 0.191 h^?1, respectively. In contrast, α-amylase synthesis in starch and maltose cultures was partially repressed by glucose as indicated by a specific activity close to basal levels and a decay constant rate (??0.065 and ??0.069 h^?1, respectively) higher than ??D. A linear dependence of the specific rate of α-amylase production on mRNA relative abundance of amy-1 gene was observed. An inhibitory effect of glucose was not observed even at a concentration of 30 g L^?1. In conclusion, the transient continuous culture is a useful tool to determine the qualitative and quantitative effects of maltose and starch on α-amylase induction and of glucose on enzyme repression, as well as to obtain a detailed understanding of the dynamic behavior of the yeast culture. Furthermore, results showed that amylaceous substrates can be very effective carbon sources for the production of α-amylase without being inhibited by glucose.     

Production of native-starch-degrading enzymes by a Bacillus firmus/lentus strain

Summary A bacterium belonging to the Bacillus firmus/lentus-complex and capable of growth on native potato starch was isolated from sludge of a pilot plant unit for potato-starch production. Utilization of a crude enzyme preparation obtained from the culture fluid after growth of the microorganism on native starch, resulted in complete degradation of native starch granules from potato, maize and wheat at a temperature of 37°C. Glucose was found as a major product. Production of maltose, maltotriose and maltotetraose was also observed. Native-starch-degrading activity (NSDA) could be selectively adsorbed on potato-starch granules, whereas soluble-starch-degrading activity (SSDA) remained mainly in solution. The use of such a starch-adsorbed enzyme preparation on native starch resulted in a completely changed product pattern. An increase in oligosaccharides concomitant with less glucose formation was observed. An increased conversion of soluble starch to maltopentaose was possible with this starch-adsorbed enzyme preparation. It is concluded that NSDA comes from -amylase(s) and SSDA from glucoamylase(s) and/or -glucosidase(s). Cultivation of B. firmus/lentus on glucose, maltose, or soluble starch resulted in substantially smaller quantities of (native) starch-degrading activity.Offprint requests to: D. J. Wijbenga     

Interaction of hydrolytic and phosphorolytic enzymes of starch metabolism in Kalanchoë daigremontiana

The degradation of starch by a protein fraction of Kalanchoë daigremontiana Hamet et Perrier, obtained by ammoniumsulfate precipitation (30–70%), was found to be catalyzed by -and -amylase (EC 3.2.1.1 and EC 3.2.1.2, respectively) and by starch phosphorylase (EC 2.4.1.1). The activity of these enzymes was determined by chromatographic analysis of the reaction products; separation and identification of -amylase was accomplished by heat-inactivation of -amylase and -glucosidase. When the interaction of amylolytic and phosphorolytic enzymes was comparatively studied, it was found that without inorganic phosphorus in the reaction mixture, ¹⁴C-starch was converted predominantly to maltose and glucose; supplementation with 1–10 mM orthophosphate (Pi) resulted in an increase in glucose-1-phosphate formation and a concomitant reduction of maltose production. Since the total volume of starch degradation remained approximately constant, Pi apparently inhibits -amylase (Ki about 3 mM Pi). Thus, free Pi in the cell participates in the regulation of starch catabolism, serving as a substrate for starch phosphorylase while simultaneously reducing the production of maltose. With respect to glucan synthesis, adenosinediphosphoglucose--1,4-glucosyltransferase (EC 2.4.1.22), maltose phosphorylase and maltoseglucosyltransferase were also found to be active. The last-named enzyme catalyzes an exchange between dextrins and is considered to provide primer carbohydrates for the synthesis of polyglucans.Abbreviations ADPG adenosinediphosphoglucose - G1P glucose-1-phosphate - PEG polyethylenglycol - PEP phosphoenolpyruvate - Pi orthophosphate     

Micropropagation and field evaluation of micropropagated plants of turmeric

A protocol was developed for in vitro propagation of turmeric cv `elite' using young vegetative buds from sprouting rhizomes. The shoot buds produced multiple shoots when cultured on MS solid medium supplemented with benzyladenine and 1-naphthalene acetic acid. The effect of various cytokinins on shoot multiplication was studied by culturing the shoot tips on MS liquid medium supplemented with benzyladenine, benzyladenine riboside, kinetin, kinetin riboside, zeatin, 6-,-dimethylallylaminopurine, adenine, adenine sulfate or metatopolin each at 10 M in combination with 1-naphthalene acetic acid (1 M). Significant differences were observed between the treatments. Liquid medium was more favourable than agar medium for shoot multiplication. Among the various concentrations of agar tested, 0.4% and 0.6% were the best and produced the highest number of shoots per explant. Among the different carbohydrates tested, sucrose, fructose, glucose, sugar cubes, maltose, levulose and market sugar were found to be equally effective for shoot multiplication and xylose, rhamnose, lactose and soluble starch were inhibitory. Ninety five percent of the micropropagated plants survived in sterilized soil in paper cups and all of them survived in the field. Among 48 plants, two plants showed variegated leaves on the tillers. The micropropagated plants showed a significant increase in shoot length, number of tillers, number and length of leaves, number of fingers and total fresh rhizome weight per plant when compared with conventionally propagated plants. RAPD analysis of 11 regenerated plants using sixteen 10-mer primers did not show any polymorphism.     

Lipid production and fatty acid composition of selected strains belonging to Mucorales

The lipid accumulation, fatty acid composition and γ-linolenic acid (GLA) production by 28 strains belonging to Mucorales were investigated. The lipid content varied from 5 to 30% on dry biomass and the percentage of γ-linolenic acid in total intracellular lipid was in a range from 2.5 to 15.4% (w/w). The best yield of γ-linolenic acid (expressed as mg GLA per 1 g biomass) was found for Mucor mucedo CCF – 1384 (28.4) and Cunninghamella echinulata CCF – 103 (25.1).     

BIOCHEMICAL STUDIES ON COCHLIOBOLUS MIYABEANUS: VII. CELLULAR CARBOHYDRATE AS AN ENDOGENOUS SUBSTRATE FOR CONIDIA GERMINATION IN COCHLIOBOLUS MIYABEANUS

The carbohydrate content in the conidia of Cochliobolus miyabeanuswas found to be about 32% of the dry conidia, approximately70 per cent of which was composed of an alkali-stable fraction,and the alkali-stable fraction decreased markedly after thegermination. The alkali-stable carbohydrate gave, on acid hydrolysis,glucose alone. Starved conidia consumed glucose, maltose, , ß-limitdextrin, and also dextran as exogenous substrates for respiration.On the other hand, germ-tube elongation proved to be stimulatedby glucose, maltose, dextran and , ß-limit dextrinas well, but not by soluble starch, glycogen and glutinous ricestarch. Accordingly it follows that an alkali-stable carbohydrateincluding terminal glucopyranose residues combined through -l,6-glucosidelinkage seems to play an essential role in the production ofendogenous energy necessary for the germination of conidia inthis mold. Moreover, it is noteworthy that dextran was not usedas a nutrient for mycerial growth, whereas soluble starch wasfound to be an efficient carbon source. (Received June 5, 1960; )     

Effect of culture conditions on mycelial growth and production of γ-linolenic acid by the fungus Mortierella ramanniana

Summary Effect of culture conditions on cell growth, lipid accumulation and -linolenic acid production is reported for four Mortierella species. The highest concentration as well as the highest productivity of -linolenic acid in lipid was determined in strains of M. ramanniana. M. ramanniana CBS 112.08 was used in the studies of the influence of medium composition, concentration of carbon- and nitrogen sources and growth temperature. Several carbon sources provided good growth and a high lipid content in biomass. The highest dry weights (11–12g/l) and lipid contents (24%, w/w), were observed if glucose or fructose was used as carbon source, whereas the highest amount of -linolenic acid (26%) was determined in starch-grown cells. The fatty acid composition in the lipid was influenced by the cultivation time, growth temperature and, to a minor extent, by the carbon source used. In fermentor cultures, both strains of Mortierella ramanniana showed relatively poor growth and incomplete consumption of glucose. M. vinacea, on the other hand, grew well in tower reactors. M. vinacea, which has a different morphology than M. ramanniana strains, also showed higher yields of biomass and lipid and higher yield coefficients than the latter.     

Deoxyribonucleic acid base composition of some members of the Micrococcaceae

Auletta, Angela E. (Catholic University, Washington, D.C.), and E. R. Kennedy. Deoxyribonucleic acid base composition of some members of the Micrococcaceae. J. Bacteriol. 92:28-34. 1966.-Thirty-seven strains from the genera Micrococcus, Staphylococcus, Gaffkya, and Sarcina were examined for deoxyribonucleic acid base composition and biochemical activity. Organisms were tested for production of catalase, coagulase, deoxyribonuclease, oxidase, phosphatase, hydrogen sulfide, indole, and acetoin; nitrate reduction; gelatin, starch, and urea hydrolysis; citrate and ammonium phosphate utilization; NaCl tolerance; growth at 10 and 45 C, and growth in litmus milk. They were tested for production of acid from dextrose and mannitol under anaerobic conditions, and for aerobic production of acid from dextrose, mannitol, lactose, sucrose, raffinose, maltose, xylose, and glycerol. Organisms could be divided into two groups on the basis of guanine-cytosine (GC) content. Group I had an average GC content of 32%, and included all organisms which produced acid from dextrose. Group II had an average GC content of 62%, and included those organisms incapable of producing acid from dextrose under anaerobic conditions. Sarcina ureae had a GC content of 43%.     

Pullulanase is a characteristic of manyKlebsiella species and functions in the degradation of starch

Summary The type strainsKlebsiella pneumoniae NCTC 9633,K.ozaenae NCTC 5050 andK.rhinoscleromatis NCTC 5046, representative for all members of the genusKlebsiella, were found to produce pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41). In addition, 58 fresh isolates ofKlebsiella sp. of human origin were screened for growth on a defined solid medium with either maltose, maltodextrin mixture, soluble starch, glycogen, or pullulan as the sole carbon source. All of the strains showed luxurious growth on maltose and maltodextrins, seven strains grew poorly or not at all on the polymeric substrates, soluble starch, pullulan or glycogen. Three fresh isolates out of the 51 strains which did grow on each carbon source tested were examined in more detail with respect to a possible involvement of pullulanase in the utilization of -glucans. The production of pullulanase was inducible by growth of the cells on -glucans, whereas cultivation on glycerol, D-glucose or lactose did not lead to enzyme formation. The level of pullulanase activity in the three strains varied under otherwise comparable culture conditions, as did the level of a co-inducible -amylase. Comparative growth experiments on linear or branched -glucans allow the conclusion that the cooperation of hydrolases specific for 1,4--glucosidic linkages (-amylase) and for 1,6--linkages (pullulanase) is an obligatory requirement for the effective utilization of starch and glycogen.     

Differential temperature effect on the production of enhanced gamma linolenic acid in <Emphasis Type="Italic">Mucor rouxii</Emphasis> CFR-G15

Mucor rouxii CFR-G15, a locally isolated phycomycetous fungus, on cultivation at room temperature produced more than 30% (w/w) lipid in their dry cell weight, in which 14.2% accounted to be GLA content of the total fatty acids. It was observed that when incubation temperature lowered at 14°C, GLA content of the mycelium increased significantly (P<0.05) from 14.2% to 21.97%. In order to optimize the cultural conditions for high biomass and lipid production with high GLA content, the fungus was grown in association of two different temperatures and supply of additional glucose in culture medium. Maximum lipid and GLA were obtained 23.56 and 19.5% respectively, when the culture was grown at 28°C for four days and followed by addition of glucose (5%), and lowered the incubation temperature to 14°C for another four days. The presence of GLA in the oil obtained from M. rouxii CFR-G15 was confirmed by the gas chromatography-mass spectrometry. Gamma linolenic acid (GLA, n-6) is gaining importance in pharmaceutical and nutraceutical industries because of clinical evidence demonstrated that it has various beneficial effects in human health. In this paper temperature played a major role in enhancing the GLA content which has been described.