Ethanol fermentation by a cellulolytic fungus Aspergillus terreus

The cellulolytic fungus Aspergillus terreus showed an additional property of fermenting glucose to ethanol. In addition to glucose, A. terreus also fermented other hexoses, pentoses and disaccharides to ethanol. Of the soluble carbon sources tested, glucose yielded maximum (2.46% (w/v)) ethanol.     

Lovastatin biosynthesis by Aspergillus terreus in a chemically defined medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.     

Production of itaconic acid from pentose sugars by Aspergillus terreus

Itaconic acid (IA), an unsaturated 5‐carbon dicarboxylic acid, is a building block platform chemical that is currently produced industrially from glucose by fermentation with Aspergillus terreus. However, lignocellulosic biomass has potential to serve as low‐cost source of sugars for production of IA. Research needs to be performed to find a suitable A. terreus strain that can use lignocellulose‐derived pentose sugars and produce IA. One hundred A. terreus strains were evaluated for the first time for production of IA from xylose and arabinose. Twenty strains showed good production of IA from the sugars. Among these, six strains (NRRL strains 1960, 1961, 1962, 1972, 66125, and DSM 23081) were selected for further study. One of these strains NRRL 1961 produced 49.8 ± 0.3, 38.9 ± 0.8, 34.8 ± 0.9, and 33.2 ± 2.4 g IA from 80 g glucose, xylose, arabinose and their mixture (1:1:1), respectively, per L at initial pH 3.1 and 33°C. This is the first report on the production of IA from arabinose and mixed sugar of glucose, xylose, and arabinose by A. terreus. The results presented in the article will be very useful in developing a process technology for production of IA from lignocellulosic feedstocks. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1059–1067, 2017     

beta-Glucosidases from cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum as typical glycosidases

By ethanol precipitation (v/v) and chromatography on Sephadex SP, DEAE (or DEAE-cellulose), and G-200 beta-glucosidases (EC 3.2.1.21) from the culture filtrates of cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum grown on the medium with cellulose containing materials were isolated. The enzymes were homogenous as shown by different techniques. The substrate specificities of the obtained enzymes were studied. beta-Glucosidases had higher affinity for p-nitrophenyl-beta-D-glucopyranoside than for cellobiose (Km 1.25, 0.34, 0.20 and 5.4, 2.0, 1.2 mM, respectively) and were able to hydrolyze both laminaribiose and gentiobiose; but they were unable to cleave cotton fiber, carboxymethylcellulose, and other glycans to reducing sugars. They showed transglycosylase activity. Ki values for arylglucosidase activity of beta-glucosidases from A. terreus, G. candidum, and T. longibrachiatum in the presence of either glucose or glucono-1,5-lactone were 12.2, 6.0, 2.1 and 0.20, 0.19, 0.07 mM, respectively. The Mr's were estimated by gel filtration and by sedimentation equilibrium centrifugation to 200,000, 200,000, 350,000, respectively. The isoelectric points of beta-glucosidases were 4.8, 5.9, and 4.2, respectively. The optimum temperatures and pH's were 60, 50, and 50 degrees C and at pH 4.5, 4.5, and 4.8-5.7, respectively. These properties appear to relate beta-glucosidases obtained in the present study to typical glycosidases.     

Removal of heavy metals by an Aspergillus terreus strain immobilized in a polyurethane matrix

AIMS: The aim was to investigate the biosorption of chromium, nickel and iron from metallurgical effluents, produced by a steel foundry, using a strain of Aspergillus terreus immobilized in polyurethane foam. METHODS AND RESULTS: A. terreus UFMG-F01 was immobilized in polyurethane foam and subjected to biosorption tests with metallurgical effluents. Maximal metal uptake values of 164.5 mg g(-1) iron, 96.5 mg g(-1) chromium and 19.6 mg g(-1) nickel were attained in a culture medium containing 100% of effluent stream supplemented with 1% of glucose, after 6 d of incubation. CONCLUSIONS: Microbial populations in metal-polluted environments include fungi that have adapted to otherwise toxic concentrations of heavy metals and have become metal resistant. In this work, a strain of A. terreus was successfully used as a metal biosorbent for the treatment of metallurgical effluents. SIGNIFICANCE AND IMPACT OF THE STUDY: A. terreus UFMG-F01 was shown to have good biosorption properties with respect to heavy metals. The low cost and simplicity of this technique make its use ideal for the treatment of effluents from steel foundries.     

Itaconate biosynthesis in Aspergillus terreus.

Itaconate biosynthesis was studied in intact cells of high-yield (RC4') and low-yield (CM85J) strains of the fungus Aspergillus terreus by methods (tracers, nuclear magnetic resonance spectroscopy, and mass spectroscopy) that did not interfere with metabolism. Itaconate formation in RC4' required de novo protein biosynthesis. Krebs cycle intermediates increased in both strains during the production of itaconic acid. The Embden-Meyerhof-Parnas pathway and the Krebs cycle were shown to be involved in this biosynthesis by using 14C- and 13C-labelled substrates and nuclear magnetic resonance spectroscopy. A metabolic pathway for itaconate formation from glucose in A. terreus is proposed.     

Continuous itaconic acid production by immobilized biocatalysts.

The continuous itaconic acid production from sucrose with Aspergillus terreus TKK 200-5-3 mycelium immobilized on polyurethane foam cubes was optimized in column bioreactors using statistical experimental design and empirical modelling. The highest itaconic acid product concentration calculated on the basis of the obtained model was 15.8 g l-1 in the investigated experimental area, when sucrose concentration was 13.5%, aeration rate 150 ml min-1 and residence time 178 h. From sucrose with immobilized A. terreus TKK 200-5-3 mycelium itaconic acid production was stable for at least 4.5 months in continuous column bioreactors. In comparison, using glucose as substrate and immobilized A. terreus TKK 200-5-1 mycelium as biocatalyst similar stability was obtained with higher product concentration. The omission of copper sulphate from the production medium gave the highest itaconic acid product concentration (26 g l-1) from 9% glucose with 0.25% ammonium nitrate and 0.095% magnesium sulphate.     

Cellulolytic activity of moulds. II. Various methods of precipitating and concentrating enzymes and their influence on the activity of cellulolytic preparation and xylanase of Aspergillus terreus F-413

Complexes of cellulolytic enzymes and xylanase were precipitated and concentrated by various methods from post-culture liquids of Aspergillus terreus F-143, containing cellucotton as carbon source. The best results in regard to the specific activity of the preparations were obtained by precipitation of enzymes with acetone-denatured ethanol. Besides high cellulolytic and xylanase activity the crude enzyme preparation showed the presence of small amounts of amylase, protease and polygalacturonase.     

Biochemical studies on acetate non-utilizing mutants of Aspergillus terreus IRRL 16043

The strain Aspergillus terreus IRRL 16043 can utilize glucose as well as acetate as a sole carbon source. Thirty-nine mutants were isolated from the wild-type by treatment with a chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNTG) which could not utilize acetate as a sole carbon source, and were designated as acetate non-utilizing (acu). By complementation and biochemical analyses they were divided into three functional groups, acu A, acu B and acu C lacking isocitrate lyase, malate synthase and acetyl-CoA synthetase activity, respectively.     

Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts.     

Effects of ethanol, fructose, and ethanol plus fructose infusions on plasma glucose concentration and glucose turnover in monkeys (Macaca fascicularis) as measured by [6-3H]glucose

Six adult, female, cynomolgus monkeys were fasted for 64 hr and then continuously infused with [6-3H]glucose to determine the rates of glucose turnover and clearance while they were also being infused with ethanol (110 mumol/min/kg), 1,3-butanediol (110 mumol/min/kg), fructose (30 mumol/min/kg) or ethanol plus fructose (110 and 30 mumol/min/kg) respectively. Both ethanol and 1,3-butanediol infusions decreased the glucose turnover rate (the steady-state input-output rate from the plasma glucose pool) and the plasma glucose concentration by halving the glucose production rate. In contrast, fructose infusions increased the glucose turnover rate and glucose concentration by increasing the glucose production rate by 20%. The plasma clearance rate of glucose was lowest when the animals were infused with ethanol plus fructose; this suggests that acetate from ethanol oxidation may have a glucose-sparing effect if normoglycemia is maintained.     

衣康酸生产菌种的定向选育和产酸条件的研究

通过紫外线—高温复合诱变处理衣康酸生产菌株土曲霉构Aspergillus terreus As3.2811,用以琥珀酸为唯一碳源的选择性乎板定向筛选高产菌株,获得产酸率较其亲株提高了5倍以上的突变株。用正交试验的方法对突变株的适宜产酸条件进行了研究,通过分批补糖发酵可提高其产酸率高达39．92％。     

Influence of L-galactonic acid γ -lactone on ascorbate production in some yeasts

L-galactonic acid -lactone appear to influence ascorbic acid production in strains of Saccharomyces cerevisiae, Clavispora lusitaniae, Cryptococcus terreus, Pichia fermentans in which this is undetected whenever glucose represents the sole carbon source. Cryptococcus terreus (strains DBVP 6012 and 6242) does not show ascorbic acid production either in presence or in the absence of L-galactonic acid -lactone. This feature is probably connected to the insensibility of the strain to the lycorine, an alkaloid which commonly inhibits cell division probably by blocking L-galactonic acid -lactone convertion into ascorbate.     

Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l-h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h^?1.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

The acid hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol sulfuric acid per liter pyrolysate using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol in 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol in 18 h. The results showed that the acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by S. cerevisiae (R) in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae

Acid-hydrolysis of cellulosic pyrolysate to glucose and its fermentation to ethanol were investigated. The maximum glucose yield (17.4%) was obtained by the hydrolysis with 0.2 mol/l sulfuric acid using autoclaving at 121 degrees C for 20 min. The fermentation by Saccharomyces cerevisiae of a hydrolysate medium containing 31.6 g/l glucose gave 14.2 g/l ethanol after 24 h, whereas the fermentation of the medium containing 31.6 g/l pure glucose gave 13.7 g/l ethanol after 18 h. The results showed that acid-hydrolyzed pyrolysate could be used for ethanol production. Different nitrogen sources were evaluated and the best ethanol concentration (15.1 g/l) was achieved by single urea. S. cerevisiae (R) was obtained by adaptation of S. cerevisiae to the hydrolysate medium for 12 times, and 40.2 g/l ethanol was produced by it in the fermentation with the hydrolysate medium containing 95.8 g/l glucose, which was about 47% increase in ethanol production compared to its parent strain.     

Application of fuzzy reasoning to control of glucose and ethanol concentrations in baker's yeast culture

Fuzzy reasoning was applied to control both ethanol and glucose concentrations in fed-batch cultures of baker's yeast. This fuzzy controller consisted of three membership functions (concentrations of dissolved oxygen (DO), ethanol and glucose) and 18 production rules. Fuzzy inference was carried out by IF {A is a and B is b,...#x007D;, THEN {C is c} from the on-line measured concentrations of DO, ethanol and glucose. When medium concentrations of ethanol and glucose in fed-batch culture of baker's yeast were set at 2 g/l and 0.2 g/l, both ethanol and glucose concentrations were controlled at 2.67±0.35 g/l and 0.27±0.25 g/l, respectively, ethanol production was reduced from 26 g/l to 34 g/l, cell yield increased from 0.38 to 0.53 g dry cell/g consumed glucose and ethanol yield decreased from 0.50 to 0.14 g ethanol/g consumed glucose, respectively, as compared with those of the glucose only control at 0.2 g/l.     

Derepression of galactose metabolism in melibiase producing bakers' and distillers' yeast

Beet molasses is widely used as a growth substrate for bakers' and distillers' yeast in the production of biomass and ethanol. Most commercial yeasts do not fully utilise the carbohydrates in molasses since they are incapable of hydrolysing the disaccharide melibiose to glucose and galactose. Also, expression of genes encoding enzymes for the utilisation of carbon sources that are alternatives to glucose is tightly regulated, sometimes rates of yeast growth and/or ethanol production. The GAL genes are regulated by specific induction by galactose and repression during growth on glucose. In an industrial distillers' yeast, two genes interacting synergistically in glucose repression of galactose utilization, MIG1 and GAL80, have been disrupted with MEL1, encoding melibiase. The physiology of the wild-type strain and the recombinant strains was investigated on mixtures of glucose and galactose and on molasses. The recombinant strain started to ferment galactose when 9.7 g 1(-1) glucose was still present during a batch fermentation, whereas the wild-type strain did not consume any galactose in the presence of glucose. The ethanol yield in the recombinant strain was 0.50 g ethanol g sugar (-1) in an ethanol fermentation on molasses, compared with 0.48 g ethanol g sugar (-1) for the wild-type strain. The increased ethanol yield was due to utilization of melibiose in the molasses.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.