Aflatoxin and cyclopiazonic acid production by a sclerotium-producing Aspergillus tamarii strain.

The production of aflatoxins B1 and B2 by Aspergillus tamarii (subgenus Circumdati section Flavi) is reported for the first time. The fungus was isolated from soil collected from a tea (Camellia sinensis) field in Miyazaki Prefecture, Japan. Three single-spore cultures, NRRL 25517, NRRL 25518, and NRRL 25519, were derived from subcultures of the original isolate 19 (MZ2). Each of these single-spore cultures of A. tamarii produced aflatoxins B1 and B2 and cyclopiazonic acid, as well as black, pear-shaped sclerotia. The demonstration of aflatoxin production by A. tamarii is examined in connection with A. tamarii phylogenetic relationships, chemical ecology, and potential use in food fermentations.     

Enzyme formation during solid-substrate fermentation in rotating vessels

Aspergillus awamori NRRL 4869 was cultured on the solid substrate, wheat bran, in a modified Rollacell apparatus to produce alpha-galactosidase and invertase. The swivel cap on the elongated bottle permits the introduction of air while the bottle rotates. Parameters of air flow rate (0.05-0.2 liter/kg/min), rpm (0.15-15 rpm), and weight of solids (150 and 300 g) were varied. At low air flow rates (0.05 liter/kg solids/min), alpha-galactosidase production was minimal independent of the rotation rate. At 0.15 rpm and 0.2 liter/kg solids/min air flow rate, invertase production ceased after five days; whereas alpha-galactosidase production continued. The modified Rollacell can be a useful apparatus for studying solid-substrate cultures.     

Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi.

The Aspergillus parasiticus aflR gene, a gene that may be involved in the regulation of aflatoxin biosynthesis, encodes a putative zinc finger DNA-binding protein. PCR and sequencing were used to examine the presence of aflR homologs in other members of Aspergillus Section Flavi. The predicted amino acid sequences indicated that the same zinc finger domain, CTSCASSKVRCTKEKPACARCIERGLAC, was present in all of the Aspergillus sojae, Aspergillus flavus, and Aspergillus parasiticus isolates examined and in some of the Aspergillus oryzae isolates examined. Unique base substitutions and a specific base deletion were found in the 5' untranslated and zinc finger region; these differences provided distinct fingerprints. A. oryzae and A. flavus had the T-G-A-A-X-C fingerprint, whereas A. parasiticus and A sojae had the C-C-C-C-C-T fingerprint at the corresponding positions. Specific nucleotides at positions -90 (C or T) and -132 (G or A) further distinguished A. flavus from A. oryzae and A. parasiticus from A. sojae, respectively. A sojae ATCC 9362, which was previously designated A. oryzae NRRL 1988, was determined to be a A. sojae strain on the basis of the presence of the characteristic fingerprint, A-C-C-C-C-C-C-T. The DNAs of other members of Aspergillus Section Flavi, such as Aspergillus nomius and Aspergillus tamarii, and some isolates of A. oryzae appeared to exhibit low levels of similarity to the A. parasiticus aflR gene since low amounts of PCR products or no PCR products were obtained when DNAs from these strains were used.     

Production of 2-Ketogluconic Acid by Serratia marcescens

Production of 2-ketogluconic acid from glucose by fermentation with Serratia marcescens NRRL B-486 was studied in 20-liter stainless-steel fermentors. Conditions for 2-ketogluconic acid production included the following: glucose-salt medium, aeration rate of 0.75 volumes per volume per minute, agitation rate of 400 rev/min, temperature of 30 C, CaCO₃ to neutralize the acid formed, and a 5% (v/v) inoculum. Foaming was controlled with an antifoam agent added at intervals during the fermentation. When 120 g per liter of glucose were supplied, 95 to 100% yields of 2-ketogluconic acid were obtained in 16 hr. Larger amounts of glucose could be used in the fermentation provided that the carbohydrate was fed continuously. Continuous feeding of glucose to a total amount of 180 g per liter gave 95 to 100% yields of 2-ketogluconic acid in 24 hr; feeding glucose to a total amount of 240 g per liter gave 85 to 90% yields in 32 to 40 hr.     

Production of 3-Hydroxypropionaldehyde from Glycerol

3-Hydroxypropionaldehyde is a precursor to acrolein, which can be used as an intermediate for making acrylic acid and a variety of other useful industrial chemicals. Conversion of glycerol, a renewable resource, to 3-hydroxypropionaldehyde was attempted via action of glycerol dehydrase isolated from Lactobacillus sp. strain NRRL B-1720. This method, however, was unsatisfactory because enzyme activity was lost within 60 to 90 min after the reaction initiation. Fermentation of glycerol by whole cells of Klebsiella pneumoniae NRRL B-199 in the presence of optimal semicarbazide hydrochloride proved more effective. Using this technique, glycerol solutions of 30 g/liter yielded 3-hydroxypropionaldehyde solutions of 13.1 g/liter. Thus, a conversion efficiency equal to 55% of the theoretical maximum was realized.     

Effect of the nitrogen source on caffeine degradation by Aspergillus tamarii

AIMS: To evaluate caffeine degradation and nitrogen requirements during Aspergillus tamarii growth in submerged culture. METHODS AND RESULTS: Aspergillus tamarii spores produced on a coffee infusion agar medium added with sucrose were used. Several caffeine and ammonium sulphate concentrations (0-1 and 0-1.36 g l-1, respectively) were tested simultaneously on fungal biomass production and caffeine degradation. An additional caffeine pulse (4 g l-1) was added for all experiments after 48 h of fermentation. Results revealed that when using 0.90 g l-1 of caffeine and 0.14 g l-1 of ammonium sulphate, biomass production and caffeine degradation were enhanced. Highest biomass production (Xmax = 9.87 g l-1) with a specific growth rate (micro) of 0.073 h-1 and caffeine degradation rate of 0.033 g l-1 h-1, was observed under these conditions. CONCLUSIONS: Caffeine degradation as well as biomass production were characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: These studies set the stage for future characterization studies of intracellular enzymes involved in caffeine degradation. Moreover, results observed may help in the biotreatment of residues from the coffee agroindustry.     

Efficient production of L-ribose with a recombinant Escherichia coli biocatalyst

A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 microM ZnCl(2) and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter(-1) day(-1). This system represents a significantly improved method for the large-scale production of l-ribose.     

Olive-mill wastewaters: a promising substrate for microbial lipase production

The present study investigated the valorization of olive-mill wastewater (OMW) by its use as a possible growth medium for the microbial production of extra-cellular lipase. To this end, strains of Geotrichum candidum (NRRL Y-552 and Y-553), Rhizopus arrhizus (NRRL 2286 and ISRIM 383), Rhizopus oryzae (NRRL 6431), Aspergillus oryzae (NRRL 1988 and 495), Aspergillus niger (NRRL 334), Candida cylindracea (NRRL Y-17506) and Penicillium citrinum (NRRL 1841 and 3754, ISRIM 118) were screened. All strains were able to grow on the undiluted OMW, producing extra-cellular lipase activity. C. cylindracea NRRL Y-17506 showed the highest lipase activity on all the typologies of OMW used. Its lipase production on OMW was markedly affected by the type of nitrogen source and was induced by the addition of olive oil. The highest activity (9.23 IU ml(-1)) of the yeast was obtained on OMW supplemented with NH(4)Cl (2.4 g l(-1)) and olive oil (3.0 g l(-1)).     

β-Carotene inhibition of aflatoxin biosynthesis amongAspergillus flavus genotypes from Illinois corn

Thirty-nineAspergillus flavus genotypes (DNA fingerprinting) isolated from corn grown in a field near Kilbourne, Illinois were evaluated for their sensitivity to β-carotene (50 μg/ml) inhibition of aflatoxin B₁ biosynthesis. Inhibition of aflatoxin was greater than 90% for 28 of the genotypes and >70% for 38 of the 39 genotypes. FiveA. flavus strains (4 fingerprint groups) isolated from molded raw peanuts, NRRL 3239, NRRL 3357, NRRL 6514, NRRL 6515 and NRRL 13135, produced greater quantities of aflatoxin than all 39 genotypes isolated from corn, and were less sensitive to β-carotene inhibition.Aspergillus flavus NRRL 3357 is commonly used as inoculum in variety trials for aflatoxin resistance. Isolate identity and sensitivity to potential inhibitors in corn can be critical in assessing corn resistance to aflatoxin.     

Optimizing aerobic conversion of glycerol to 3-hydroxypropionaldehyde

When cells of Klebsiella pneumoniae NRRL B-199 (ATCC 8724) were grown aerobically on a rich glycerol medium and then suspended in buffer supplemented with semicarbazide and glycerol, aerobic conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) ensued. Depending on conditions, 0.38 to 0.67 g of 3-HPA were formed per gram of glycerol consumed. This means that up to 83.8% of the carbon invested as glycerol could potentially be recovered as the target product, 3-HPA. Production of 3-HPA was sensitive to the age of cells harvested for resuspension and was nonexistent if cells were cultivated on glucose instead of glycerol as the sole carbon source. Compared with 24- and 72-h cells, 48-h cells produced 3-HPA at the highest rate and with the greatest yield. The cell biomass concentration present during the fermentation was never particularly critical to the 3-HPA yield, but initial fermentation rates and 3-HPA accumulation displayed a linear dependence on biomass concentration that faded when biomass exceeded 3 g/liter. Fermentation performance was a function of temperature, and an optimum initial specific 3-HPA productivity occurred at 32 degrees C, although the overall 3-HPA yield increased continuously within the 25 to 37 degrees C range studied. The pH optimum based on fermentation rate was different from that based on overall yield; 8 versus 7, respectively. Initial glycerol concentrations in the 20 to 50 g/liter range optimized initial 3-HPA productivity and yield.     

l-leucine aminopeptidase production by filamentous Aspergillus fungi

AIMS: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. METHODS AND RESULTS: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2.61, 2.59 and 1.30 IU ml(-1) for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11.39, 17.40 and 13.02 IU g(-1) dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65-75 degrees C at pH 8.0-9.0 for all three fungi. Metal ions, Co(2+) and Fe(2+) in 2 mmol l(-1) concentration apparently enhanced the relative enzyme activity and heat stability. CONCLUSIONS: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Optimizing aerobic conversion of glycerol to 3-hydroxypropionaldehyde.

When cells of Klebsiella pneumoniae NRRL B-199 (ATCC 8724) were grown aerobically on a rich glycerol medium and then suspended in buffer supplemented with semicarbazide and glycerol, aerobic conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) ensued. Depending on conditions, 0.38 to 0.67 g of 3-HPA were formed per gram of glycerol consumed. This means that up to 83.8% of the carbon invested as glycerol could potentially be recovered as the target product, 3-HPA. Production of 3-HPA was sensitive to the age of cells harvested for resuspension and was nonexistent if cells were cultivated on glucose instead of glycerol as the sole carbon source. Compared with 24- and 72-h cells, 48-h cells produced 3-HPA at the highest rate and with the greatest yield. The cell biomass concentration present during the fermentation was never particularly critical to the 3-HPA yield, but initial fermentation rates and 3-HPA accumulation displayed a linear dependence on biomass concentration that faded when biomass exceeded 3 g/liter. Fermentation performance was a function of temperature, and an optimum initial specific 3-HPA productivity occurred at 32 degrees C, although the overall 3-HPA yield increased continuously within the 25 to 37 degrees C range studied. The pH optimum based on fermentation rate was different from that based on overall yield; 8 versus 7, respectively. Initial glycerol concentrations in the 20 to 50 g/liter range optimized initial 3-HPA productivity and yield.     

N—乙酰氨基己糖苷酶产生菌的筛选与产酶条件

About 1200 strains of microorganisms were screened including fungi, actinomyces, and bacteria, in which 237 strains producing the enzyme desired. The results showed that the beta-GlcNAcase and beta-GalNAcase always co-existed in one strain, though may be in different ratio. From strains mentioned above the authors screened out a potent beta-N-acetylhexosaminidase producing strain, Aspergillus tamarii S215, from the soil sample. The optimal conditions for enzyme production were as follows: the microorganisms was inoculated in a 5% wheat bran suspension, cultured at 28-30 degrees C on shaker for 5-6 days. The productivity can be moderately enhanced by the addition of cellobiose or glucosamine or galactosamine or by the extra supplement of (NH4)2SO4 and NH4NO3 as N sources. In the culture filtrate of Asp. tamarii, the alpha, (beta)-galactosidase, beta-glucosidase, alpha-mannosidase and beta-fucosidase were also found.     

Blastobotrys serpentis sp. nov., isolated from the intestine of a Trinket snake (Elaphe sp., Colubridae)

Asporogenus yeast strains W113AT and W113B were isolated from the intestine of a dead Trinket snake. The two isolates showed 100% sequence similarity in the D1/D2 domain of the large-subunit (LSU) rRNA gene, internal transcribed spacer (ITS) 1-5.8S rRNA gene-ITS2 region and mitochondrial small-subunit rRNA gene and the cytochrome oxidase II gene sequence and also showed similar phenotypic characteristics. The nearest phylogenetic neighbors of W113AT and W113B based on the sequence of the D1/D2 domain of the LSU rRNA gene were Blastobotrys chiropterorum NRRL Y-17017T and Blastobotrys terrestris NRRL Y-17704T with about 98% similarity. The close affiliation of W113AT and W113B with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T was also evident from the high similarity observed in the nucleotide sequences of the mitochondrial small subunit rRNA (96-97.8%) and the cytochrome oxidase II (95.5-95.6%) genes. In the neighbor-joining phylogenetic trees constructed based on the D1/D2 domain or cytochrome oxidase gene, the isolates clustered with the above-mentioned species. However, the isolates showed a number of differences in their phenotypic properties with B. chiropterorum NRRL Y-17017T and B. terrestris NRRL Y-17704T and hence are regarded as representing a novel member of the genus Blastobotrys, for which the name Blastobotrys serpentis sp. nov. is proposed.     

Integrated enzymatic production of specific structured lipid and phytosterol ester compositions

Mixtures of specific structured lipids and phytosterol esters, valuable food components, were synthesized by an enzymatic one-pot process in organic-solvent-free medium starting from a mixture of phytosterol, caprylic acid and sunflower oil. Nine biocatalysts, seven commercially available lipases and two air-dried solid state (SSF) fermentation preparations of Aspergillus oryzae NRRL 6270 (AoSSF) and Aspergillus sojae NRRL 6271 (AsSSF), were screened for lipase activity in the transesterification reactions of sunflower oil with caprylic acid and for sterol esterase activity in the direct esterification of phytosterols with free fatty acids. The best process variant using a sequence of sterol esterase (AoSSF)-catalyzed esterification reaction of the free fatty acids and phytosterols, followed by water removal in vacuum and lipase-catalyzed transesterification with immobilized lipase from Rhizomucor miehei (Lipozyme) resulted in 92.1% conversion to phytosterol esters and 44.1% conversion to triacylglycerols containing two caprylic esters.     

Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by Azotobacter vinelandii UWD.

Azotobacter vinelandii UWD formed polyhydroxyalkanoate (PHA) copolymers containing beta-hydroxybutyrate and beta-hydroxyvalerate (HV) when grown in a medium containing glucose as the primary C source and valerate (pentanoate) as a precursor. Copolymer was not formed when propionate was added to the glucose medium but was formed when heptanoate, nonanoate, or trans-2-pentenoate was present. Optimal levels of HV were formed when valerate was added at the time of maximum PHA synthesis, although HV incorporation was not dependent on glucose catabolism. HV content in the polymer was increased from 17 to 24 mol% by adding 10 to 40 mM valerate to glucose medium, but HV insertion into the polymer occurred at a fixed rate. Similarly, the addition of valerate to a fed-batch culture of strain UWD in beet molasses in a fermentor produced 19 to 22 g of polymer per liter, containing 8.5 to 23 mol% HV after 38 to 40 h. The synthesis of HV in these cultures also occurred at a fixed rate (2.3 to 2.8 mol% h-1), while the maximum PHA production rate was 1.1 g liter-1 h-1. During synthesis of copolymer in batch or fed-batch culture, the yield from conversion of glucose into PHA (YP/S) remained at maximum theoretical efficiency (greater than or equal to 0.33 g of PHA per g of glucose consumed). Up to 45 mol% C source, but the PHA produced amounted to less than 1 g/liter. The combination of 30 mM valerate as a sole C source and 0.5 mM 4-pentenoate increased the HV content in the polymer to 52 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of high concentrations of ethanol from inulin by simultaneous saccharification and fermentation using Aspergillus niger and Saccharomyces cerevisiae.

Pure nonhydrolyzed inulin was directly converted to ethanol in a simultaneous saccharification and fermentation process. An inulinase-hyperproducing mutant, Aspergillus niger 817, was grown in a submerged culture at 30 degrees C for 5 days. The inulin-digestive liquid culture (150 ml) was supplemented with 45 g of inulin, 0.45 g of (NH4)2SO4, and 0.15 g of KH2PO4. The medium (pH 5.0) was inoculated with an ethanol-tolerant strain, Saccharomyces cerevisiae 1200, and fermentation was conducted at 30 degrees C. An additional 20 g of inulin was added to the culture after 15 h of fermentation. S. cerevisiae 1200 utilized 99% of the 65 g of inulin during the fermentation, and produced 20.4 and 21.0% (vol/vol) ethanol from chicory and dahlia inulins, respectively, within 3 days of fermentation. The maximum volumetric productivities of ethanol were 6.2 and 6.0 g/liter/h for chicory and dahlia inulins, respectively. The conversion efficiency of inulin to ethanol was 83 to 84% of the theoretical ethanol yield.     

Carbon Concentration and Carbon-to-Nitrogen Ratio Influence Submerged-Culture Conidiation by the Potential Bioherbicide Colletotrichum truncatum NRRL 13737

We assessed the influence of various carbon concentrations and carbon-to-nitrogen (C:N) ratios on Colletotrichum truncatum NRRL 13737 conidium formation in submerged cultures grown in a basal salts medium containing various amounts of glucose and Casamino Acids. Under the nutritional conditions tested, the highest conidium concentrations were produced in media with carbon concentrations of 4.0 to 15.3 g/liter. High carbon concentrations (20.4 to 40.8 g/liter) inhibited sporulation and enhanced the formation of microsclerotiumlike hyphal masses. At all the carbon concentrations tested, a culture grown in a medium with a C:N ratio of 15:1 produced more conidia than cultures grown in media with C:N ratios of 40:1 or 5:1. While glucose exhaustion was often coincident with conidium formation, cultures containing residual glucose sporulated and those with high carbon concentrations (>25 g/liter) exhausted glucose without sporulation. Nitrogen source studies showed that the levels of C. truncatum NRRL 13737 conidiation were similar for all protein hydrolysates tested. Reduced conidiation occurred when amino acid and inorganic nitrogen sources were used. Of the nine carbon sources evaluated, acetate as the sole carbon source resulted in the lowest level of sporulation.     

Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by Azotobacter vinelandii UWD.

Azotobacter vinelandii UWD formed polyhydroxyalkanoate (PHA) copolymers containing beta-hydroxybutyrate and beta-hydroxyvalerate (HV) when grown in a medium containing glucose as the primary C source and valerate (pentanoate) as a precursor. Copolymer was not formed when propionate was added to the glucose medium but was formed when heptanoate, nonanoate, or trans-2-pentenoate was present. Optimal levels of HV were formed when valerate was added at the time of maximum PHA synthesis, although HV incorporation was not dependent on glucose catabolism. HV content in the polymer was increased from 17 to 24 mol% by adding 10 to 40 mM valerate to glucose medium, but HV insertion into the polymer occurred at a fixed rate. Similarly, the addition of valerate to a fed-batch culture of strain UWD in beet molasses in a fermentor produced 19 to 22 g of polymer per liter, containing 8.5 to 23 mol% HV after 38 to 40 h. The synthesis of HV in these cultures also occurred at a fixed rate (2.3 to 2.8 mol% h-1), while the maximum PHA production rate was 1.1 g liter-1 h-1. During synthesis of copolymer in batch or fed-batch culture, the yield from conversion of glucose into PHA (YP/S) remained at maximum theoretical efficiency (greater than or equal to 0.33 g of PHA per g of glucose consumed). Up to 45 mol% C source, but the PHA produced amounted to less than 1 g/liter. The combination of 30 mM valerate as a sole C source and 0.5 mM 4-pentenoate increased the HV content in the polymer to 52 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum, and Zymomonas mobilis

Saccharomyces cerevisiae NRRL Y-2034, S, uvarum NRRL Y-1347, and Zymomonas mobilis NRRL B-806 each were separately immobilized in a Ca-alginate matrix and incubated in the presence of a free-flowing and continuous 1, 3, 5, 10, or 20% (w/w) glucose solution. In general, the yeast cells, converted 100percnt; of the 1, 3, and 5% glucose to alcohol within 48 h and maintained such a conversion rate for at least two weeks. The bacterium converted ca. 90% (w/w) of the 1, 3, and 5% glucose to alcohol continuously for one week. However, both the yeast and bacterium were inhibited in the highest glucose (20% w/w) solution. All of the immobilized cultures produced some alcohol for at least 14 days. Immobilized S. cerevisiae was the best alcohol producer of all of the glucose concentrations; the yeast yielded 4.7 g ethanol/100 g solution within 72 h in the 10% glucose solution. After 7-8 days in the 10% solution, S. cerevisiae produced ethanol at 100% of theoretical yield (5.0 g ethanol/100 g solution), with a gradual decrease in alcohol production by 14 days. Immobillized S. uvarum produced a maximum of 4.0 g ethanol/100 g solution within 2 days and then declined to ca. 1.0 g ethanol/100 g solution after 7 days continuous fermentation in the 10% glucose solution. Zymomonas mobilis reached its maximum ethanol production at 4 days (4.7 g/100 g solution), and then diminished similarly to S. uvarum. The development of a multiple disk shaft eliminated the problem both of uneven distribution of alginate-encapsulated cells and of glucose channeling within the continuous-flow fermentor column. This invention improved alcohol production about threefold for the yeast cells.