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Corn fiber, a by-product of the corn wet-milling industry, represents a renewable resource that is readily available in significant quantities and could potentially serve as a low-cost feedstock for the production of fuel-grade alcohol. In this study, we used a batch reactor to steam explode corn fiber at various degrees of severity to evaluate the potential of using this feedstock in the bioconversion process. The results indicated that maximum sugar yields (soluble and following enzymatic hydrolysis) were recovered from corn fiber that was pretreated at 190°C for 5 min with 6% SO2. Sequential SO2-catalyzed steam explosion and enzymatic hydrolysis resulted in very high conversion (81%) of all polysaccharides in the corn fiber to monomeric sugars. Subsequently, Saccharomyces cerevisiae was able to convert the resultant corn fiber hydrolysates to ethanol very efficiently, yielding 90–96% of theoretical conversion during the fermentation process. [ABSTRACT FROM AUTHOR]

Two new ethanologenic strains (FBR4 and FBR5) of Escherichia coli were constructed and used to ferment corn fiber hydrolysate. The strains carry the plasmid pLO1297, which contains the genes from Zymomonas mobilis necessary for efficiently converting pyruvate into ethanol. Both strains selectively maintained the plasmid when grown anaerobically. Each culture was serially transferred 10 times in anaerobic culture with sugar-limited medium containing xylose, but noselective antibiotic. An average of 93 and 95% of the FBR4 and FBR5 cells, respectively, maintained pLO1297 in anaerobic culture. The fermentation performances of the repeatedly transferred cultures were compared with those of cultures freshly revived from stock in pH-controlled batch fermentations with 10% (w/v) xylose. Fermentation results were similar for all the cultures. Fermentations were completed within 60 h and ethanol yields were 86–92% of theoretical. Maximal ethanol concentrations were 3.9–4.2% (w/v). The strains were also tested for their ability to ferment corn fiber hydrolysate, which contained 8.5% (w/v) total sugars (2.0% arabinose, 2.8% glucose, and 3.7% xylose). E. coli FBR5 produced more ethanol than FBR4 from the corn fiber hydrolysate. E. coli FBR5 fermented all but 0.4% (w/v) of the available sugar, whereas strain FBR4 left 1.6% unconsumed. The fermentation with FBR5 was completed within 55 h and yielded 0.46 g of ethanol/g of available sugar, 90% of the maximum obtainable. [ABSTRACT FROM AUTHOR]

Lack of effective chemicals impedes control of Fusarium dry rot of stored potato tubers destined for processed and table stock use. Biological control of dry rot incited by Gibberella pulicaris (anamorph=Fusarium sambucinum) has been demonstrated in laboratory studies but not in commercial storage environments. Several Gram-negative bacterial strains that were efficacious and amenable to production in liquid culture in laboratory studies were selected for pilot studies in Idaho and for bin trials at commercial storage houses in Idaho and North Dakota. In the first year of pilot studies, Pseudomonas fluorescens S22:T:04 (≈1 x 108 cfu/ml) decreased dry rot caused by G. pulicaris by 19% when coinoculated with the pathogen compared to controls and to the fungicide thiabendazole (TBZ) applied at label rates. In second year pilot studies, P. fluorescens P22:Y:05 and Enterobacter cloacae S11:T:07 (≈4 x 108 cfu/ml) reduced the severity of disease incited by G. pulicaris (25% and 17% average disease decrease, respectively) when antagonists were applied after pathogen inoculum. Treatment effects on dry rot that developed from inoculation with Nectria haematococca (anamorph=Fusarium solani var. coeruleum) were variable and influenced by interactions between antagonists, a wetting agent, and TBZ. In commercial storage bin trials, E. cloacae S11:T:07 reduced naturally occurring levels of dry rot by an average of 21% compared to 14% for TBZ, demonstrating that this antagonist was effective when produced using a liquid culture medium and methodology that approximates commercial practices for producing biomass. [ABSTRACT FROM AUTHOR]

Corn fiber consists of about 20% starch, 14% cellulose, and 35% hemicellulose, and has the potential to serve as a low-cost feedstock for production of fuel ethanol. Several pretreatments (hot water, alkali, and dilute, acid) and enzymatic saccharification procedures were evaluated for the conversion of corn fiber starch, cellulose, and hemicellulose to monomeric sugars. Hot water pretreatment (121°C, 1 h) facilitated the enzymatic sacch arification of starch and cellulose but not hemicellulose. Hydrolysis of corn fiber pretreated with alkali un dersimilar conditions by enzymatic means gave similar results. Hemicellulose and starch components were converted to monomeric sugars by dilute H2SO4 pretreatment (0.5–1.0%, v/v) at 121°C. Based on these findings, a method for pretreatment and enzymatic saccharification of corn fiber is presented. It in volves the pretreatment of corn fiber (15% solid, w/v) with dilute acid (0.5% H2SO4, v/v) at 121°C for 1 h, neutralization to pH 5.0, then saccharification of the pretreated corn fiber material with commercial cellulase and β-glucosidase preparations The yield of monomeric sugars from corn fiber was typically 85–100% of the theoretical yield. [ABSTRACT FROM AUTHOR]

Utilization of pentose sugars ( d-xylose and l-arabinose) derived from hemi-cellulose is essential for the economic conversion of biomass to ethanol. Xylose-fermenting yeasts were discovered in the 1980s, but to date, no yeasts have been found that ferment l-arabinose to ethanol in significant quantities. We have screened 116 different yeasts for the ability to ferment l-arabinose and have found the following species able to ferment the sugar: Candida auringiensis, Candida succiphila, Ambrosiozyma monospora, and Candida sp. (YB-2248). Though these yeasts produced ethanol concentrations of 4.1 g/L or less, they are potential candidates for mutational enhancement of l-arabinose fermentation. These yeasts were also found to ferment d-xylose. [ABSTRACT FROM AUTHOR]

Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. During aerobic growth, Rhizopus oryzae produces L-lactic acid from lactate dehydrogenase mediated reduction of pyruvate, while O2 limiting conditions yield primarily ethanol. A mutant was isolated that expressed only 5% of the wild type alcohol dehydrogenase activity under O2 limiting conditions and produced nearly 40 g lactic acid/l in 70 h. This is almost a ten-fold increase in lactic acid production when compared to the parent strain. [ABSTRACT FROM AUTHOR]

Of nineteen Aspergilli and ten Rhizopus strains examined for their ability to ferment simple sugars (glucose, xylose, and arabinose) as well as complex substrates (cellulose, oat-spelt xylan, corn fiber, and corn germ pressing), three Rhizopus strains were identified that could produce more than 31 g ethanol/l under anaerobic stress. By 72 h, glucose , xylose, cellobiose, and corn fiber were fermented with perspective yields of 100, 47, 80, and 40 percent, of theoretical. [ABSTRACT FROM AUTHOR]

Pseudomonas fluorescens strain 2-79 (NRRL-15132) produces a fluorescent yellow-green pyoverdine when cultured on Fe(III)-poor medium. When cultured on Fe(III)-rich medium, strain 2-79 produces an antibiotic, phenazine 1-carboxylic acid, which is effective in suppressing plant fungal diseases such as take-all of wheat. A 2 factorial design was used to examine pyoverdine production as a function of the presence or absence of Bacto casamino acids, purines-pyrimidines and vitamins in an iron-deficient medium. Amino acids were found to be an important factor ( P=0.0002). A Plackett-Burman design was used to identity eight amino acids, out of the 19 present in casamino acids, that were responsible for the increased pyoverdine production: methionine, valine, isoleucine, tyrosine, proline, phenylalanine, glutamic acid, and glycine. Biomass was enhanced only by glutamic acid. [ABSTRACT FROM AUTHOR]

Corn fiber, which consists of about 20% starch, 14% cellulose, and 35% hemicellulose, has the potential to serve as a low cost feedstock for production of fuel ethanol. Currently, the use of corn fiber to produce fuel ethanol faces significant technical and economic challenges. Its success depends largely on the development of environmentally friendly pretreatment procedures, highly effective enzyme systems for conversion of pretreated corn fiber to fermentable sugars, and efficient microorganisms to convert multiple sugars to ethanol. Several promising pretreatment and enzymatic processes for conversion of corn fiber cellulose, hemicellulose, and remaining starch to fermentable sugars were evaluated. These hydrolyzates were then examined for ethanol production in bioreactors, using genetically modified bacteria and yeast. Several novel enzymes were also developed for use in pretreated corn fiber saccharification. [ABSTRACT FROM AUTHOR]

Corn fiber is a coproduct produced during the corn wet-milling process and is similar to other high hemicellulose/cellulose-containing biomass such as grasses, straws, or bagasse, all of which represent potential fermentation feedstock for conversion into biofuels or other products. Corn fiber was subjected to ammonia-explosion (AFEX) treatment to increase degradability and then enzymatically digested with a combined mixture of commercial amylase, xylanase, and cellulase enzyme preparations. Whereas the starch and cellulose components were converted solely to glucose, oligosaccharides represented 30–40% of the xylan degradation products. This enzyme mixture also produced substantial oligosaccharides with xylans purified from corn fiber, corn germ, beechwood, oatspelt, or wheat germ. Commercial xylan-degrading enzyme preparations containing xylanase, xylosidase, and arabinosidase activities were then used alone or in varying combinations to attempt to maximize degradation of these isolated xylans of differing chemical compositions. The results showed that oatspelt and beechwood xylans were degraded most extensively (40–60%) with substantial amounts of xylose, xylobiose, and xylotriose as products depending on the enzyme combination used. Corn fiber and wheat germ xylans, which contain large amounts of arabinose and uronic acid sidechains, were poorly degraded and only small amounts of arabinose and xylose and large amounts of pentamer or longer oligosaccharides were produced by enzymatic degradation. The data suggest that whereas enzymatic digestion of biomass hemicellulose does not produce toxic products, the process is not effective in producing a suitable fermentable substrate stream because of the low levels of monosaccharides and high levels of oligosaccharides produced. [ABSTRACT FROM AUTHOR]

The yeast Candida wickerhamii exports a cell-associated β-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active β-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-µ replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the α-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity. [ABSTRACT FROM AUTHOR]

A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced α-L-arabinofuranosidase (α-L-AFase) when grown in liquid culture on sugar beet arabinan, wheat arabinoxylan, L-arabinose, L-arabitol, xylose, xylitol, oat spelt xylan, corn fiber, or arabinogalactan. L-Arabinose was most effective for production of both whole-broth and extracellular α-L-AFase activity, followed by L-arabitol. Oat spelt xylan, sugar beet arabinan, xylose, xylitol, and wheat arabinoxylan were intermediate in their ability to support α-L-AFase production. Lower amounts of enzyme activity were detected in corn fiber- and arabinogalactan-grown cultures. [ABSTRACT FROM AUTHOR]

Aureobasidium pullulans produced an intracellular α-glucosidase. The enzyme was purified 124-fold by solubilization with Triton X-100, Q-Sepharose treatment, hydroxylapatite, octyl-Sepharose column chromatography, and gel filtration on Sephacryl S-200, and had a specific activity of 316.82 U/mg protein. The enzyme displayed an optimum pH for its action at 4.0 and was fully stable at pH 3.0-6.0 at 50°C. The α-glucosidase was completely stable up to 60°C and had an optimum activity at 60°C. The partially purified enzyme preparation hydrolyzed maltose, isomaltose, sucrose, and trehalose at relative rates of 100, 60, 47, and 50, respectively, and had little or no activity on polysaccharides. The K value for maltose hydrolysis at pH 4.0 and 50°C was 1.85 mm. The enzyme was not adsorbed onto raw corn starch and showed little raw starch degradation. The α-glucosidase did not require any metal ion for activity. This represents the first characterization of intracellular α-glucosidase from A. pullulans. [ABSTRACT FROM AUTHOR]

A color variant strain of Aureobasidium pullulans (NRRL Y-12974) produced amylase and α-glucosidase activities when grown at 28°C for 4 days in liquid culture on a wide variety of carbon sources such as starch, pullulan, glucose, maltose, cyclodextrins, sucrose, xylose, and xylan. An α-glucosidase was separated by Q-Sepharose adsorption from the cell-free culture broth and partially purified by hydroxylapatite and octyl-Sepharose chromatography. After ammonium sulfate treatment of the culture supernatant (obtained after Q-Sepharose adsorption), the amylase fraction was separated into three active fractions by hydroxylapatite column chromatography, which were identified as α-amylase, glucoamylase A, and glucoamylase B. The glucoamylase A was further purified by octyl-Sepharose column chromatography. The pH optima for the action of α-amylase, glucoamylase A, glucoamylase B, and α-glucosidase were 5.0, 4.5, 4.0-4.5, and 4.5, respectively. The α-amylase and glucoamylase B were fully stable at pH 3.0-6.0, glucoamylase A at pH 4.5-5.5, and α-glucosidase at pH 3.5-7.0 for 1 h at 50°C. The optimum temperatures for the action of these enzymes were 55°, 50°-60°, 65°, and 65°C, respectively. The α-amylase, glucoamylase A, and glucoamylase B were adsorbed onto raw corn starch and degraded it. Glucoamylase B readily cleaved pullulan. The α-glucosidase was not adsorbed onto raw starch and did not degrade it at all. It hydrolyzed both α-1,4 and α-1,6 linkages in oligosaccharides. All four enzymes did not require any metal ion for activity and were inhibited by cyclodextrins (α-and β-, 10 mm). [ABSTRACT FROM AUTHOR]

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