Enhanced conversion of starch to cyclodextrins in ethanolic solutions by bacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase

Improved formation of cyclodextrins (CDs) from starch in ethanolic solutions byBacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase was studied. The β- and γ-CD yields increased and α-CD yield gradually decreased as the ethanol concentration was raised. The ethanol concentration required for maximal CD yield depended essentially on starch concentration. The ethanol's effect was pronounced at high starch concentrations. For example, with 30% (w/v) starch, the CD yield was 2.4-fold (146.5 g/L) in the presence of 15% (v/v) ethanol. The effect of dimethylsulfoxide on the formation of CDs was similar to that of ethanol. The disintegration of β- and γ-CDs were narrowly interdependent on the formation of a α-CD and malto-sugars. The amount of reducing sugars decreased from a dextrose equivalent value of roughly 7.5 to 4.5 in the presence of ethanol at starch concentrations 1-30% (w/v). The effect of ethanol on starchy materials from various sources was similar. It was concluded that ethanol retards the decomposition of β-CD by a general mechanism involving a decreased activity of water.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.     

Consolidated Conversion of Hulled Barley into Fermentable Sugars Using Chemical,Thermal, and Enzymatic (CTE) Treatment

A novel process using chemical, thermal, and enzymatic treatment for conversion of hulled barley into fermentable sugars was developed. The purpose of this process is to convert both lignocellulosic polysaccharides and starch in hulled barley grains into fermentable sugars simultaneously without a need for grinding and hull separation. In this study, hulled barley grains were treated with 0.1 and 1.0 wt.-% sulfuric acid at various temperatures ranging from 110 to 170 °C in a 63-ml flow-through packed-bed stainless steel reactor. After sulfuric acid pretreatment, simultaneous conversion of lignocellulose and starch in the barley grains into fermentable sugars was performed using an enzyme cocktail, which included α-amylase, glucoamylase, cellulase, and β-glucosidase. Both starch and non-starch polysaccharides in the pre-treated barley grains were readily converted to fermentable sugars. The treated hulled barley grains, including their hull, were completely hydrolyzed to fermentable sugars with recovery of almost 100% of the available glucose and xylose. The pretreatment conditions of this chemical, thermal, and enzymatic (CTE) process for achieving maximum yield of fermentable sugars were 1.0 wt.% sulfuric acid and 110 °C. In addition to starch, the acid pretreatment also retained most of the available proteins in solid form, which is essential for subsequent production of fuel ethanol and high protein distiller’s dried grains with solubles co-product.     

Composition and ethanol production potential of cotton gin residues

Cotton gin residue (CGR) collected from five cotton gins was fractionated and characterized for summative composition. The major fractions of the CGR varied widely between cotton gins and consisted of clean lint (5–12%), hulls (16–48%), seeds (6–24%), motes (16–24%), and leaves (14–30%). The summative composition varied within and between cotton gins and consisted of ash (7.9–14.6%), acid-insoluble material (18–26%), xylan (4–15%), and cellulose (20–38%). Overlimed steam-exploded cotton gin waste was readily fermented to ethanol by Escherichia coli KO11. Ethanol yields were feedstock and severity dependent and ranged from 58 to 92.5% of the theoretical yields. The highest ethanol yield was 191 L (50 gal)/t, and the lowest was 120 L (32 gal)/t.     

Effects of Brewers’ condensed solubles (bcs) on the production of ethanol from low-grade starch materials

Yeast fermentation was performed on grain and bakery byproducts with and without adding the same volume of brewers’ condensed solubles (BCS). Starch material in the grain and bakery byproducts effectively was converted to fermentable sugars with conversion ratios of 93–97% by successive treatments of samples with bacterial αamylase and fungal glucoamylase. The yeast fermentation of these enzyme-digested byproducts alone showed that ethanol concentrations of 16.4–42.7 mL/100 g dry solid in the broth were achieved with fermentation efficiencies of 87–96%. Addition of BCS to the grain byproducts increased ethanol concentration by 10–86% by increasing the potential glucose content of the broth. The rates of fermentation measured by CO₂ gas production demonstrated that BCS addition to bakery byproducts reduced the fermentation time from 62–72 h to 34–35 h. In bakery byproducts that were low in amino nitrogen, exhaustion of nitrogenous compounds in substrates was found to be a limiting factor for yeast growth. Because BCS is a rich source of nitrogen, adding BCS to these substrates markedly increased the fermentation rate.     

Pretreatment and enzymatic saccharification of corn fiber

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. 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.     

Dilute acid pretreatment of softwoods

Selective thinning of forests in the western United States will generate a large, sustainable quantity of softwood residues that can be an attractive feedstock for fuel ethanol production. The major species available from thinning of forests in northern California and the eastern Rocky Mountains include white fir (Abies concolor), Douglas fir (Pseudotsuga menziesii), and Ponderosa pine (Pinus ponderosa). Douglas fir chips were soaked in 0.4% sulfuric acid solution, then pretreated with steam at 200 – 230°C for 1 – 5 min. After pretreatment, 90 – 95% of the hemicellulose and as much as 20% of the cellulose was solubilized in water, and 90% of the remaining cellulose can be hydrolyzed to glucose by cellulase enzyme. The prehydrolysates, at as high as 10% total solid concentration, can be readily fermented by the unadapted yeastSaccharomyces cerevisiae D₅A.     

Pretreatment of Whole-Crop Harvested, Ensiled Maize for Ethanol Production

To have all-year-round available feedstock, whole-crop maize is harvested premature, when it still contains enough moisture for the anaerobic ensiling process. Silage preparation is a well-known procedure for preserving plant material. At first, this method was applied to obtain high-quality animal feed. However, it was found that such ensiled crops are very suitable for bioenergy production. Maize silage, which consists of hardly degradable lignocellulosic material, hemicellulosic material, and starch, was evaluated for its potential as a feedstock in the production of bioethanol. It was pretreated at low severity (185 °C, 15 min) giving very high glucan (∼100%) and hemicellulose recoveries (<80%)—as well as very high ethanol yield in simultaneous saccharification and fermentation experiments (98% of the theoretical production based on available glucan in the medium). The theoretical ethanol production of maize silage pretreated at 185 °C for 15 min without oxygen or catalyst was 392 kg ethanol per ton of dry maize silage.     

Feasibility of Hydrothermal Pretreatment on Maize Silage for Bioethanol Production

The potential of maize silage as a feedstock to produce bioethanol was evaluated in the present study. The hydrothermal pretreatment with five different pretreatment severity factors (PSF) was employed to pretreat the maize silage and compared in terms of sugar recovery, toxic test, and ethanol production by prehydrolysis and simultaneous saccharification and fermentation. After pretreatment, most of the cellulose remained in the residue, ranging between 85.87% by the highest PSF (185°C, 15 min) and 92.90% obtained at the lowest PSF (185°C, 3 min). A larger part of starch, varying from 71.64% by the highest PSF to 78.28% by the lowest, was liberated into liquor part, leaving 8.05–11.74% in the residues. Xylan recovery in the residues increased from 44.25% at the highest PSF to 82.95% at the lowest. The recovery of xylan in liquor changed from 20.13% to 50.33%. Toxic test indicated that all the liquors from the five conditions were not toxic to the Baker’s yeast. Pretreatment under 195°C for 7 min had the similar PSF with that of 185°C for 15 min, and both gave the higher ethanol concentration of 19.92 and 19.98 g/L, respectively. The ethanol concentration from untreated maize silage was only 7.67 g/L.     

Two high-throughput techniques for determining wood properties as part of a molecular genetics analysis of hybrid poplar and loblolly pine

Two new high-through put techniques, computer tomography X-ray densitometry (CT scan) and pyrolysis molecular beam mass spectrometry (pyMBMS), coupled with quantitative trait loci (QTL) analysis, were tested as a means to overcome the time and cost associated with conventional characterization of biomass feedstock components. Applications of these two techniques were evaluated using hybrid poplar for the CT scan and loblolly pine for the pyMBMS. Segregating progeny from hybrid poplar varied in specific gravity, with individual mean estimates ranging from 0.21–0.41. Progeny from loblolly pine varied in lignin, α cellulose, and mannan contents, with individual mean estimates of lignin content ranging from 28.7–33.1%, α cellulose content from 28.8–43.5% and mannan content from 4.2–10.1%. QTL analysis of the loblolly pine data suggested that eleven QTLs were associated with individual feedstock characteristics and that two QTLs for several feedstock components were linked to the same position on the loblolly pine genetic map. Each QTL individually accounted for 7–13% of the total phenotypic variation in associated loblolly pine feedstock components.     

Role of CH, CH3, and OH Radicals in Organic Compound Decomposition by Water Plasmas

Decomposition of acetone, methanol, ethanol, and glycerine by water plasmas at atmospheric pressure has been investigated using a direct current discharge. At torch powers of 910–1,050 W and organic compound concentrations of 1–10 mol%, the decomposition rate of methanol and glycerine was over 99%, while those of acetone and ethanol was 95.4–99%. The concentrations of H₂ obtained were 60–80% in the effluent gas for any compounds by pyrolysis. Based on the experimental results, the decomposition mechanism of organic compounds in water plasmas was proposed and the roles of intermediate species such as CH, CH₃, and OH have been investigated; CH radical generated from organic compounds decomposition was easily oxidized to form CO; incomplete oxidation of CH₃ leads to C₂H₂ generation as well as soot formation; and negligible amount of soot observed from glycerine decomposition even at high concentration indicated that oxidation of CH_×(×:1–3) was enhanced by OH radical.     

Production of ethanol from starch by co-immobilizedZymomonas mobilis-glucoamylase in a fluidized-bed reactor

The production of ethanol from starch was studied in a fluidized-bed reactor (FBR) using co-immobilizedZymomonas mobilis and glucoamylase. The FBR was a glass column of 2.54 cm in diameter and 120 cm in length. TheZ. mobilis and glucoamylase were co-immobilized within small uniform beads (1.2-2.5 mm diameter) of κ-carrageenan. The substrate for ethanol production was a soluble starch. Light steep water was used as the complex nutrient source. The experiments were performed at 35κC and pH range of 4.0-5.5. The substrate concentrations ranged from 40 to 185 g/L, and the feed rates from 10 to 37 mL/min. Under relaxed sterility conditions, the FBR was successfully operated for a period of 22 d, during which no contamination or structural failure of the biocatalyst beads was observed. Volumetric productivity as high as 38 g ethanol/(Lh), which was 74% of the maximum expected value, was obtained. Typical ethanol volumetric productivity was in the range of 15-20 g/(Lh). The average yield was 0.49 g ethanol/g substrate consumed, which was 90% of the theoretical yield. Very low levels of glucose were observed in the reactor, indicating that starch hydrolysis was the rate-limiting step.     

Sorption of aliphatic alcohols, acetates, and their mixtures by corn starch cryotextures depending on concentrations of sorbates and starch in initial sols

The sorption of a mixture ofn-hexanol,n-octanol, andn-hexyl andn-octyl acetates from aqueous solutions by corn starch cryotextures was studied using capillary gas chromatography at different initial concentrations of the sorbates (1–25 mmol L⁻¹) and corn starch (2–6%). The amounts of compounds sorbed by cryotextures are proportional to the increase in their concentration in the initial sol and the length of the alkyl substituent. Linear equations describing the concentration dependence were proposed. The sorption ofn-hexanol from a mixture of substances containingn-octanol increases as compared to that from the individual alcohol. It was shown that the degree of sorption of aroma by cryotextures was independent of the content of starch in the initial sol. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1496–1501, August, 1999.     

Dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor

Corn stover is a domestic feedstock that has potential to produce significant quantities of fuel ethanol and other bioenergy and biobased products. However, comprehensive yield and carbon mass balance information and validated kinetic models for dilute-sulfuric acid (H₂SO₄) pretreatment of corn stover have not been available. This has hindered the estimation of process economics and also limited the ability to perform technoeconomic modeling to guide research. To better characterize pretreatment and assess its kinetics, we pretreated corn stover in a continuous 1 t/d reactor. Corn stover was pretreated at 20% (w/w) solids concentration over a range of conditions encompassing residence times of 3–12 min, temperatures of 165–195°C, and H₂SO₄ concentrations of 0.5–1.4% (w/w). Xylan conversion yield and carbon mass balance data were collected at each run condition. Performance results were used to estimate kinetic model parameters assuming biphasic hemicellulose hydrolysis and a hydrolysis mechanism incorporating formation of intermediate xylo-oligomers. In addition, some of the pretreated solids were tested in a simultaneous saccharification and fermentation (SSF) process to measure the reactivity of their cellulose component to enzymatic digestion by cellulase enzymes. Monomeric xylose yields of 69–71% and total xylose yields (monomers and oligomers) of 70–77% were achieved with performance level depending on pretreatment severity. Cellulose conversion yields in SSF of 80–87% were obtained for some of the most digestible pretreated solids.     

Atmospheric Pressure Liquefaction of Dried Distillers Grains (DDG) and Making Polyurethane Foams from Liquefied DDG

In this study, dried distillers grains (DDG) was liquefied in acidic conditions at atmospheric pressure, and polyurethane foams were subsequently prepared from the liquefied DDG. Liquefaction was examined over a range of conditions including liquefaction time of 1–3 h, temperature of 150–170 °C, sulfuric acid (as catalyst) concentration of 1.0–3.0 wt%, and liquefaction solvent (ethylene carbonate) to DDG ratio of 3:1–5:1. The bio-polyols in the liquefied DDG were rich in hydroxyl groups, which can react with methylene diphenyl diisocyanate (MDI) to form cross-linked polyurethane networks. The biodegradability of the prepared polyurethane foams was also evaluated. This study strives to broaden the application of DDG as a feedstock for bio-polyurethane preparation.     

β-Cyclodextrin production by simultaneous fermentation and cyclization

Production of β-cyclodextrin (CD) with high-dextrose equivalent (DE) starch hydrolysates by simultaneous fermentation and cyclization (SFC) gives higher yields than using only the enzyme CGTase, because fermentation eliminates glucose and maltose that inhibit CD production, while at the same time, produces ethanol that increases yield. A 10% (w/v) solution of cassava starch, liquefied with α-amylase, was incubated with CGTase using: only the enzyme, added ethanol (from 1 to 5%), and added yeast,S. cerevisiae (12% w/v), plus nutrients, the latter being the SFC process. Reaction conditions were: 38αC, pH 6.0, DE from 2 to 25, and 3.3 mL of CGTase/L. The yield of β-CD has decreased with an increase in DE, and maximum reaction yields were found for DE equal to 3.54, reaching 5.6, 14.7, and 11.5 mM β-CD, respectively. For an increase of DE, of approx 6 times (from 3.54 to 23.79), β-CD yield decreased 6 times for the first, and second reaction media with 3% (v/v) ethanol, and only approx 3 times for SFC (from 11.5 to 3.73 mM), showing that this process is less sensitive to variations in the DE     

Conversion of distiller's grain into fuel alcohol and a higher-value animal feed by dilute-acid pretreatment

Over the past three decades ethanol production in the United States has increased more than 10-fold, to approx 2.9 billion gal/yr (mid-2003), with ethanol production expected to reach 5 billion gal/yr by 2005. The simultaneous coproduction of 7 million t/yr of distiller's grain (DG) may potentially drive down the price of DG as a cattle feed supplement. The sale of residual DG for animal feed is an important part of corn dry-grind ethanol production economics; therefore, dry-grind ethanol producers are seeking ways to improve the quality of DG to increase market penetration and help stabilize prices. One possible improvement is to increase the protein content of DG by converting the residual starch and fiber into ethanol. We have developed methods for steam explosion, SO₂, and dilute-sulfuric acid pretreatment of DG for evaluation as a feedstock for ethanol production. The highest soluble sugar yields (∼77% of available carbohydrate) were obtained by pretreatment of DG at 140°C for 20 min with 3.27 wt% H₂SO₄. Fermentation protocols for pretreated DG were developed at the bench scale and scaled to a working volume of 809 L for production of hydrolyzed distiller's grain (HDG) for feeding trials. The pretreated DG was fermented with Saccharomyces cerevisiae D₅A, with ethanol yields of 73% of theoretical from available glucans. The HDG was air-dried and used for turkey-feeding trials. The inclusion of HDG into turkey poult (as a model non-ruminant animal) diets at 5 and 10% levels, replacing corn and soybean meal, showed weight gains in the birds similar to controls, whereas 15 and 20% inclusion levels showed slight decreases (−6%) in weight gain. At the conclusion of the trial, no negative effects on internal organs or morphology, and no mortality among the poults, was found. The high protein levels (58–61%) available in HDG show promising economics for incorporation of this process into corn dry-grind ethanol plants.     

Effects of chemical modification on the structure and mechanical properties of starch-based biofilms

Abstract  

In this work, chemically modified corn starch and plasticized corn starch biofilms were obtained and characterized in four steps: (1) preparation of corn starch microparticles, (2) preparation of malic acid-modified corn starch microparticles (MA–SM), (3) preparation of corn starch biofilms and MA–SM-plasticized corn starch biofilms, and (4) characterization of the biofilms. The effects of MA–SM concentration (4, 8, and 12% based on the amount of corn starch) on the structural characteristics and mechanical properties of the biofilms were investigated. Changes in the starch granules after chemical modification were studied by X-ray diffraction, FT-IR spectroscopy, and scanning electron microscopy. The presence of ester carbonyl group stretching vibration at 1,720 cm⁻¹ in FT-IR spectra was evidence of reaction of the starch microparticles with malic acid. The tensile yield strength and Young’s modulus of the films increased with increasing MA–SM content. Water uptake decreased from 69.8% for biofilm without MA–SM to 52.7% for biofilm with MA–SM. The improvement of these properties in the plasticized product could be attributed to the good interaction between the MA–SM filler and the corn starch.     

Production of fuel ethanol from rye and triticale by very-high-gravity (VHG) fermentation

Very-high-gravity (VHG) rye and triticale mashes, containing about 28.5 g dissolved solids/100 mL of mash supernatant, were prepared by adjusting water:grain ratios to 2:1. Because of high viscosity, which develops during mashing, it was necessary to pretreat ground rye-water slurries with viscosity-reducing enzymes. There were no viscosity problems during the preparation of triticale mashes. Fermentations were conducted at 20°C, with and without 16 mM urea as a nitrogenous supplement. All fermentations were completed within 120–144 h. Supplementation with urea shortened the times required for completion of fermentation by 33% for triticale and by 40% for rye. The fermentation efficiencies for both grains ranged between 90 and 93%. These values are comparable to those reported for wheat, implying competitiveness of rye and triticale as fermentation feedstocks to replace wheat. The final ethanol yields were 409 L for rye and 417–435 L for triticale/t (dry basis). For a given size of fermentation vessel, 33% more grain was used in the VHG fermentation process than in normal gravity fermentation. This resulted in a 35–56% increase in ethanol concentration in the beer, when fermentors were filled to a constant volume. The corresponding reduction in water use by about one-third would result in savings in energy consumption in mash heating, mash cooling, and ethanol distillation. Fermentation efficiencies and final ethanol yields obtained per unit weight of grain fermented were not significantly different from the normal gravity fermentations.     

Sodium meta bisulfite and ph tolerance ofPleurotus sajor caju under submerged cultivation

To develop a procedure for submerged cultivation of the edible fungusPleurotus sajor caju, we investigated the organism’s tolerance to sodium meta bisulfite and acidic pH levels. These factors were evaluated as means of controlling bacterial and/or fungal contamination. Trials were conducted in 500 mL Erlenmeyer flasks that contained 200–300 mL of a 3% glucose, 0.5% yeast extract medium, and either 0-0.225% SMB (wt/wt) added, or initially adjusted to pH levels between 1.8–6.5. Inoculated flasks were incubated 7–10 d at 30°C and 200 rpm in an environmental shaker, with samples removed daily to determine mycelial dry weights. Results showed that SMB levels up to 0.05% significantly lengthened the lag phase (from 27 to 79 h) but had no effect on productivity. Maximal productivity varied between 0.054–0.057 g/L/h, whereas overall productivity was 0.042–0.045 g/L/h. Biomass concentrations ranged from 7.1–8.4 g/L. Higher SMB levels rapidly reduced productivities and yields, eventually inactivating the inoculum above 0.1% SMB. In one instance the SMB tolerance of P.sajor caju was increased to 0.075% by repeatedly exposing the organism to sublethal levels of the chemical; however, this trait was not maintained in later trials. Bacterial contaminants were detected at SMB concentrations of 0.02–0.07%, while fungal contaminants were found up to 0.125% SMB. Thus it appears that SMB might be useful in controlling bacterial contamination, but may not be as effective against fungal contaminants. The optimum pH range in terms of lag phase length, biomass yield, and productivity was 4.5–5.5, however, in certain trialsP. sajor caju still exhibited good growth parameters at pH levels as low as 3.8–4.0. pH levels below 3.8 and above 5.8 greatly reduced both growth rates and yields. Acidic pH levels (3.8–4.5) were also effective in controlling the majority of bacterial and fungal contaminants encountered. Therefore low pH or perhaps a combination of low pH and SMB should be useful in developing a large scale system for submerged cultivation ofP. sajor caju.