Alcohol fermentation of corn starch digested by Chalara paradoxa amylase without cooking

Alcohol fermentation of corn starch without cooking was performed by using Chalara paradoxa glucoamylase preparation, which had stronger raw starch digesting activity than those of the conventionally known glucoamylases. A raw corn starch-enzyme-yeast mixture was fermented optimally at pH 5.0 and 30 degrees C for five days and produced ethanol. The yields of ethanol were between 63.5 and 86.8% of the theoretical value by baker's yeast (Saccharomyces cerevisiae), and between 81.1 and 92.1% of the theoretical value by sake yeast (Saccharomyces sake).     

Improved method for preparing high maltose conversion syrups

An improved method is presented for producing high maltose conversion syrups from liquefied and raw starch. It comprises saccharifying the starch at higher temperatures than presently used with environmentally compatible thermostable beta-amylase and other thermostable enzymes.     

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and Alpha‐amylase to produce ethanol

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α‐amylase from Aspergillus oryzae as well as α‐amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:338–347, 2014     

Direct fermentation of potato starch to ethanol by cocultures of Aspergillus niger and Saccharomyces cerevisiae

Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomyces cerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in coculture versus the monoculture due to the synergistic metabolic interactions between the species. Optimal ethanol yields were obtained in the pH range 5 to 6 and amylolytic activity was obtained in the pH range 5 to 8. Ethanol yields were maximal when fermentations were conducted anaerobically. Increasing S. cerevisiae inoculum in the coculture from 4 to 12% gave a dramatic increase in the rate of ethanol production, and ethanol yields of greater than 96% of the theoretical maximum were obtained within 2 days of fermentation. These results indicate that simultaneous fermentation of starch to ethanol can be conducted efficiently by using cocultures of the amylolytic fungus A. niger and a nonamylolytic sugar fermenter, S. cerevisiae.     

Direct fermentation of potato starch to ethanol by cocultures of Aspergillus niger and Saccharomyces cerevisiae.

Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomyces cerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in coculture versus the monoculture due to the synergistic metabolic interactions between the species. Optimal ethanol yields were obtained in the pH range 5 to 6 and amylolytic activity was obtained in the pH range 5 to 8. Ethanol yields were maximal when fermentations were conducted anaerobically. Increasing S. cerevisiae inoculum in the coculture from 4 to 12% gave a dramatic increase in the rate of ethanol production, and ethanol yields of greater than 96% of the theoretical maximum were obtained within 2 days of fermentation. These results indicate that simultaneous fermentation of starch to ethanol can be conducted efficiently by using cocultures of the amylolytic fungus A. niger and a nonamylolytic sugar fermenter, S. cerevisiae.     

Continuous ethanol production from raw starch using a reversibly soluble-autoprecipitating amylase and flocculating yeast cells

Direct ethanol production from raw starch was performed continuously using a combination of a reversibly soluble-autoprecipitating amylase (D-AS) in which Dabiase K-27 was immobilized covalently on an enteric coating polymer (hydroxypropyl methylcellulose acetate succinate, AS) as a carrier, and a flocculating yeast. Continuous production was carried out using a reactor equipped with a mixing vessel and a separation vessel. D-AS and the yeast were separated continuously from the product solution by self-sedimentation in the separation vessel and they were utilized repeatedly. In the continuous saccharification of raw starch by D-AS alone, the glucose productivity was about 3.6 g/l/h at a dilution rate (D) of 0.1 h⁻¹. In the continuous ethanol production from raw starch by a combination of D-AS and flocculating yeast cells, high ethanol productivity up to 2.0 g/l/h was achieved at D=0.1 h⁻¹. Although the enzymatic activity of D-AS is inactivated due to insolubilization of the enzyme by the accumulation of NaCl produced in controlling the pH in the reactor, it is possible to recover the D-AS enzymatic activity by removing the NaCl. This continuous fermentation system suggests a potential for effective ethanol production from raw starch, and it may be widely applicable in heterogeneous culture systems using solid substrates other than raw starch.     

High-concentration alcoholic production from hydrolysate of raw ground corn by a tetraploid yeast strain

Summary The suitable conditions for high-concentration ethanol production from raw ground corn by a tetraploid yeast strain were examined. We found that the glucoamylase preparation which ia usually employed for alcoholic fermentation of cooked starch could effectively saccharify raw ground corn starch.     

Ethanol production from raw starch by simultaneous fermentation using Schizosaccharomyces pombe and a raw starch saccharifying enzyme from Corticium rolfsii

Alcoholic fermentation from raw corn starch using Schizosaccharomyces pombe AHU 3179 and a raw starch saccharifying enzyme (RSSE) from Corticium rolfsii AHU 9627 was investigated. The optimum ethanol production was achieved at pH 3.5, 27°C and under the yeast cell concentration of 2.7 × 10⁹ cells/ml. Addition of RSSE 5 units (as glucoamylase)/g raw corn starch was found sufficient. Under these optimum conditions, 18.5% (v/v, at 15°C) ethanol was obtained from 30% raw corn starch (30.8% as glucose) after incubation for 48 h.     

Direct fermentation of starch to lactic acid byLactobacillus amylovorus

Summary Fermentation production of lactic acid directly from starch was studied in a batch fermentor usingLactobacillus amylovorus. At an initial concentration of 120 g/L starch, 96.2 g/L of lactic acid was produced from liquefied starch in 20 hours while 92.5 g/L of lactate was produced from the raw starch in 39 hours. High initial glucose levels (100 g/L) in the medium inhibited the organism, unless it had been adapted by growing it in a low-glucose medium. The direct production of lactic acid from starch could reduce overall production costs significantly.     

Soluble and immobilized enzyme technology in bioconversion of barley starch

Homogeneous and heterogeneous biocatalysis were both investigated as tools for barley starch syrup production. Barley starch was first liquefied by soluble heat-stable Bacillus sp. α-amylase EC 3.2.1.1 (1,4-α-d-glucan glucanohydrolase) Termamyl 60 L at 95°C, pH 6.5, to obtain slurries of varying DE-values up to ≈37. Alternatively, it was extruded with a Creusot-Loire BC 45 twin-screw extruder at 25% moisture, 150°C, for denaturation. After cooling and adjusting the pH to 4.5 or grinding, respectively, the pretreated starch was saccharified either by soluble or by immobilized Aspergillus niger glucoamylase EC 3.2.1.3 (1,4-α-d-glucan glucohydrolase) at 60°C, pH 4.5, to obtain glucose syrup of up to DE 96. The course of hydrolysis was followed by automated Biogel P-2 chromatographic analysis. Glucoamylase was immobilized either on a phenol-formaldehyde resin Duolite S 761 or on silanized Spherosil porous silica beads. Barley glucose syrup obtained was further continuously converted to high fructose syrup by a packed bed reactor of Actinoplanes missouriensis whole cell glucose isomerase (EC 5.3.1.5) Maxazyme entrapped within α-cellulose beads. We could conclude that barley starch may be used as an alternative raw material for biocatalytic starch syrup production.     

Conversion of starch into ethanol by Clostridium thermohydrosulfuricum

Summary Production of ethanol from starch by Clostridium thermohydrosulfuricum was compared with that from glucose, fructose or maltose in batch fermentations. Optimal substrate concentration and pH for ethanol production were determined. The rate of ethanol production on starch was about the same as that on glucose or fructose and overall yields were also similar (about 1.6 mol ethanol per mol glucose or glucose equivalent). Maltose was not an effective substrate for growth and ethanol production.When a mixture of starch and glucose in equal amounts was used, breakdown of starch and utilization of glucose were simultaneous. When starch and fructose were supplied together, the fructose was utilized but no hydrolysis of starch was observed. With a mixture of glucose and fructose, uptake of fructose preceeded that of glucose.     

Physicochemical changes to starch granules during micronisation and extrusion processing of wheat, and their implications for starch digestibility in the newly weaned piglet

Two trials were performed to assess changes in the physicochemical properties of precisely processed (micronised v. extruded) wheats, prior to inclusion in piglet diets. The in vitro data obtained were subsequently related to biological responses of newly weaned piglets over 14 days. The effects of the severity of micronisation (Trial 1) or extrusion (Trial 2) on the nutritional value of two wheats (varying in endosperm texture) were examined. Extrusion, in contrast to micronisation, drastically disrupted the structural properties of wheat starch granules through melting of crystallites and macromolecular degradation of starch polysaccharides. These structural changes strongly improved the hydration characteristics of starch and its digestibility. The amount of starch digested in vitro was approximately 0.20, 0.70 and 0.90 for the unprocessed, micronised and extruded samples, respectively. This enhanced in vitro digestibility correlated well with, and helped to explain, the significant improvement in the apparent digestibility of starch at both the 0.5 region (mean coefficients for extruded wheat were 0.869 and 0.959 v. raw 0.392; P = 0.017) and 0.75 region (extruded 0.973 v. raw 0.809; P = 0.009) of the small intestine, when compared with piglets fed an unprocessed wheat diet. Extrusion and, to a lesser extent, micronisation lessened the reduction in apparent starch digestibility on day 4 post-weaning, typically seen at the 0.5 intestinal region in piglets fed an unprocessed wheat diet. Processing variables influenced both in vitro and in vivo data, with for instance, a positive relationship between specific mechanical energy (SME) input during extrusion and starch digestibility at the 0.5 region. The higher digestibility coefficient observed at the 0.5 region for the high SME diet suggests enhanced digestion and more rapid release of starch. However, it remains to be seen whether a diet containing rapidly digestible, as opposed to slowly digestible, starch is more beneficial for piglets. This rate of starch breakdown in the piglet is an important finding, which may have implications in helping to alleviate the post-weaning growth check, particularly in the absence of in-feed antibiotic growth promoters. Processing did not appear to offer any benefit over unprocessed wheat with regard to daily live-weight gain or the apparent digestibility of nitrogen in the small intestine over the 14-day period. Based on the enhanced in vivo starch digestibility, performance might be improved over a longer period, although future studies are required to confirm this. Precise processing variables for raw materials must be stated in all animal trials.     

Selected morphological and functional properties of extruded acetylated starch-cellulose foams

Starch acetates with degrees of substitution (DS) of 1.68 and 2.3 were extruded with 10%, 20% and 30% (w/w) cellulose and 20% (w/w) ethanol in a twin screw extruder at 150, 160 and 170 degrees C barrel temperatures and 170, 200 and 230 rpm screw speeds. X-ray diffractogram (XRD), differential scanning calormetry (DSC) and Fourier transform infrared spectroscopy (FTIR) were used to analyze the morphological properties of extruded foams. A central composite response surface design was applied to analyze the effects of starch type, cellulose content, barrel temperature and screw speed on specific mechanical energy requirement of extruding foams and the radial expansion ratio and compressibility of the extruded foams. XRD showed losses of DS starch and cellulose crystallinity and the formation of new complexes. FTIR spectra revealed that functional groups and chemical bonds were maintained after extrusion. Melting temperatures changed significantly when higher DS starch acetate was used. Cellulose content, barrel temperature and screw speed showed significant effects on thermal, physical and mechanical properties of extruded foams and the specific mechanical energy requirement.     

Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase

Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.     

Production of ethanol by raw starch hydrolysis and fermentation of damaged grains of wheat and sorghum

The simultaneous saccharification and fermentation was used to produce ethanol from raw starch of damaged quality wheat and sorghum grains by utilising crude amylase preparation from B. subtilis VB2 and an amylolytic yeast strain S. cerevisiae VSJ4. Various concentrations of damaged wheat and sorghum starch from 10% to 30%W/V were used and 25% was found to be optimum for damaged wheat and sorghum starch yielding 4.40%V/V and 3.50%V/V ethanol respectively. Whereas 25% raw starch of fine quality wheat and sorghum grains gave an yield of 5.60%V/V and 5.00%V/V respectively. The process was carried out at 35v°C, 5.8 pH and 200 rpm for 4 days.     

Extrusion of corn for ethanol fermentation

Summary Extrusion and conventional cooking of corn for ethanol production were compared. Extrusion processing requires less energy and water than conventional cooking methods. Optimal autogenous extrusion conditions were determined as: feed rate=5.2kg/min, moisture=15% (wet basis) and extrusion discharge temperature=1600 C. Improved yields of glucose and ethanol from the extruded samples were found over conventionally cooked samples.     

Repeated fermentation from raw starch using Saccharomyces cerevisiae displaying both glucoamylase and α-amylase

A diploid yeast strain displaying both α-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both glucoamylase and α-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100g/l of raw starch for 23 cycles without the loss of α-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.     

Direct and efficient ethanol production from high-yielding rice using a Saccharomyces cerevisiae strain that express amylases

Efficient ethanol producing yeast Saccharomyces cerevisiae cannot produce ethanol from raw starch directly. Thus the conventional ethanol production required expensive and complex process. In this study, we developed a direct and efficient ethanol production process from high-yielding rice harvested in Japan by using amylase expressing yeast without any pretreatment or addition of enzymes or nutrients. Ethanol productivity from high-yielding brown rice (1.1g/L/h) was about 5-fold higher than that obtained from purified raw corn starch (0.2g/L/h) when nutrients were added. Using an inoculum volume equivalent to 10% of the fermentation volume without any nutrient supplementation resulted in ethanol productivity and yield reaching 1.2g/L/h and 101%, respectively, in a 24-h period. High-yielding rice was demonstrated to be a suitable feedstock for bioethanol production. In addition, our polyploid amylase-expressing yeast was sufficiently robust to produce ethanol efficiently from real biomass. This is first report of direct ethanol production on real biomass using an amylase-expressing yeast strain without any pretreatment or commercial enzyme addition.     

Effect of flocculation on performance of arming yeast in direct ethanol fermentation

In the direct ethanol fermentation of raw starch by arming yeast with α-amylase and glucoamylase, it is preferable to use a flocculent yeast because it can be recovered without centrifugation. Three types of arming yeast system, I (nonflocculent), II (mildly flocculent), and III (heavily flocculent), were constructed and their fermentation performances were compared. With an increase in the degree of flocculation, specific ethanol production rate for soluble starch decreased (0.19, 0.17, and 0.12 g g-dry-cell⁻¹ h⁻¹ for systems I, II, and III, respectively), but that for raw starch did not decrease as much as expected (0.06, 0.06, and 0.04 g g-dry-cell⁻¹ h⁻¹ for systems I, II and III, respectively). Microscopic observation revealed that many starch granules were captured in the yeast flocs in system III during the direct ethanol fermentation of raw starch. It was suggested that the capture of starch granules increases apparent substrate concentration for amylolytic enzymes in arming yeast cell flocs; thus, the specific ethanol production rate of system III was kept at a level comparable to those of the other systems.     

Efficient and direct fermentation of starch to ethanol by sake yeast strains displaying fungal glucoamylases

Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.