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     

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.     

Selection and characterization of a newly isolated thermotolerant Pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate

Pichia kudriavzevii DMKU 3-ET15 was isolated from traditional fermented pork sausage by an enrichment technique in a yeast extract peptone dextrose (YPD) broth, supplemented with 4 % (v/v) ethanol at 40 °C and selected based on its ethanol fermentation ability at 40 °C in YPD broth composed of 16 % glucose, and in a cassava starch hydrolysate medium composed of cassava starch hydrolysate adjusted to 16 % glucose. The strain produced ethanol from cassava starch hydrolysate at a high temperature up to 45 °C, but the optimal temperature for ethanol production was at 40 °C. Ethanol production by this strain using shaking flask cultivation was the highest in a medium containing cassava starch hydrolysate adjusted to 18 % glucose, 0.05 % (NH₄)₂SO₄, 0.09 % yeast extract, 0.05 % KH₂PO₄, and 0.05 % MgSO₄·7H₂O, with a pH of 5.0 at 40 °C. The highest ethanol concentration reached 7.86 % (w/v) after 24 h, with productivity of 3.28 g/l/h and yield of 85.4 % of the theoretical yield. At 42 °C, ethanol production by this strain became slightly lower, while at 45 °C only 3.82 % (w/v) of ethanol, 1.27 g/l/h productivity and 41.5 % of the theoretical yield were attained. In a study on ethanol production in a 2.5-l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.1 vvm throughout the fermentation, P. kudriavzevii DMKU 3-ET15 yielded a final ethanol concentration of 7.35 % (w/v) after 33 h, a productivity of 2.23 g/l/h and a yield of 79.9 % of the theoretical yield.     

Simultaneous saccharification and ethanol fermentation of sago starch using immobilized Zymomonas mobilis

Simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and immobilized Zymomonas mobilis ZM4 on sodium alginate was studied. The immobilized Zymomonas cells were more thermo-stable than free Zymomonas cells in this system. The optimum temperature in the SSF system was 40°C, and 0.5% (v/w) AMG concentration was adopted for the economical operation of the system. The final ethanol concentration obtained was 68.3 g/l and the ethanol yield, Y_p/s, was 0.49 g/g (96% of the theoretical yield). After 6 cycles of reuse at 40°C with 15% sago starch hydrolysate, the immobilized Z. mobilis retained about 50% of its ethanol fermenting ability.     

Production of a novel raw-starch-digesting glucoamylase by <Emphasis Type="Italic">Penicillium</Emphasis> sp. X-1 under solid state fermentation and its use in direct hydrolysis of raw starch

Penicillium sp. X−1, isolated from decayed raw corn, produced high level of raw-starch-digesting glucoamylase (RSDG) under solid state fermentation (SSF). Maximum enzyme yield of 306.2 U g⁻¹ dry mouldy bran (DMB) was obtained after 36 h of culture upon optimized production. The enzyme could hydrolyse both small and large granule starches but did not adsorb on raw starch. The enzyme exhibited maximum activity at 65°C and pH 6.5, which provided an opportunity of synergism with α-amylase. It significantly hydrolysed 15% (w/v) raw corn starch slurry in synergism with the commercial α-amylase and a degree of hydrolysis of 92.4% was obtained after 2 h of incubation.     

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.     

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8?g:10?g:2?g yielded the highest enzyme production of 201.6?U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5?×?10⁶ spores/mL inoculum, which gave the highest enzyme activity of 389.5?U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2?g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300?g raw cassava chips/L with cane molasses.     

Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence

Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.     

Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus

A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K _m and k _cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from 150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and 0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation. Received: 25 September 1998 / Received revision: 15 December 1998 / Accepted: 21 December 1998     

Characterization of an organic solvent‐tolerant thermostable glucoamylase from a halophilic isolate,Halolactibacillus sp. SK71 and its application in raw starch hydrolysis for bioethanol production

A halophilic bacterium Halolactibacillus sp. SK71 producing extracellular glucoamylase was isolated from saline soil of Yuncheng Salt Lake, China. Enzyme production was strongly influenced by the salinity of growth medium with maximum in the presence of 5% NaCl. The glucoamylase was purified to homogeneity with a molecular mass of 78.5 kDa. It showed broad substrate specificity and raw starch hydrolyzing activity. Analysis of hydrolysis products from soluble starch by thin‐layer chromatography revealed that glucose was the sole end‐product, indicating the enzyme was a true glucoamylase. Optimal enzyme activity was found to be at 70°C, pH 8.0, and 7.5% NaCl. In addition, it was highly active and stable over broad ranges of temperature (0–100°C), pH (7.0–12.0), and NaCl concentration (0–20%), showing excellent thermostable, alkali stable, and halotolerant properties. Furthermore, it displayed high stability in the presence of hydrophobic organic solvents. The purified glucoamylase was applied for raw corn starch hydrolysis and subsequent bioethanol production using Saccharomyces cerevisiae. The yield in terms of grams of ethanol produced per gram of sugar consumed was 0.365 g/g, with 71.6% of theoretical yield from raw corn starch. This study demonstrated the feasibility of using enzymes from halophiles for further application in bioenergy production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1262–1268, 2014     

Production of an amylase-degrading raw starch by Gibberella pulicaris

An endophytic fungus, Gibberella pulicaris, produced an amylase which degraded raw starches from cereals and other crops including raw potato, sago, tapioca, corn, wheat and rice starch. In each case, glucose was the main product. Among the raw starches used, raw potato starch gave the highest enzyme activity (85 units mg^–1 protein) and raw wheat starch the lowest (49 units mg^–1 protein). The highest amylase production (260 units mg^–1 protein) was achieved when the concentration of raw potato starch was increased to 60 g l^–1. Optimum hydrolysis was at 40°C and pH 5.5.     

Domain C of thermostable α-amylase of Geobacillus thermoleovorans mediates raw starch adsorption

The gene (1,542 bp) encoding thermostable Ca²⁺-independent and raw starch hydrolyzing α-amylase of the extremely thermophilic bacterium Geobacillus thermoleovorans encodes for a protein of 50 kDa (Gt-amyII) with 488 amino acids. The enzyme is optimally active at pH 7.0 and 60 °C with a t _1/2 of 19.4 h at 60 and 4 h at 70 °C. Gt-amyII hydrolyses corn and tapioca raw starches efficiently and therefore finds application in starch saccharification at industrial sub-gelatinisation temperatures. The starch hydrolysis is facilitated following adsorption of the enzyme to starch at the C-terminal domain, as confirmed by the truncation analysis. The adsorption rate constant of Gt-amyII to raw corn starch is 37.6-fold greater than that for the C-terminus truncated enzyme (Gt-amyII-T). Langmuir–Hinshelwood kinetic analysis in terms of equilibrium parameter (K _R) suggested that the adsorption of Gt-amyII to corn starch is more favourable than that of Gt-amyII-T. Thermodynamics of temperature inactivation indicated a decrease in thermostabilisation of Gt-amyII upon truncation of its C-terminus. The addition of raw corn starch increased t _1/2 of Gt-amyII, but it has no such effect on Gt-amyII-T. It can, therefore, be stated that Gt-amyII binds to raw corn starch via C-terminal region that contributes to its thermostability. Phylogenetic analysis confirmed that starch binding region of Gt-amyII is, in fact, the non-catalytic domain C, and not the typical SBD of CBM families. The role of domain C in raw starch binding throws light on the evolutionary path of the known SBDs.     

Cloning and overexpression of raw starch digesting α-amylase gene from Bacillus subtilis strain AS01a in Escherichia coli and application of the purified recombinant α-amylase (AmyBS-I) in raw starch digestion and baking industry

Considering the economic and industrial relevance of α-amylases used in food and starch industries, a raw starch digesting α-amylase gene (amyBS-I) from Bacillus subtilis strain AS01a was cloned and expressed in Escherichia coli BL21 cells. The gene also includes its signal peptide sequence (SPS) for facilitating the efficient extracellular expression of recombinant α-amylase (AmyBS-I) in correctly folded (enzymatically active) form. The native AmyBS-I consists of 659 amino acids with a molecular mass and pI of 72,387 Da and 5.8, respectively. The extracellular secretion of AmyBS-I after response surface optimization of culture conditions was found to be 7-fold higher as compared to its production under non-optimized conditions. Purified AmyBS-I demonstrated optimum activity at 70 °C and pH 6.0. It shows K_m and V_max values toward soluble starch as 2.7 mg/ml and 454 U/ml, respectively. Further, it does not require Ca²⁺ ion for its α-amylase activity/thermo-stability, which is an added advantage for its use in the starch industry. The AmyBS-I also hydrolyzed a wide variety of raw starches and produced maltose and glucose as main hydrolyzed products. The bread dough supplemented with AmyBS-I showed better amelioration of the bread quality as compared to the bread supplemented with commercial α-amylase.     

Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalose purification

In this study, corn starch was used as the substrate for cell growth and trehalose accumulation by Saccharomycopsis fibuligera A11. Effect of different aeration rates, agitation speeds, and concentrations of corn starch on direct conversion of corn starch to trehalose by S. fibuligera A11 were examined using a Biostat B2 2-l fermentor. We found that the optimal conditions for direct conversion of corn starch to trehalose by this yeast strain were that agitation speed was 200 rpm, aeration rate was 4.0 l/min, concentration of corn starch was 2.0% (w/v), initial pH was 5.5, fermentation temperature was 30°C. Under these conditions, over 22.9 g of trehalose per 100 g of cell dry weight was accumulated in the yeast cells, cell mass was 15.2 g/l of the fermentation medium, 0.12% (w/v) of reducing sugar, and 0.21% (w/v) of total sugar were left in the fermented medium within 48 h of the fermentation. It was found that trehalose in the yeast cells could be efficiently extracted by the hot distilled water (80°C). After isolation and purification, the crystal trehalose was obtained from the extract of the cells.     

Effect of C/N ratio and starch concentration on ethanol production from sago starch using recombinant yeast

Recombinant Saccharomyces cerevisiae YKU 131 (capable of expressing glucoamylase) was used to produce ethanol from sago starch. The optimum C/N ratio for ethanol production by the recombinant yeast was 7.9, where 4.7 and 10.1 g/l ethanol was produced from 20 and 40 g/l sago starch, respectively. At sago starch concentration higher than 40 g/l and C/N ratio higher than 10.4, glucoamylase production and rate of starch hydrolysis were reduced, which in turn, reduced ethanol production significantly. The theoretical yield of ethanol based on sago starch consumed in fermentation using 40 g/l was 72.6%. This yield was slightly lower than those obtained in fermentation using soluble starch such as potato and corn starch, which ranged from 80–90% as reported in the literature. However, S. cerevisiae YKU 131 could only utilize 62% of the total amount of starch added to a medium.     

Behaviour of Endomycopsis fibuligera glucoamylase towards raw starch

An extracellular glucoamylase [exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] of Endomycopsis fibuligera has been purified and some of its properties studied. It had a very high debranching activity (0.63). The enzyme was completely adsorbed onto raw starch at all the pH values tested (pH 2.0–7.6). Amylase inhibitor from Streptomyces sp. did not prevent the adsorption of glucoamylase onto raw starch although the enzyme did not digest raw starch in the presence of amylase inhibitor. Sodium borate (0.1 m) eluted only 35% of the adsorbed enzyme from raw starch. The optimum pH for raw starch digestion was 4.5 whereas that of boiled soluble starch hydrolysis was 5.5. Waxy starches were more easily digested than non-waxy starches, and root starches were slowly digested by this enzyme.     

Polyhydroxybutyrate production by Saccharophagus degradans using raw starch as carbon source

Biosynthesis of poly(3‐hydroxybutyrate) (PHB) from raw starch as the carbon source by the polysaccharide‐digesting bacteria Saccharophagus degradans was investigated in a fed‐batch culture. The production and properties of the PHB synthesized from starch were compared to those obtained using glucose as carbon source. In fed‐batch cultures, S. degradans accumulated 21.35 and 17.46% of PHB, using glucose or starch as carbon source, respectively. The physical properties of the biopolymer produced from each carbon source were similar between them. Molecular mass, melting temperature and heat of fusion were 54.23 kDa, 165.61°C and 59.59 J/g, respectively, using glucose; and 57.07 kDa, 174.31°C and 67.66 J/g, respectively, using starch. This is the first work describing the capability of S. degradans to utilize raw starch as the sole carbon source for the production of PHB.     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

Purification,biochemical characterization and gene sequencing of a thermostable raw starch digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov.

This study reports the purification and biochemical characterization of a raw starch-digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov. (strain Pizzo^T). The molecular weight was estimated to be 58 kDa by SDS–PAGE. The enzyme was highly active over a wide range of pH from 4.0–10.0. The optimum temperature of the enzyme was 70°C. It showed extreme thermostability in the presence of Ca²⁺, retaining 50% of its initial activity after 90 h at 70°C. The enzyme efficiently hydrolyzed 20% (w/v) of raw starches, concentration normally used in starch industries. The α-amylase showed an high stability in presence of many organic solvents. In particular the residual activity was of 73% in presence of 15% (v/v) ethyl alcohol, which corresponds to ethanol yield in yeast fermentation process. By analyzing its complete amyA gene sequence (1,542 bp), the enzyme was proposed to be a new α-amylase.     

Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

A gene encoding the thermostable raw starch digesting α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% of the original activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 48-h treatment, the DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules displayed deep cavities.