Carbohydrate degrading enzyme production by plant pathogenic mycelia and microsclerotia isolates of Macrophomina phaseolina through koji fermentation

The ability of hydrolytic enzyme production by two different isolates of Macrophomina phaseolina was studied on apple pomace as a substrate for solid state fermentation (SSF). Initial moisture level, temperature and fermentation period was optimized so as to achieve higher output. Among the two different isolates, microsclerotial (MphP) and mycelial (MphM), MphP was observed as a potential source of different hydrolytic enzymes as compared to MphM. MphP gave higher enzyme activities (IU/gram dry substrate (gds): filter paper cellulase (FPase) activity 196.21 ± 16.3 (120 h), carboxymethyl cellulase (CMCase) 279.34 ± 28.25 (72 h), β-glucosidase (BGL) 129.82 ± 12.41 (96 h), xylanase 2527.88 ± 46.15 (120 h), and amylase 2780.72 ± 38.13 (96 h), respectively at 70% (v/w) IML. The incubation temperature was also found to have impact on the enzyme production ability of Macrophomina strains. The higher enzyme activities were achieved (IU/gds) as follows FPase 276.13 ± 25.02 (40 °C, 120 h), CMCase 278.11 ± 24.47 (35 °C, 144 h), BGL 189.47 ± 15.05 (30 °C, 144 h), xylanase 3845.77 ± 43.38 (35 °C, 144 h) and amylase activity of 3309.45 ± 29.22 (35 °C, 120 h), respectively using MphP at 70% (v/w) IML. This study reports for the first time the potential of carbohydrate degrading enzyme bioproduction by different isolates of M. phaseolina.     

Solid state fermentation of agricultural wastes for endoglucanase production

The lignocellulosic biomass (especially agricultural wastes) is known to be an excellent carbon source for microbial enzyme production. In this paper, the cellulase production from lignocellulosic materials under solid state fermentation (SSF) was investigated. The effects of fermentation conditions, such as moisture content, initial pH, temperature, and composition of mixed substrate (wheat straw and wheat bran) on endoglucanase production by Aspergillus niger 38 were studied. With a moisture content of 74% a pH range of 4.5–5.5 on mixed substrate containing wheat straw:wheat bran of 9:1, 14.80 international units (UI) endoglucanase activity/ml were obtained in 96 h.     

Optimization of ethanol production by Saccharomyces cerevisiae UFPEDA 1238 in simultaneous saccharification and fermentation of delignified sugarcane bagasse

Ethanol production by Saccharomyces cerevisiae UFPEDA1238 was performed in simultaneous saccharification and fermentation of delignified sugarcane bagasse. Temperature (32 °C, 37 °C), agitation (80; 100 rpm), enzymatic load (20 FPU/g cellulose and 10%, v/v β-glucosidase or 10 FPU/g cellulose and 5% β-glucosidase) and composition of culture medium were evaluated. Ethanol concentration, enzymatic convertibility of cellulose and volumetric productivity were higher than 25 g/L, 72% and 0.70 g/L h, respectively, after 30 h, when the culture medium 1 and 20 FPU/g cellulose/10%, v/v β-glucosidase or the culture medium 2 and 10 FPU/g cellulose/5% β-glucosidase were used in SSF at 37 °C and 80 rpm. In the SSF with culture medium 2 (supplemented with ammonium, phosphate, potassium and magnesium), 150 L ethanol/t bagasse was achieved, with minimum enzyme loading (10 FPU/g cellulose and 5%, v/v β-glucosidase) for 8%, w/v of solids, which is often an important requirement to provide cost-efficient second generation ethanol processes.     

Ethanol production from alkali- and ozone-treated cotton stalks using thermotolerant Pichia kudriavzevii HOP-1

In the present study, milled cotton stalks were subjected to alkali pretreatment with NaOH at 1-4% (w/v) concentrations at 121 °C for time ranging from 30 to 90 min. Ozone pretreatment was performed by passing 45 mg/L of ozone gas over 2 mm cotton stalks for 150 min at a flow rate of 0.37 L/min. The residual biomass from 4% alkali pretreatment for 60 min showed 46.6% lignin degradation accompanied by 83.2% increase in glucan content, compared with the untreated biomass. Hydrolysis of 4% alkali-treated and ozone-treated cotton stalks was conducted using enzyme combination of 20 filter paper cellulase units/gram dried substrate (FPU/g-ds), 45 IU/g-ds β-glucosidase and 15 IU/g-ds pectinase. Enzymatic hydrolysis of alkali-treated and ozone-treated biomass after 48 h resulted in 42.29 g/L glucose, 6.82 g/L xylose and 24.13 g/L glucose, 8.3 g/L xylose, respectively. About 99% of glucose was consumed in 24 h by Pichia kudriavzevii HOP-1 cells resulting in 19.82 g/L of ethanol from alkali-treated cotton stalks and 10.96 g/L of ethanol from ozone-treated cotton stalks. Simultaneous saccharification and fermentation of the alkali-treated cotton stalks after 12-h pre-hydrolysis resulted in ethanol concentration, ethanol yield on dry biomass basis and ethanol productivity of 19.48 g/L, 0.21 g/g and 0.41 g/L/h, respectively which holds promise for further scale-up studies. To the best of our knowledge, this is the first study employing SSF for ethanol production from cotton stalks.     

Utilization of pretreated bagasse for the sustainable bioproduction of cellulase by Aspergillus nidulans MTCC344 using response surface methodology

Response surface methodology (RSM) was used to optimize the conditions for the production of endo β-1,4 glucanase, a component of cellulase by Aspergillus nidulans MTCC344 under solid state fermentation, using bagasse as the chief substrate. A four-factor-five-level central composite design was employed for experimental design and analysis of the results. Maximum cellulase activity (CMCase was 28.96 U g⁻¹) can be attained at the optimum conditions, 16.8 mm bagasse bed height, 60% moisture content, pH 4.25 and temperature 40 °C in the solid state fermenter. These data were rather close to the experimental results obtained (CMCase was 28.84 U g⁻¹). A. nidulans MTCC344 was able to hydrolyze pretreated bagasse completely after 8 days of incubation with significant endo β-1,4 glucanase activities. The results of Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) of bagasse showed structural changes through pretreatment, in favor of enzymatic hydrolysis. Bagasse with alkali pretreatment using sodium hydroxide is a source of lignocelluloses able to improve the yield of endo β-1,4 glucanase by the strain of A. nidulans. The endo β-1,4 glucanase produced during the bioconversion of cellulose to glucose by A. nidulans MTCC344 is strongly dependent on the pretreatment given before hydrolysis.     

Modeling the effects of solid state fermentation operating conditions on endoglucanase production using an instrumented bioreactor

Cellulase production is one of the most critical steps in the economics of second generation ethanol. Although solid-state fermentation (SSF) is an attractive process for the production of enzymes, SSF is highly limited by the difficulty in controlling the operating variables which affect microbial growth and metabolites production. In this context, this work evaluates the effects of operational conditions on endoglucanase production by a selected strain of Aspergillus niger cultivated under SSF using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of air flow rate, inlet air relative humidity and substrate initial moisture on endoglucanase production were evaluated using a statistical design methodology. A correlation coefficient of 0.9106 and a calculated value of F, 5.46 folds higher than the listed value (P-value < 0.05) allowed the modeling of endoglucanase production under different process conditions. Higher endoglucanase production (56.1 U/g) was achieved for a selected condition of substrate initial moisture of 72%, air inlet humidity of 70%, and flow rate of 20 mL/min. A significant increase in endoglucanase production was also found to be achieved under forced aeration conditions (50.2 IU/g) compared to static conditions (29.8 IU/g) after 72 h of cultivation. Besides, respirometric analysis revealed that the total amount of CO₂ produced was linearly correlated with enzyme production (R² of 0.988). The bioreactor system used, as well as the methodology employed herein, was very effective in evaluating the influence of operational variables on enzymes production under SSF.     

Bioethanol production from sweet sorghum bagasse by Mucor hiemalis

The present work deals with production of ethanol from sweet sorghum bagasse by a zygomycetes fungus Mucor hiemalis. The bagasse was treated with phosphoric acid and sodium hydroxide, with or without ultrasonication, prior to enzymatic hydrolysis by commercial cellulase and β-glucosidase enzymes. The phosphoric acid pretreatment was performed at 50 °C for 30 min, while the alkali treatment performed with 12% NaOH at 0 °C for 3 h. The pretreatments resulted in improving the subsequent enzymatic hydrolysis to 79-92% of the theoretical yield. The best hydrolysis performance was obtained after pretreatment by NaOH assisted with ultrasonication. The fungus showed promising results in fermentation of the hydrolyzates. In the best case, the hydrolyzate of NaOH-ultrasound pretreated bagasse followed by 24 h fermentation resulted in about 81% of the corresponding theoretical ethanol yield. Furthermore, the highest volumetric ethanol productivity was observed in the hydrolyzates of NaOH pretreated bagasse, especially after ultrasonication in pretreatment stage.     

Evidence for the Presence of a Pentosanase Inhibitor in Wheat Flours

The solubilisation, by a pentosanase preparation fromAspergillus niger, of arabinoxylans from water-unextractable pentosans (WUP) isolated from wheat flour was much reduced when carried out in flour aqueous extracts as medium, instead of pure buffer. When flour extracts were previously heated at 100°C, the extent of arabinoxylan solubilisation was almost restored. The heating at 100°C and centrifugation of the flour extracts removed approximately one-third of the soluble protein but very low amounts of arabinoxylan. Increasing the concentration of extracts decreased the extent of WUP arabinoxylan solubilisation. There was slight variability between wheat cultivars Apollo, Soissons and Thésée in the extent of the inhibitory effect. Compounds responsible for this effect were mainly present in wheat grain endosperm but also in bran. Different microbial xylanases fromA. niger(Grindamyl S 100 and E1, an endoxylanase purified from this commercial preparation) andTrichodermastrains (C1, a partially purified cellulase/hemicellulase complex, and the commercial preparations Veron HE and Multifect XL) were strongly inhibited. Also the arabinofuranosidase activity present in Grindamyl S 100 was inhibited but to a lower extent than xylanases. Pronase treatment and protein addition in the extracts had no effect on the level of inhibition.     

Jatropha curcas seed cake as substrate for production of xylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentation

Jatropha curcas seed-cake was evaluated for use as a solid state fermentation substrate for production of cellulolytic and xylanolytic enzymes by Aspergillus niger. Supplementation of the seedcake with 10% thatch grass (Hyperrhaenia sp.) resulted in a fivefold increase in xylanase production. Ammonium chloride supplementation increased production of xylanase by 13%. Under the same conditions, cellulase production was not influenced by supplementation with grass or the nitrogen sources used. Maximum xylanase was produced at 25 °C whilst cellulase was maximally produced at 40 °C. Highest xylanase activity was obtained when the cultures had an initial pH of 3 whereas cellulase was maximally produced at an initial pH of 5. Under optimised conditions, 6087 U and 3974 U of xylanase and cellulase respectively were obtained per gram of substrate. Zymograms of crude enzyme extracts showed six active bands ranging from 20 kDa to 43 kDa for cellulase and a 31 kDa active band for xylanase.     

Dephytinization of wheat bran by fermentation with bakers' yeast,incubation with barley malt flour and autoclaving at different pH levels

Wheat bran is an important source of dietary fiber but also contains considerable amounts of phytic acid, which is known to impair mineral absorption. The present study was conducted to investigate the phytic acid reduction in coarse and fine wheat bran by fermentation with the different levels of bakers' yeast (3, 6 and 9%) for 8 h at 30 °C, incubation with the different levels of barley malt flour (2.5, 5.0, 7.5 and 10.0%) for 8 h at pH 5.2 and 55 °C, and autoclaving at the different pH levels (pH 5.0, 4.5, 4.0 and 3.5) adjusted with acetic acid for 2 h. The phytic acid content of the wheat bran was effectively reduced by all treatments, and the phytic acid lost was in the range of 88.4–96.9%. Without addition of yeast or malt flour, or autoclaving without pH adjustment, the phytic acid content of the bran samples was reduced at most to 44.9% of the initial amounts under the investigated conditions. Increasing the concentration of yeast or malt flour or decreasing the pH towards 3.5 did not enhance the phytic acid reduction. The most reduction occurred after 2 h of yeast fermentation and malt flour incubation, and after 30 min of autoclaving, which made up 92–98% of the total phytic acid loss. Extending the treatment periods contributed nominally to further increase in the phytic acid reduction, and the rate of the phytic acid loss decreased progressively.     

Enhancing three phenolic fractions of oats (Avena sativa L.) and their antioxidant activities by solid-state fermentation with Monascus anka and Bacillus subtilis

This work aims to improve phenolic fractions of oats and their antioxidant activities by solid-state fermentation (SSF) combining Monascus anka with Bacillus subtilis. Results revealed that the fermentation system significantly (p < 0.05) increased the total phenolic content (TPC), which was 23-fold over that in unfermented oats. The hydrolytic enzyme activities of α-amylase, β-glucosidase, and cellulase were significantly (r > 0.75, p < 0.05) correlated to the TPC, indicated an important role in the release of phenolic in oats. The proportion of free phenolic fraction in oats was increased from 39.76% to 61.62%, while that of bound phenolic fraction decreased from 42.17% to 22.78%. Moreover, the phenolic compositions of free, conjugated and bound phenolic fractions varied with fermentation. It implied that the phenolic compounds in different forms could be converted and transformed in the SSF system. Meanwhile, the DPPH· and ABTS^·+ scavenging activities of the free and conjugated phenolic fractions in oats became stronger after fermentation. It indicated that the changes of antioxidant activities of phenolic fractions might be closely related to the structural modification by microbial enzymes in the SSF system. Consequently, this study provides a prospect fermentation process for improving the phenolic release and antioxidant activities in oats.     

Induction of cellulases and hemicellulases from Neurospora crassa under solid-state cultivation for bioconversion of sorghum bagasse into ethanol

The cellulolytic and hemicellulolytic system from the mesophilic fungus Neurospora crassa was produced under solid-state cultivation (SSC) on wheat straw and wheat bran mixtures. Following optimization of nitrogen source, pH and initial moisture of the growth medium, yields as high as 492.8, 1.08, 26.7, 297.8 and 0.132 (in U g^?1 of carbon source) were obtained for endoglucanase, exoglucanase, β-glucosidase, xylanase and β-xylosidase, respectively. The potential of the multienzyme system was demonstrated for hydrolysis of sorghum bagasse (SB) into fermentable carbohydrates. N. crassa cells were found able to assimilate the majority of the released sugars and generated limited levels of other metabolic products during simultaneous saccharification and fermentation of this valuable substrate into ethanol.     

Changes in bran structure by bioprocessing with enzymes and yeast modifies the in vitro digestibility and fermentability of bran protein and dietary fibre complex

Bran is a good source of dietary fibre, phytochemicals, and also protein, but highly insoluble and recalcitrant structure of bran hinders accessibility of these components for gastrointestinal digestion. In the present work, influence of bioprocessing on the microstructure and chemical properties of rye bran and wheat bread fortified with the rye bran were studied. In vitro protein digestibility, and release of short chain fatty acids (SCFA) and ferulic acid in a gut model were studied. Bioprocessing of rye bran was performed with subsequent treatments with cell-wall hydrolysing enzymes (40 °C, 4 h) and yeast fermentation (20 °C, 20 h). Bioprocessing of rye bran resulted in reduced total dietary fibre content, caused mainly by degradation of fructan and β-glucan, and increased soluble fibre content, caused mainly by solubilisation of arabinoxylans. Microscopic analysis revealed degradation of aleurone cell wall structure of the bioprocessed rye bran. Bioprocessing caused release of protein from aleurone cells, assessed as a larger content of soluble protein in bran and a higher hydrolysis rate in vitro. Bioprocessed bran had also faster SCFA formation and ferulic acid release in the colon fermentation in vitro as compared to native bran.     

Effect of efficient pectinolytic bacterial isolates on retting and fibre quality of jute

Four bacterial strains with high balanced polygalacturonase (PG) and pectin lyase (PNL), and xylanase with minimal cellulase activity were used in jute retting. The 16S rDNA analysis revealed that the organisms were: Bacillus sp. L6 (GQ891097), Bacillus pumilus strain EK-17 (GQ891098), B. pumilus strain Geo-03-422 (GQ891103) and B. pumilus strain IK-MB12-518F (GQ891105). The microorganisms in different combinations of consortia showed synergistic effect resulting in increased PG (35.52-46.61 IU/g cell wet weight), PNL (39.79-72.12 U/ml), xylanase (0.705-0.840 μmol/ml/min) and little cellulase (0-0.153 μmol/ml/min) activities. The organisms in different combinations of consortia reduced the retting period from 11 to 13 days as compared to 19 days in the control. Microbial inoculation produced remarkable improvement in jute fibre strength (26.62-28.91 g/tex) and fineness (2.76-2.92 tex) over control. The pH of the post-retting water samples became acidic, and the electrical conductivity (Ec), chemical oxygen demand (COD) and hardness increased. The organisms could be adopted in industrial application for extraction of jute fibre.     

Production of gluten-free wheat starch by peptidase treatment

The potential of peptidase-containing bran extracts from germinated cereals (wheat, emmer, barley) and a peptidase preparation from Aspergillus niger (AN-PEP) to degrade gluten in wheat starch below the threshold for gluten-free foods of 20 mg/kg was compared. The gluten-specific peptidase activity of the peptidases was determined by using gliadin as a protein-based substrate as well as the two celiac-active peptides PQPQLPYPQPQLPY (α-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein). The peptidase activity of AN-PEP exceeded the activities of bran from germinated cereals by a factor up to 690,000. Three wheat starches with initial gluten contents of 110, 1679, and 2070 mg/kg, respectively, were incubated with bran extracts and AN-PEP, lyophilized, and residual gluten was quantitated by a competitive ELISA. Unlike peptidases from bran extracts, AN-PEP was capable of degrading gluten below 20 mg/kg in all starches. The absence of gluten in AN-PEP-treated starches was confirmed by liquid-chromatography-mass spectrometry. The properties of gluten-free starches were comparable to the native starches with the exception of a reduced viscosity after AN-PEP treatment. This problem could be overcome by using higher enzyme concentrations and shorter incubation times or by optimizing AN-PEP production for lower residual α-amylase activity.     

Sourdough fermentation of wholemeal wheat bread increases solubility of arabinoxylan and protein and decreases postprandial glucose and insulin responses

Glycemic responses to most of the conventional breads are high, including breads made of wholemeal flour. Baking technology is known to affect these responses. The aim of the present study was to investigate effects of xylanase enzyme treatment and sourdough fermentation in wholemeal wheat bread baking on postprandial glucose and insulin responses and on in vitro protein digestibility. The wheat breads were made of 100% flour from peeled kernels by a straight dough or sourdough fermentation method, and with or without using xylanase during mixing of dough. Standard white wheat bread was used as a reference. All test bread portions contained 50 g available carbohydrate and were served in random order to eleven insulin resistant subjects. Blood samples for measuring glucose and insulin concentrations were drawn over 4 h. The sourdough wholemeal wheat bread resulted in the lowest postprandial glucose and insulin responses among the four tested breads (treatment × time; p = 0.000 and p = 0.022, respectively). There were differences in solubility and depolymerisation of protein and arabinoxylan among the breads but these did not fully explain the in vivo findings. In conclusion, the health effects of wholemeal wheat bread can be further improved by using sourdough process in breadmaking.     

Value added waste of Jatropha curcas residue: Optimization of protease production in solid state fermentation by Taguchi DOE methodology

The oil extraction of Jatropha curcas created the large amount of the by-product from its seeds. An application of solid-state fermentation (SSF) was considered to be of value to these raw materials. This study investigated the potential of a utilization of deoiled J. curcas seed cake as substrate for protease productions by Aspergillus oryzae. While various parameters for SSF was conventionally individually optimized, five parameters were simultaneously examined based on Taguchi method. The effect of three different levels of five factors, including moisture content of substrate, inoculums size, incubation temperature, type of porous substrate and incubation time were examined. The optimum conditions for the protease production by A. oryzae obtained from this experiment were 45% moisture content of substrate, 10% inoculums size, 30 °C incubation temperature, deoiled J. curcas seed cake mixed with cassava bagasse ratio 4:1 as porous substrate at 84 h of incubation time. By adjusting the conditions to these optimum levels, the protease production increased up to 4.6 times as many as the protease yield from the non-optimizing experiment. The use of statistical approach, Taguchi method, provided a satisfactory outcome in defining the optimum conditions for protease production by A. oryzae. Further, the utilization of deoiled J. curcas seed cake as substrate for SSF was proven as the suitable practice for this agricultural waste, in order to develop for an industrial use.     

Rice bran, a potential source of biodiesel production in Indonesia

Biodiesel is a biodegradable, renewable, non-toxic and environmentally friendly alternative fuel. The cost of raw materials comprises 60-88% of the production cost in commercial biodiesel (fatty acid methyl esters, FAMEs) production. Therefore, the use of low-cost raw material as a substrate and an in situ process for biodiesel production are being preferred. In this case, rice bran, which contains 13.5% oil, was an interesting substrate. In situ esterification of high-acidity rice bran with methanol and sulfuric acid catalyst was investigated. The individual and interaction effects of methanol to rice bran ratio, sulfuric acid catalyst concentration and reaction time on purity and recovery of biodiesel were discussed. Our results suggest that under the following operation conditions: methanol to rice bran ratio of 5 mL/g, sulfuric acid concentration in methanol of 1.5 vol.%, and reaction time of 60 min, an in situ esterification operated on rice bran could yield FAMEs with a high purity and recovery. By applying an in situ esterification with n-hexane/water extractions, Indonesia will be succesfull in obtaining biodiesel from rice bran up to 96,000 ton per year.     

Enzymatic hydrolysis of arabinoxylans from spelt bran and hull

Spelt bran and hull were submitted to enzymatic treatment during 1 h, 4 h and 24 h to release arabinoxylans (AXs). Several enzymatic commercial preparations with mainly endo-xylanase activity from Trichoderma reesei (Rohalase WL), Thermomyces lanuginosus (Pentopan Mono conc. BG), Bacillus subtilis (Belfeed B MP and Grindamyl Powerbake 900), Humicola insolens (Ultraflo L), Aspergillus aculeatus (Shearzyme 2×), A. aculeatus plus T. reesei (Shearzyme Plus) were tested and combined with a commercial cellulolytic preparation from T. reesei (Celluclast 1.5L). The xylanolytic activity of the commercial enzyme preparations was heterogeneous. The production of monomers by the different enzymes was evaluated on oat spelt xylan. Spelt bran and hull hydrolysates were analysed in regard to their content of total and free sugars. Monomers of glucose were mainly released with Celluclast, Ultraflo L and Shearzyme 2×. The percentage of released AXs ranged from 18.1% to 69.7% for bran and 0.5% to 6.4% for hull (% of the AXs originally present in the substrate). The solution of AXs hydrolysed by endoxylanases from spelt bran contained polysaccharides with a degree of polymerisation (DP) of 2–71 while those hydrolysed with endoxylanases plus Celluclast had DPs were 1–1164. The hydrolysates from spelt hull had much lower DPs (between 2 and 17).     

Field-based evaluation of vernalization requirement,photoperiod response and earliness per se in bread wheat (Triticum aestivum L.)

Vernalization requirement, photoperiod response and earliness per se (EPS) of bread wheat cultivars are often determined using controlled environments. However, use of non-field conditions may reduce the applicability of results for predicting field performance as well as increase the cost of evaluations. This research was undertaken, therefore, to determine whether field experiments could replace controlled environment studies and provide accurate characterization of these three traits among winter wheat cultivars. Twenty-six cultivars were evaluated under field conditions using two natural photoperiod regimes (from different transplanting dates) and vernalization pre-treatments. Relative responses to vernalization (RRV_GDD) and photoperiod (RRP_GDD) were quantified using the reciprocal of thermal time to end of ear emergence, whereas earliness per se was estimated by calculating thermal time from seedling emergence until end of ear emergence for fully vernalized and lately planted material. An additional index based on final leaf numbers was also calculated to characterize response to vernalization (RRV_FLN). To test whether the obtained indices have predictive power, results were compared with cultivar parameters estimated for the CSM-Cropsim-CERES-Wheat model Version 4.0.2.0. For vernalization requirement, RRV_GDD was compared with the vernalization parameter P1V, for photoperiod (RRP_GDD), with P1D, and for earliness per se, EPS was compared with the sum of the component phase durations. Allowing for variation in EPS in the calibration improved the relation between observed versus simulated data substantially: correlations of RRP_GDD with P1D increased from r² = .34 (p < .01), to .82 (p < .001), and of RRV_GDD with P1V, from r² = .88 (p < .001), to .94 (p < .001). In comparisons of observed versus simulated anthesis dates for independent field experiments, the estimated model coefficients resulted in an r² of .98 (p < .001) and root mean square error of 1d. Overall, the results indicated that combining planting dates with vernalization pre-treatments can permit reliable, quantitative characterization of vernalization requirement, photoperiod response and EPS of wheat cultivars. Furthermore, emphasize the need for further study to clarify aspects that determine EPS, including whether measured EPS varies with temperature or other factors.