The effect of temperature on the ethanol tolerance of the yeast,Saccharomyces uvarum

The optimum temperature for fermentation by Saccharomyces uvarum was found to be higher than that for its growth. Fermentation continued at temperatures above the growth maximum (40°C). S.uvarum was most resistant to growth inhibition by ethanol at temperatures 5°C and 10°C below its growth optimum (35°C). Fermentation became more resistant to ethanol inhibition with increasing temperature.     

Simultaneous saccharification and fermentation by mixed cultures ofBrettanomyces clausenii andPichia spipitis R of SO2-prehydrolysed wood

Summary Using pilot scale Wenger and Stake II reactors for prehydrolysing aspen and coniferous wood chips in the presence of SO₂ catalyst, highly digestible lignocellulosic substrates were generated from which about 90% yields of hemicellulose mostly in monomeric form could be recovered. Simultaneous saccharification and fermentation (SSF) of these SO₂ feedstocks by a mixed culture ofBrettanomyces clausenii andPichia stipitis R resulted in rapid and efficient fermentation giving a final yield of 369 and 360 L ethanol/tonne of the prehydrolysed woods, respectively. BecauseB. clausenii is an excellent cellobiose fermenter, no -glucosidase was needed during SSF.     

Enhanced rate of ethanol production from D-xylose using recycled or immobilized cells ofPachysolen tannophilus

Summary Enhanced rates of ethanol production byPachysolen tannophilus from D-xylose were obtained by performing the fermentation with recycled cells in suspension culture or immobilized in a Ca-alginate gel. Fermentation under these conditions did not require aeration. Increasing temperature from 30 to 37°C enhanced the amount of ethanol produced in 24 hours from the recycled or the immobilized cells.Issued as National Research Council of Canada Publication Number 19475.     

Ethanol production in simultaneous saccharification and fermentation of cellulose with temperature profiling

The effects of temperature on enzymatic saccharification of cellulose and simulataneous saccharification and fermentation (SSF) were investigated with 100 g·l⁻¹ Solka Floc, 5g·l⁻¹Trichoderma reesei cellulase, and Zymomonas mobilis ATCC 29191. The following results were obtained: 1) Ethanol fermentation under glucose dificient conditions can proceed for more than 100 h at 30°C but gradually ceases after 50 h of operation at 40°C. 2) Equivalent glucose yield based on cellulose for SSF operated at its optimum temperature (37°C) is higher than that for enzymatic saccharification of cellulose at the same temperature by 32%. However, the same equivalent glucose yields were obtained for both processes if they were operated at their respective optimum temperature. 3) SSF with temperature cycling increased the ethanol productivity but gave similar ethanol yield to SSF at 37°C. 4) SSF with temperature profiling gave an ethanol yield of 0.32 g·g⁻¹ and cellulose use of 0.86 g·g⁻¹ which were increased by 39% and 34% over SSF with temperature cycling and at 37°C.     

Optimization of fermentation conditions for the production of ethanol from sago starch using response surface methodology

The quantitative effects of temperature, pH and time of fermentation were investigated on simultaneous saccharification and fermentation (SSF) of ethanol from sago starch with glucoamylase (AMG) and Zymomonas mobilis ZM4 using a Box–Wilson central composite design protocol. The SSF process was studied using free enzyme and free cells and it was found that with sago starch, maximum ethanol concentration of 70.68 g/l was obtained using a starch concentration of 140 g/l, which represents an ethanol yield of 97.08%. The optimum conditions for the above yield were found to be a temperature of 36.74 °C, pH of 5.02 and time of fermentation of 17 h. Thus by using the central composite design, it is possible to determine the accurate values of the fermentation parameters where maximum production of ethanol occurs.     

Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4

A thermotolerant yeast strain named Kluyveromyces marxianus IMB4 was used in a simultaneous saccharification and fermentation (SSF) process using Kanlow switchgrass as a feedstock. Switchgrass was pretreated using hydrothermolysis at 200 degrees C for 10 min. After pretreatment, insoluble solids were separated from the liquid prehydrolyzate by filtration and washed with deionized water to remove soluble sugars and inhibitors. Insoluble solids were then hydrolyzed using a commercial cellulase preparation and the released glucose was fermented to ethanol by K. marxianus IMB4 in an SSF process. SSF temperature was 37, 41, or 45 degrees C and pH was 4.8 or 5.5. SSF was conducted for 7 days. Results were compared with a control of Saccharomyces cerevisiae D(5)A at 37 degrees C and pH 4.8. Fermentation by IMB4 at 45 and 41 degrees C ceased after 3 and 4 days, respectively, when a pH 4.8 citrate buffer was used. Fermentation continued for all 7 days using IMB4 at 37 degrees C and the control. When pH 5.5 citrate buffer was used, fermentation ceased after 96 h using IMB4 at 45 degrees C, and ethanol yield was greater than when pH 4.8 citrate buffer was used (78% theoretical). Ethanol yield using IMB4 at 45 degrees C, pH 5.5 was greater than the control after 48, 72, and 96 h (P < 0.05).     

Fermentation pattern of Zymomonas mobilis at high sucrose concentrations

Summary Zymomonas mobilis was grown in batch concentrations between 200 and 400 g/l sucrose. The fermentation pattern revealed that the efficiency of sucrose hydrolysis dropped only from 94 to 78.6% whereas the efficiency with which the hydrolyzed products were converted to ethanol decreased from 94 to 43%. The ethanol yields were relatively constant for final concentrations which lay between 80 and 132 g/l. Fermentation times increased to 72 hours at the higher sucrose concentrations. Sorbitol and fructose were identified as the major by-products. Preliminary evidence suggests that the ratio between the two by-products depends on the pH of the culture medium. Results suggest the possibility of processes producing ethanol plus fructose, ethanol plus fructose and sorbitol, or ethanol plus sorbitol in a single-stage batch fermentation.     

Optimization of fermentation conditions for the production of ethanol from sago starch by co-immobilized amyloglucosidase and cells of Zymomonas mobilis using response surface methodology

Statistical experimental design was used to optimize the conditions of simultaneous saccharification and fermentation (SSF), viz. temperature, pH and time of fermentation of ethanol from sago starch with co-immobilized amyloglucosidase (AMG) and Zymomonas mobilis MTCC 92 by submerged fermentation. Maximum ethanol concentration of 55.3 g/l was obtained using a starch concentration of 150 g/l. The optimum conditions were found to be a temperature of 32.4 °C, pH of 4.93 and time of fermentation of 17.24 h. Thus, by using SSF process with co-immobilized AMG and Z. mobilis cells MTCC 92, the central composite design (CCD) was found to be the most favourable strategy investigated with respect to ethanol production and enzyme recovery.     

A Rapid Simultaneous Saccharification and Fermentation (SSF) Technique to Determine Ethanol Yields

We have developed a relatively simple simultaneous saccharification and fermentation (SSF) technique to determine the ethanol production potential for large sets of biomass samples. The technique is based on soaking approximately 0.5 grams of a biomass sample in aqueous ammonia at room temperature and at atmospheric pressure for 24 h, then fermenting with Saccharomyces cerevisiae D₅A for 24 h using Spezyme CP, for enzymatic hydrolysis of structural polysaccharides. We have tested the technique on a set of corn stover samples representing much of the genetic variability in the commercial corn hybrid population. The samples were weighed into modified Ankom filter bags (F57) before soaking to avoid biomass loss during the process. Fermentation samples were analyzed for ethanol after 24 h by HPLC. Percentages of theoretical maximum ethanol yields of the samples ranged between 44.9 and 73%. We observed that percentages of theoretical maximum ethanol yields were highly correlated (r ²?=?0.90) with acid detergent lignin concentration while a low correlation was observed between cellulose concentration and ethanol yield. Near infrared spectra of corn stover samples were also examined. The coefficient of determination (r ²) from regression of predicted versus measured percent theoretical maximum ethanol yield was 0.96. This result suggests that using NIRS is a promising method for predicting ethanol yield, but larger calibration sets are necessary for obtaining improved accuracy for larger sample populations. We conclude that the developed SSF technique could be applied to large numbers of biomass samples to rapidly estimate ethanol yields and to compare different biomass samples in terms of ethanol yields.     

Red oak wood derived inhibitors in the ethanol fermentation of xylose byPichia stipitis CBS 5776

Summary Xylose, the predominant sugar in red oak prehydrolysate, is fermented to ethanol byPichia stipitis CBS 5776. Toxic model compounds derived from red oak hemicelluloses, lignin, and extractives inhibited the fermentation. Treatment of the prehydrolysate with molecular sieve and mixed bed ion resins facilitated the ethanol fermentation giving about 10 g/l ethanol from 32 g/l initial xylose. Fermentation inhibitors derived from red oak lignin and extractives were identified.     

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.     

Simultaneous Saccharification and Ethanol Fermentation of Corn Stover at High Temperature and High Solids Loading by a Thermotolerant Strain Saccharomyces cerevisiae DQ1

The thermotolerant strain Saccharomyces cerevisiae DQ1 was applied to the simultaneous saccharification and fermentation (SSF) at high temperature and high solids loading of the dilute acid-pretreated corn stover in the present study. The SSF using S. cerevisiae DQ1 was operated at 30?% solids loading of the pretreated corn stover with three-step SSF mode and achieved up to ethanol titer of 48?g/L and yield of 65.6?%. S. cerevisiae DQ1 showed strong thermotolerance in both the regular one-step SSF and the three-step SSF with changing temperature in each step. The three-step SSF at 40°C using S. cerevisiae DQ1 tolerated the greater cellulase dosage and solids loading of the pretreated corn stover and resulted in increased ethanol production. The present study provided a practical potential for the future SSF of lignocellulose feedstock at high temperature to reach high ethanol titer.     

Ethanol fermentation of hexose and pentose wood sugars produced by hydrogen-fluoride solvolysis of aspen chips

Summary Hexose and pentose sugars, produced by hydrogen-fluoride solvolysis of aspen wood chips, were totally consumed in a coculture fermentation by Zymomonas mobilis and a mutant of Clostridium saccharolyticum. Z. mobilis converted the glucose to ethanol, while the mutant, which was improved in both ethanol production and tolerance, converted the xylose component to ethanol. A high conversion efficiency of wood sugars to ethanol was obtained, and the cells after the fermentation were successfully used for cell recycle.NRCC no. 23211     

Evaluation of steam-treated giant bamboo for production of fermentable sugars

Giant bamboo plantations are currently being established in the Southern Africa region and can be considered as potential lignocellulosic feedstock for the production of second generation bioethanol. In this study, giant bamboo internodal material was subjected to sulphur dioxide (SO(2)) impregnated steam pretreatment prior to enzymatic hydrolysis. The effect of temperature, residence time, and acidity on the overall sugar recovery and byproduct formation was studied using response surface response technology according to a central composite experimental design (CCD) at a fixed SO(2) concentration of 2.5% (w/w liquid) after impregnation. The results showed that pretreatment conditions with combined severity factor (CSF) values and enzyme dosages greater than 1.72 and 30 FPU/g water insoluble solid, respectively, were required to obtain an efficient glucan digestibility and a good overall glucose recovery. Up to 81.2% of the sugar in the raw material was recovered for a CSF of 2.25. However, considering overall sugar yield and byproducts concentration, the pretreated material obtained with a CSF of 1.62 can be considered as the most appropriate for SSF experiments using a xylose-utilizing yeast. At these conditions, it could be possible to obtain up to 247 L of ethanol per dry ton of giant bamboo considering hexose and pentose sugars fermentation. This amount could be increased up to 292 L of ethanol per dry ton of giant bamboo with the maximum sugar yield obtained (CSF = 2.25) if the microorganism possesses robust fermentative characteristics as well as a high resistance to pretreatment by-products.     

Ethanol production from paper material using a simultaneous saccharification and fermentation system in a fed-batch basis

In this work, a recycled paper-derived feedstock was used to produce ethanol by the simultaneous saccharification and fermentation (SSF) process using the thermotolerant yeast Kluyveromyces marxianus CECT 10875. At standard SSF conditions, the highest yield (about 80% of theoretical) was obtained at low substrate concentration and high enzyme loading. With increasing substrate concentration, mixing difficulties appeared which prevented an adequate SSF process performance and limited ethanol production. An SSF fed-batch procedure was then used which permitted an increase in substrate concentrations while maintaining SSF yields similar to that obtained at standard SSF, thus allowing an increased final ethanol production (about 18 g/l).     

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.     

Simultaneous saccharification and fermentation of steam-pretreated bagasse using Saccharomyces cerevisiae TMB3400 and Pichia stipitis CBS6054

Sugarcane bagasse--a residue from sugar and ethanol production from sugar cane--is a potential raw material for lignocellulosic ethanol production. This material is high in xylan content. A prerequisite for bioethanol production from bagasse is therefore that xylose is efficiently fermented to ethanol. In the current study, ethanolic fermentation of steam-pretreated sugarcane bagasse was assessed in a simultaneous saccharification and fermentation (SSF) set-up using either Saccharomyces cerevisiae TMB3400, a recombinant xylose utilizing yeast strain, or Pichia stipitis CBS6054, a naturally xylose utilizing yeast strain. Commercial cellulolytic enzymes were used and the content of water insoluble solids (WIS) was 5% or 7.5%. S. cerevisiae TMB3400 consumed all glucose and large fraction of the xylose in SSF. Almost complete xylose conversion could be achieved at 5% WIS and 32 degrees C. Fermentation did not occur with P. stipitis CBS6054 at pH 5.0. However, at pH 6.0, complete glucose conversion and high xylose conversion (>70%) was obtained. Microaeration was required for P. stipitis CBS6054. This was not necessary for S. cerevisiae TMB3400.     

Effects of by-products of fermentation of banana pseudostem on ethanol separation by pervaporation

In this work, we performed recovery of ethanol from a fermentation broth of banana pseudostem by pervaporation (PV) as a lower-energy-cost alternative to traditional separation processes such as distillation. As real fermentation systems generally contain by-products, it was investigated the effects of different components from the fermentation broth of banana pseudostem on PV performance for ethanol recovery through commercial flat sheet polydimethylsiloxane (PDMS) membrane. The experiments were compared to a binary solution (ethanol/water) to determine differences in the results due to the presence of fermentation by-products. A real fermented broth of banana pseudostem was also used as feed for the PV experiments. Seven by-products from fermented broth were identified: propanol, isobutanol, methanol, isoamyl alcohol, 1-pentanol, acetic acid, and succinic acid. Moreover, the residual sugar content of 3.02 g/L¹ was obtained. The presence of methanol showed the best results for total permeate flux (0.1626 kg·m⁻²·h⁻¹) and ethanol permeate flux (0.0391 kg·m⁻²·h⁻¹) during PV at 25°C and 3 wt% ethanol, also demonstrated by the selectivity and enrichment factor. The lowest total fluxes of permeate were observed in the experiments containing the acids. Better permeance of 0.1171 from 0.0796 kg·m⁻²·h⁻¹ and membrane selectivity of 9.77 from 9.30 were obtained with real fermentation broth than with synthetic solutions, possibly due to the presence of by-products in the multicomponent mixtures, which contributed to ethanol permeation. The results of this work indicate that by-products influence pervaporation of ethanol with hydrophobic flat sheet membrane produced from the fermented broth of banana pseudostem.     

Comparison of SHF and SSF processes for the bioconversion of steam-exploded wheat straw

Two processes for ethanol production from wheat straw have been evaluated — separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). The study compares the ethanol yield for biomass subjected to varying steam explosion pretreatment conditions: temperature and time of pretreatment was 200°C or 217°C and at 3 or 10 min. A rinsing procedure with water and NaOH solutions was employed for removing lignin residues and the products of hemicellulose degradation from the biomass, resulting in a final structure that facilitated enzymatic hydrolysis. Biomass loading in the bioreactor ranged from 25 to 100 g l⁻¹ (dry weight). The enzyme-to-biomass mass ratio was 0.06. Ethanol yields close to 81% of theoretical were achieved in the two-step process (SHF) at hydrolysis and fermentation temperatures of 45°C and 37°C, respectively. The broth required addition of nutrients. Sterilisation of the biomass hydrolysate in SHF and of reaction medium in SSF can be avoided as can the use of different buffers in the two stages. The optimum temperature for the single-step process (SSF) was found to be 37°C and ethanol yields close to 68% of theoretical were achieved. The SSF process required a much shorter overall process time (≈30 h) than the SHF process (96 h) and resulted in a large increase in ethanol productivity (0.837 g l⁻¹ h⁻¹ for SSF compared to 0.313 g l⁻¹ h⁻¹ for SHF). Journal of Industrial Microbiology & Biotechnology (2000) 25, 184–192. Received 02 December 1999/ Accepted in revised form 20 July 2000     

Enzymatic hydrolysis and simultaneous saccharification and fermentation of alkali/peracetic acid-pretreated sugarcane bagasse for ethanol and 2,3-butanediol production

The enzymatic digestibility of alkali/peracetic acid (PAA)-pretreated bagasse was systematically investigated. The effects of initial solid consistency, cellulase loading and addition of supplemental β-glucosidase on the enzymatic conversion of glycan were studied. It was found the alkali-PAA pulp showed excellent enzymatic digestibility. The enzymatic glycan conversion could reach about 80% after 24 h incubation when enzyme loading was 10 FPU/g solid. Simultaneous saccharification and fermentation (SSF) results indicated that the pulp could be well converted to ethanol. Compared with dilute acid pretreated bagasse (DAPB), alkali-PAA pulp could obtain much higher ethanol and xylose concentrations. The fermentation broth still showed some cellulase activity so that the fed pulp could be further converted to sugars and ethanol. After the second batch SSF, the fermentation broth of alkali-PAA pulp still kept about 50% of initial cellulase activity. However, only 21% of initial cellulase activity was kept in the fermentation broth of DAPB. The xylose syrup obtained in SSF of alkali-PAA pulp could be well converted to 2,3-butanediol by Klebsiella pneumoniae CGMCC 1.9131.