Ethanol production from mixtures of wheat straw and wheat meal
© Erdei et al; licensee BioMed Central Ltd. 2010
Received: 11 February 2010
Accepted: 2 July 2010
Published: 2 July 2010
Bioethanol can be produced from sugar-rich, starch-rich (first generation; 1G) or lignocellulosic (second generation; 2G) raw materials. Integration of 2G ethanol with 1G could facilitate the introduction of the 2G technology. The capital cost per ton of fuel produced would be diminished and better utilization of the biomass can be achieved. It would, furthermore, decrease the energy demand of 2G ethanol production and also provide both 1G and 2G plants with heat and electricity. In the current study, steam-pretreated wheat straw (SPWS) was mixed with presaccharified wheat meal (PWM) and converted to ethanol in simultaneous saccharification and fermentation (SSF).
Both the ethanol concentration and the ethanol yield increased with increasing amounts of PWM in mixtures with SPWS. The maximum ethanol yield (99% of the theoretical yield, based on the available C6 sugars) was obtained with a mixture of SPWS containing 2.5% water-insoluble solids (WIS) and PWM containing 2.5% WIS, resulting in an ethanol concentration of 56.5 g/L. This yield was higher than those obtained with SSF of either SPWS (68%) or PWM alone (91%).
Mixing wheat straw with wheat meal would be beneficial for both 1G and 2G ethanol production. However, increasing the proportion of WIS as wheat straw and the possibility of consuming the xylose fraction with a pentose-fermenting yeast should be further investigated.
The use of bioethanol can reduce our dependence on fossil fuels, while at the same time decreasing net emissions of carbon dioxide, the main greenhouse gas [1, 2]. However, large-scale production of bioethanol is being increasingly criticized for its use of food sources as raw material. Brazil's bioethanol production consumes large quantities of sugar cane, while in the USA, corn is used . Other starch-rich grains, such as wheat and barley, are mostly used in Europe . The use of such sugar-rich feedstock causes the escalation of food prices, owing to competition on the market [5, 6]. Therefore, future expansion of biofuel production must be increasingly based on bioethanol from lignocellulosic materials, such as agricultural byproducts, forest residues, industrial waste streams or energy crops [7, 8]. These feedstocks, which are being used in second-generation (2G) bioethanol production, are abundant, and their cost is lower than that of food crops . In Europe, wheat straw has the greatest potential of all agricultural residues because of its wide availability and low cost .
To efficiently utilize lignocellulosic products, pretreatment is required to hydrolyse the hemicelluloses to make the celluloses more accessible to the enzymes. One of the most suitable kinds of pretreatment for lignocellulosic material is steam explosion . Combining steam explosion with acid catalysts is considered one of the most promising techniques for the commercialization of the process . Several studies have shown that impregnation of wheat straw with small amounts of H2SO4 before steam pretreatment results in improved sugar yields [13, 14].
During pretreatment, several sugar degradation products such as 5-hydroxymethyl-furfural (HMF) and furfural (degradation products of hexoses and pentoses, respectively), weak organic acids and phenolic compounds from lignin degradation are released into the hydrolysate, and have been shown to inhibit both yeast [15, 16] and enzymes ; however, these compounds affect cell growth more than ethanol formation. It has also been shown by Larsson et al. that the ethanol yield in the presence of several inhibitors decreased only slightly compared with the reference fermentation . Furthermore, the addition of weak acids has an intense inhibitory effect on growth of Saccharomyces cerevisiae, but leads to increased ethanol yield at low concentrations [19, 20]. Therefore, we hypothesized that mixing starch hydrolysate with the lignocellulosic stream would dilute the inhibitor concentration in the cellulose hydrolysate and probably improve the fermentation, and at the same time, the presence of inhibitors might also improve the ethanol yield from the starch fraction.
To obtain efficient ethanol fermentation with S. cerevisiae, numerous nutrients, including trace metals and vitamins, are required during the process. Chemicals contribute significantly to the cost of large-scale production ; although it was not in the scope of this study to investigate this, their use should thus be minimized. Wheat hydrolysate, which is relatively cheap compared with chemicals, has been proven to be a potential supplement for lignocellulosic hydrolysate, because it is not only a sugar-containing material, but is also a complex nutrient source [22, 23].
The production cost of ethanol is not only dependent on the yield but also on the concentration of ethanol in the fermentation broth, because of the high energy demand in the distillation step. In this step, the ethanol concentration in the broth after fermentation is increased to 94% using two stripper columns and a rectification column, which are heat-integrated by operating at different pressures. A significant increase in energy demand is observed at an ethanol concentration below 4% . A higher ethanol concentration can be achieved in the broth by adding starch-rich material to the lignocellulosic process, leading to a lower energy demand in distillation, thus reducing the production cost.
The aim of this study was to evaluate the simultaneous saccharification and fermentation (SSF) of mixtures of cellulosic material (steam pretreated wheat straw; SPWS and presaccharified wheat meal (PWM). The effect on ethanol concentration and ethanol yield of varying the proportions of starch and cellulose fraction in SSF was investigated and compared with the pure starch and pure cellulose alternatives.
Wheat straw was kindly provided by Lunds Civila Ryttarförening (Lund, Sweden). It was chopped in a hammer mill, sieved to obtain pieces of 2-10 mm, and then stored at room temperature before pretreatment. Wheat meal (dry-milled grain) with an average particle size of 2.5 mm was kindly provided by Sileco (Halland, Sweden) and stored at 5°C before use.
α-Amylase (Termamyl® SC; Novozymes A/S, Bagsværd, Denmark) and amyloglucosidase (Spirizyme® Fuel; Novozymes) amylolytic enzymes were used for starch liquefaction and saccharification, respectively. The amylolytic activity of these enzymes were not measured, because they were loaded based on their weight, as it is recommended by the manufacturer . In the SSF experiments, cellulase (Celluclast 1.5 L) and β-glucosidase (Novozym 188) enzyme preparations (both Novozymes) were used. Celluclast 1.5 L had an activity of 65 filter paper units (FPU)/g, measured using the IUPAC protocol , and 33 IU/g β-glucosidase activity according to the method of Berghem and Petterson . Novozym 188 had a β-glucosidase activity of 350 IU/g.
The carbohydrate and lignin contents of the raw wheat straw, the starch-free fibre, and the solid fraction of the pretreated wheat straw were determined according to the standard National Renewable Energy Laboratory (NREL) method [7, 28]. Finely ground samples were treated with 72% H2SO4 for 1 h at 30°C, then diluted to 4% H2SO4 and autoclaved for 1 hour at 121°C. Sugar contents were analysed with high performance liquid chromatography (HPLC) (LC-10AD; Shimadzu, Kyoto, Japan), acid-insoluble lignin was measured by weighing after overnight drying at 105°C, and acid-soluble lignin was determined by spectrophotometry using a wavelength of 240 nm. Each sample was analysed in duplicate.
The liquid fraction of the SPWS and the supernatant after fermentation were analysed for total sugar content according to an NREL procedure . In this method, the sample is treated with 4% H2SO4 at 121°C for 1 h, and then analysed by HPLC.
The fraction of acid-insoluble ash was determined after the two-step acid hydrolysis described above, and again on the ash of the residue. Both samples were heated at 550°C until the sample weight remained constant. Total ash refers to the inorganic part of raw material or solid fraction after pretreatment.
To determine the starch content, the wheat meal was subjected to a two-step enzymatic hydrolysis consisting of liquefaction and saccharification. All batches were hydrolysed using a 7 L evaporator (Rotavapor® R-153; Büchi Labortechnik AG, Flawil, Switzerland). The dry matter content was set to 35%. In the first step, wheat meal slurry supplemented with 0.5 g/kg dry matter (DM) and Termamyl® SC was liquefied at 85°C, pH 5.5, for 3 h. In the second step, Spirizyme® Fuel was added at a ratio of 0.5 mL/kg DM at pH 4.2, and the slurry was treated at 60°C for 24 h to ensure total starch hydrolysis. The wort was filtered and the glucose content of the supernatant was measured using HPLC. The washed solid residue is referred to as the starch-free residue (SFR).
Wheat meal was presaccharified as described above, except that the duration of saccharification was 2 h instead of 24 h. PWM was then used in SSF.
The wheat straw was immersed in an aqueous solution of 0.2% H2SO4 at a liquid:dry straw weight ratio of 20. It was stored in sealed buckets for 1 h, and was then squeezed in a manual 3 L press (Fisher Maschinenfabrik Gmbh, Burgkunstadt, Germany) to an average dry matter content of 43%. Steam pretreatment was performed in a unit (described previously ) comprising a 10 L pressurized vessel, with a flash cyclone in which the pretreated material was released and collected. Previously optimized conditions for wheat straw  were used; that is, the temperature was maintained at 190°C for 10 min using saturated steam. Each batch that was fed into the reactor was 600 g wet weight. The steam-pretreated wheat straw (SPWS) was then subjected to SSF.
Details of the substrates used in the SSF experiments
Analysis of sugars, ethanol and byproducts
The content of reducing sugars was measured colorimetrically using dinitrosalicylic acid, according to Miller's method . The liquid fractions from pretreatment, samples from acid hydrolysis and the supernatants of SSF broth were analysed by HPLC, in a chromatograph equipped with a refractive index detector. Cellobiose, glucose, mannose, xylose, galactose and arabinose were separated on an ion-exchange column (Aminex HPX-87P; Bio-Rad Laboratories, Hercules, CA, USA) at 85°C. Ultrapure water was used as eluent at a flow rate of 0.6 mL/min. Lactic acid, glycerol, acetic acid, ethanol, HMF and furfural were separated (Aminex HPX-87H column; Bio-Rad Laboratories) at 65°C. The eluent was 0.005 M H2SO4 at a flow rate of 0.5 mL/min.
Results and Discussion
The ethanol yields were calculated as a percentage of the maximal theoretical yield for glucose (0.51 g/g) that could have been produced if all the glucose present in the slurry and the PWM, including both monomers and oligomers in the liquid and glucan fibres in the WIS, had been converted to ethanol. The theoretical amount of glucose released during the hydrolysis was calculated by multiplying the amount of glucan by 1.11.
Material composition and pretreatment
Composition of raw wheat straw and wheat meal, in % of DM, including breakdown of the starch-free residue.
Percentage of DM, mean ± SD
Raw wheat straw
% of SFRa
17.5 ± 0.1
38.8 ± 0.5
1.7 ± 0.2
14.4 ± 0.0
22.2 ± 0.3
1.6 ± 0.0
2.7 ± 0.1
8.5 ± 0.0
4.7 ± 0.1
3.1 ± 0.0
2.4 ± 0.0
15.1 ± 3.0
16.1 ± 0.1
2.3 ± 0.3
5.8 ± 0.1
2.4 ± 0.4
Composition of WIS and liquid (prehydrolysate) fractions in steam-pretreated wheat straw slurry.
Steam-pretreated wheat straw
Percentage of DM
67.6 ± 0.5
0.9 ± 0.1
0.7 ± 0.0
4.1 ± 0.1
2.9 ± 0.7
0.4 ± 0.0
5.1 ± 0.2
23.1 ± 0.2
1.0 ± 0.1
0.4 ± 0.2
Total ash, % of prehydrated material
0.3 ± 0.2
Enzymatic hydrolysis of wheat meal
It has been shown previously that the amyloglucosidase dosage can be reduced by 5-10% when saccharification is carried out before fermentation . However, a high glucose concentration at the beginning of SSF with lignocellulosics should be avoided to prevent end-product inhibition of the enzymes and osmotic stress to the yeast cells. β-glucosidase activity is reduced by 80% in the presence of only 10 g/L glucose when p-nitrophenyl-β-D-glicopyronoside is used as substrate, and less significantly with cellobiose . In that study also, a high degree of inhibition of cellulase activity was observed at a glucose concentration range of 0 to 100 g/L. Osmotic stress affects the yeast cell when the glucose in the solution is > 150 g/L [25, 36]. Therefore, instead of completely saccharifying the wheat meal, we chose to perform partial saccharification (presaccharification). Optimum presaccharification in a starch-based material is about 50-70 dextrose equivalents (DE), which is an indication of the total amount of reducing sugars, expressed as D-glucose, present in the solution [25, 37].
Effect of PWM on the ethanol concentration
Significant differences, in terms of the initial rate of ethanol formation, were observed between SSF on pure SPWS and SSF on mixtures containing different proportions of PWM. During the first 2 hours of SSF, the ethanol productivity was 1.6 g/L/hour in the case of pure SPWS, whereas it was ≥4.7 g/L/hour for PWM alone or PWM mixed with SPWS. This could be due to the high water-soluble sugar content of PWM (Table 1) present at the beginning of fermentation, mainly as glucose, which was consumed rapidly (data not shown), resulting in an increased rate of ethanol formation. In pure SPWS, the major part of the glucose is in polymeric form bound in the solid phase, and this had to be hydrolysed before fermentation. However, glucose was measured in the solution during the initial 8 hours, which means that hydrolysis is not the rate-limiting step in this reaction. However, furfural and HMF may cause a lag-phase in ethanol fermentation , because ethanol production is inhibited by the degradation of these compounds to furfuryl alcohol and HMF alcohol, respectively. The most rapid ethanol formation (6.7 g/L/hour) was obtained with pure PWM.
Effect of PWM on the yield
The ethanol yield is usually reported as g EtOH/g DM of the raw material. However, this means of expressing the yield was not appropriate for this study because mixtures of materials were used. Therefore, the yields are expressed as a percentage of the theoretical maximum, considering only the glucose available in the substrates, as galactose and the pentoses are not usually fermented to ethanol by S. cerevisiae. These sugars were not consumed in any of the SSF experiments, which validates this assumption (data not shown).
0.46 g/g is 90% of the maximum theoretical ethanol yield for hexose sugars. After applying this correction for lactic acid, the yield slightly exceeds the theoretical maximum for mixture D (Figure 5).
Specific raw material demand
Raw material required (kg total DM per L EtOH)
Wheat straw only
Wheat meal only
The residue:crop ratio for wheat is typically about 1.3:1.0 (w/w) . Approximately 30-40% of the straw is left on the field for soil protection, leaving the same amount of residue for biomass utilization when the crop is harvested (that is, the straw:wheat ratio is 1.0:1.0). In the case of mixture D, which gave the highest yield, the proportion of wheat meal was double that of the wheat straw. However, the yield was still rather high (87% of theoretical yield) when straw and meal were used in the proportions at harvest, as in mixture B. By comparison, the total yield obtained if the fermentations are carried out separately is 78%.
In this study, we investigated the effects on ethanol yield of mixing different proportions of PWM and SPWS before SSF. The highest yield was obtained when equal amounts of PWM and SPWS (based on WIS) were used. Thus, a mixed substrate is favourable in terms of final ethanol yield, probably due to the stress on S. cerevisiae caused by weak acids present in SPWS. At the same time, it is also easier to reach a high ethanol concentration using such as mixture than when using wheat straw only as a raw material.
Increasing the proportion of WIS of the lignocellulosic material should be studied further in an attempt to improve the ethanol production from mainly lignocellulosics. Bearing in mind the significant proportion of hemicelluloses in wheat straw, a pentose-fermenting yeast should also be considered as a potential alternative. Assuming 70% ethanol yield from pentoses, the final ethanol concentration in the fermentation broth could be further improved by 3-5 g/L ethanol. To decrease the cost of chemicals, decreasing the amount of added nutrients is an option to consider and further investigate when wheat hydrolysate is used as a supplement to SSF with lignocellulosic substrate.
We gratefully acknowledge the Swedish Energy Agency for its financial support, and the European Community Vocational Training Programme (Leonardo da Vinci) for mobility support.
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