This study investigated the potential use of alkali pretreatment of sawdust from Australian timber mills to produce bioethanol. Sawdust was treated using 3–10% w/w NaOH at temperatures of 60, 121, and −20°C. Two pathways of production were trialled to see the impact on the bioethanol potential, enzymatic hydrolysis for glucose production, and simultaneous saccharification and fermentation (SSF) for ethanol production. The maximum yields obtained were at 121°C and −20°C using 7% NaOH, with 29.3% and 30.6% ethanol yields after 0.5 and 24 hr, respectively, these treatments yielded 233% and 137% increase from the 60°C counter parts. A notable trend of increased ethanol yields with increased NaOH concentration was observed for samples treated at 60°C; for example, samples treated using 10% NaOH produced 1.92–2.07 times more than those treated using 3% NaOH. FTIR analysis showed reduction in crystallinity correlating with increased ethanol yields with the largest reduction in crystallinity in the sample treated at −20°C for 24 hr with 7% NaOH.
Waste lignocellulose is the most abundant potential source for biofuels on the planet [
Hardwoods are a lignocellulosic biomass, with a significant proportion of cellulose between 45 and 55% w/w. Hemicellulose also composes a large proportion of the mass accounting for 24–40% w/w [
The lignin-hemicellulose matrix surrounding the cellulose microfibril provides the first hurdle in enzymatic hydrolysis, as it prevents the enzymes’ access to the cellulose, inhibiting the hydrolysis of the cellulose. This can be overcome by the removal or modifications of lignin and or hemicellulose, increasing the pore space allowing enzyme access to the cellulose microfibrils [
It has been shown that alkali pretreatment increases access to the cellulose by saponification of the intermolecular ester bonds between hemicellulose and lignin. The cleaving of these links can cause solubilisation of lignin and hemicellulose [
Alkali pretreatment has been observed to decrease cellulose crystallinity and the degree of polymerisation, which is considered to occur due to swelling of the internal cellulose microfibrils increasing the amount of amorphous regions of cellulose microfibrils [
The use of moderate temperatures in alkali pretreatment can prevent the creation of furfural, hydroxymethylfurfural, and organic acids, reducing the loss of usable sugars [
The effect of pretreatment conditions, for example, temperature, time, and alkali dosage, has been studied by many researchers [
A large number of research studies have looked into the feasibility of pretreatment of lignocellulosic materials with Ca(OH)2 and NaOH [
Simultaneous saccharification and fermentation (SSF) systems for the production of ethanol from lignocellulosic material have been thoroughly investigated. SSF has the ability to prevent end-product inhibition by conversion of glucose to ethanol, resulting in improved yields [
This investigation will be into the possibility of pretreatment at 60°C over 0.5–2 hr with 3, 7, and 10% NaOH. Three comparative treatments were also assessed with 7% NaOH at 121°C for 0.5 hr and −20°C for 2 and 24 hr.
The sawdust mixture utilised in this investigation was collected from a timber mill located in regional Victoria. The sawdust mixture used contained a mixture of eucalyptus hardwoods and nonnative softwoods; the waste wood samples were ground utilising a bladed mill (IKA analytical mill) and separated with a ASTM number 18 mesh 1 mm sized sieve.
The cellulase enzyme complex Accellerase 1500 (enzyme solutions, Australia) was used in the hydrolysis and SSF systems. The cellulase activity in filter paper units (FPU) was 45.6/mL measured as described in [
A freeze dried thermo-tolerant
The first phase of alkali pretreatment of sawdust samples was carried out using 3%, 7%, and 10% NaOH solutions (w/w) at a temperature of 60°C and exposure times of 0.5, 1, and 2 hr(s) with a solid to liquid ratio of 1 : 10 w/v, based on the works previously undertaken by [
Hydrolysis of cellulose in the pretreated sawdust was undertaken using 118 mL reactors with a working mass of 20 g. Substrate loadings of 3
SSF was carried out in 250 mL reactors with a working mass of 50 g, and a substrate loading of 7.5
Measurements of TS and volatile solids (VS) were carried out according to the standard methods as described in [
FTIR analysis was undertaken utilising a Perkin Elmer Spectrum 100 FTIR within a spectrum of 600–4000 cm−1. Before analysis samples were dried in a 35°C oven [
The composition of the sawdust before and after alkali pretreatment is given in Table
Composition of sawdust before and after pretreatment with NaOH.
Dose | Temp. (°C) | Time (hr) | Cellulose (%) w/w | Xylan (%) w/w | Galactans (%) w/w | ASL |
AIL |
Extractives (%) w/w |
---|---|---|---|---|---|---|---|---|
Untreated sawdust | 41.0 ± 2.8 | 6.0 ± 3.0 | 1.1 ± 1.5 | 5.7 ± 0.3 | 25.5 ± 4.2 | 8.05% | ||
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3% |
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39.9 ± 1.4 | 4.76 ± 0.4 | 0.5 ± 0.2 | 4.9 ± 0.6 | 26.5 ± 1 | 0.96% |
|
44.6 ± 1 | 4.76 ± 0.7 | 0.3 ± 0.4 | 4.7 ± 0.4 | 31.7 ± 0.3 | 1.16% | ||
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48.3 ± 7.3 | 4.18 ± 0.3 | 1.3 ± 0.8 | 4.7 ± 0.7 | 31.8 ± 0.8 | 0.75% | ||
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7% |
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45.2 ± 3.9 | 4.08 ± 0.1 | 0.9 ± 0.9 | 4.1 ± 0.3 | 28.2 ± 4.9 | 0.52% | |
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47.8 ± 7.4 | 3.81 ± 0.1 | 0.3 ± 0.1 | 4.0 ± 0.1 | 29.6 ± 0.8 | 0.69% | ||
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50.9 ± 6.0 | 4.5 ± 1.0 | 0.3 ± 0.0 | 3.8 ± 0.4 | 28.8 ± 4.3 | 0.87% | ||
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10% |
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51.7 ± 3.6 | 6.4 ± 2.1 | 1.4 ± 1.2 | 4.3 ± 0.4 | 26.9 ± 3.2 | 0.72% | |
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48.3 ± 0.8 | 5.9 ± 0.9 | 0.5 ± 0.1 | 3.8 ± 0.1 | 27.7 ± 5.3 | 0.87% | ||
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48.1 ± 4.0 | 4.9 ± 0.4 | 0.3 ± 0.1 | 4.5 ± 0.2 | 31.7 ± 0.6 | 0.85% | ||
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7% | − |
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51.0 ± 2.0 | 4.3 ± 0.7 | 0.4 ± 0.0 | 4.4 ± 0.2 | 24.0 ± 2.7 | 0.59% |
− |
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49.5 ± 4.9 | 4.7 ± 1.2 | 0.4 ± 0.3 | 4.4 ± 0.1 | 26.8 ± 1.2 | 0.72% | |
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51.8 ± 3.5 | 4.4 ± 1.0 | 0.0 ± 0.1 | 3.8 ± 0.2 | 23.1 ± 2.6 | 0.51% |
Impact of pretreatment on structural composition ratios.
Dose | Temp. (°C) | Time (hr) | Cellulose/hemicellulose | Lignin/cellulose | Lignin/hemicellulose |
---|---|---|---|---|---|
Untreated sawdust | 5.81 | 0.76 | 4.42 | ||
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3% |
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7.52 | 0.79 | 5.92 |
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7.40 | 0.82 | 6.04 | ||
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5.49 | 0.76 | 4.15 | ||
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7% |
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6.21 | 0.72 | 4.44 | |
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9.77 | 0.71 | 6.87 | ||
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10.70 | 0.64 | 6.85 | ||
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10% |
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6.61 | 0.61 | 3.99 | |
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7.51 | 0.65 | 4.90 | ||
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9.17 | 0.76 | 6.89 | ||
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7% |
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10.97 | 0.56 | 6.10 |
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9.71 | 0.63 | 6.13 | |
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11.57 | 0.52 | 6.01 |
Significant extractive removal was observed for all alkali pretreated sawdust, reduced from 8.05% down to approximately ~1% or less in treated samples (Table
Lignin removal occurred in all pretreated samples with ASL content reduced from the 6.2% of the untreated samples to ≤5% w/w. AIL content of pretreatments of 60°C with 3% NaOH did not show significant reduction of total lignin due to the lack of reduction in the lignin/cellulose ratio as shown in Table
The FTIR analysis displayed in Figure
FTIR analysis of a verity of NaOH pretreated sawdust between 800 and 1800 cm−1.
The reductions in peaks at 1250 cm−1 may relate to the removal of xyloglucan acetyl groups or reduction in COOH groups in the lignin hemicellulose matrix [
Pretreatment of the sawdust at 60°C showed two apparent trends in relation to the composition of the sawdust. Firstly as pretreatment time increased levels of xylose decreased, secondly concentrations of 7 and 10% NaOH produced a greater reduction in xylose and lignin removal with exception of 10% NaOH for 2 hr and 3% NaOH for 0.5 hr. Notably samples treated with 7% NaOH produced a larger cellulose/xylan ratio suggesting that more xylans had been removed in these treatments than that of the 10% solutions; conversely the 10% solution displayed a reduction in the lignin/cellulose ratio suggesting greater lignin removal than the 7% counter parts.
The hydrolysis of the sawdust pretreated at 60°C yielded between 5.8 and 16.2% of potential glucose production compared to untreated sawdust which could only produce 0.5 ± 0.2% as shown in Table
Peak glucose production in cellulose hydrolysis systems of sawdust pretreated with NaOH at 60°C.
3% NaOH 60°C | Glucose yield (%) | 7% NaOH 60°C | Glucose yield (%) | 10% NaOH 60°C | Glucose yield (%) |
---|---|---|---|---|---|
0.5 hr | 8.1 ± 3.6% | 0.5 hr | 11.6 ± 3.2% | 0.5 hr | 16.2 ± 1.4% |
1 hr | 5.8 ± 0.6% | 1 hr | 9.4 ± 3.6% | 1 hr | 9.9 ± 7.0% |
2 hr | 7.0 ± 1.5% | 2 hr | 11.2 ± 3.4% | 2 hr | 12.9 ± 1.4% |
It is possible that the lower yields encountered using the hydrolysis system were due to end-product inhibition [
Preliminary analysis showed that greater yields were achieved for glucose fermentation through hydration followed by in situ addition from the acclimatised inoculums. In addition, preliminary testing of the inoculation of the reactors showed that 0.5 hr of hydration and direct inoculation into the reactor was favourable.
SSF systems of the sawdust pretreated with NaOH at 60°C produced ethanol yields exceeding the 2.6 ± 2.0% produced by the untreated sawdust. Highest yields were observed after 96 hr and 120 hr for the majority of pretreatments conditions as shown in Figure
Ethanol yield from sawdust pretreated for 2 hr at 60°C in NaOH solution, with 20
The highest yield of ethanol from a 60°C pretreatment was 25.2%; this result was obtained from samples treated with 10% NaOH solution for 1 hr, using an enzyme dosage of 20.0
Low variance between many of the reactors operated at cellulase dosages of 9.1 and 20.0
Ethanol yield from sawdust pretreated with NaOH at 60°C with enzyme dosage of (a) 9.1
No significant correlation was observed with variation in pretreatment times for the reactors that received 9.1
When pretreated at 121°C improved yields were observed at a faster rate, ethanol yield of 30.6% was obtained after 72 hr of SSF as shown in Figure
Ethanol yield from sawdust samples pretreated with 7% NaOH at 121°C and −20°C using SSF system with 20.0
Ethanol production from sawdust exposed to high temperature of 121°C showed significant increased yields, where sawdust treated at 121°C produced 82–123% more ethanol than sawdust pretreated using the same NaOH concentration and exposure time at 60°C. In a similar way −20°C for 2 hr pretreatment produced 37–49% greater yields than samples pretreated at 60°C for 2 hr, as shown in Figure
A proportional relationship between the cellulose concentration, decrease in xylans content, and increase in ethanol production was observed, as predicted. This is likely due to increased enzyme access to cell interior with the removal of hemicelluloses and extractives [
The absorbance ratio
When assessing
Absorbance ratios of
Considering the total crystallinity for samples pretreated at 60°C with NaOH, it was observed that only those pretreated with 10% NaOH had ratios below the 1.69 of untreated sawdust with Abs
Total crystallinity of sawdust pretreated with NaOH at 60°C.
The total crystallinity consistently decreased with increasing pretreatment times, although no apparent correlation exists relative to the ethanol yields. The largest increase in total crystallinity was 32%, observed for samples pretreated with 7% NaOH and 0.5 hr. From these results it appears that both time and concentration impact had an effect on total crystallinity. Increased concentrations of NaOH pretreatment at 60°C produced improved yields for the majority of conditions tested as discussed above (Figure
Absorption ratios
Crystallinity analysis of 7% NaOH extreme conditions: (a) comparison of absorbance ratios; (b) comparison of total crystallinity index.
Reduction in total crystallinity was observed for all 7% NaOH pretreated sawdust exposed to 121°C or −20°C. It was noted that pretreatments at 121°C for 0.5 hr produced a 5% reduction in crystallinity which unlike analysis of absorbance ratios of
FTIR analysis of sawdust pretreated with 7% NaOH for 0.5 hr at 121 and −20°C for 24 hr showed peaks signifying bending in CH2 at C-6 in glucose shifted and was located at 1431 cm−1 associated with cellulose II instead of the cellulose I
The high yields of samples pretreated at 121°C are likely due to the highest level of hemicellulose and lignin reduction as well as the most significant conversion of cellulose I
Similarly, samples that had been exposed to −20°C showed increased yields due to the greater increase in amorphous cellulose, microfibril swelling, and the large reduction of xylans and galactans. The greater levels of amorphous cellulose would likely lead to reduction in crystallinity and likely larger internal areas [
Pretreatment at 60°C with 3–10% NaOH could be used as a pretreatment to increase ethanol yields of sawdust produced in Australian timber mills. It was observed that the increased concentration of NaOH leads to an increase of ethanol yield, with exposure time not being the major factor between 0.5 and 2 hr. The increase in yield with enzyme dosage is likely linked to the increase in amorphous regions in the cellulose structure, the increased presence of cellulose II, and removal of extractives and hemicellulose. These combined factors increase enzyme ability to hydrolyse cellulose and remove inhibitors.
It was observed that the moderate temperatures of 60°C and short exposure time alkali pretreatment yielded less ethanol than samples exposed to either −20°C or 121°C temperatures. This is likely due to the lower impact on crystallinity and cellulose state in conjunction with the lower levels of hemicellulose and lignin removal within these samples.
The most significant observed difference in the pretreatments at −20°C and 121°C was the greater conversion to cellulose II and an improved removal of ASL observed in samples pretreated at 121°C. Alternatively total reduction in crystallinity was greater in samples pretreated at −20°C, suggesting that although cellulose crystalline morphology had not been converted, the internal surface area of the cellulose microfibrils had likely been increased due to swelling and amorphous region production.
The use of combined Ca(OH)2 and NaOH or oxygenated alkali pretreatments may prove to be capable of pretreating timbers to a level of economic viability, but this requires further research. Alternatively, assessing the potential for reuse of the NaOH solutions used for pretreatment could potentially lead to an economically viable system, as NaOH recovery would account for a significant portion of the operational costs.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank Smartwood and Business Victoria for funding this study, Enzyme Solutions Australia for providing the enzymes, and Lallemand Biofuels & Distilled Spirits for providing yeast to undertake this research.