Scholarly article on topic 'Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation'

Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Adam Elliston, David R. Wilson, Nikolaus Wellner, Samuel R.A. Collins, Ian N. Roberts, et al.

Abstract This study evaluated steam (SE) explosion on the saccharification and simultaneous saccharification and fermentation (SSF) of waste copier paper. SE resulted in a colouration, a reduction in fibre thickness and increased water absorption. Changes in chemical composition were evident at severities greater than 4.24 resulting in a loss of xylose and the production of breakdown products known to inhibit fermentation (particularly formic acid and acetic acid). SE did not improve final yields of glucose or ethanol, and at severities 4.53 and 4.83 reduced yields probably due to the effect of breakdown products and fermentation inhibitors. However, at moderate severities of 3.6 and 3.9 there was an increase in initial rates of hydrolysis which may provide a basis for reducing processing times. Co-steam explosion of waste copier paper and wheat straw attenuated the production of breakdown products, and may also provide a basis for improving SSF of lignocellulose.

Academic research paper on topic "Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation"

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Bioresource Technology

journal homepage: www.elsevier.com/locate/biortech

BIORESOURCE TECHNOLOGY

Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation

Adam Ellistona, David R. Wilson a, Nikolaus Wellnera, Samuel R.A. Collins a, Ian N. Roberts b, Keith W. Waldron a'*

a The Biorefinery Centre, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom b The National Collection of Yeast Cultures, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom

HIGHLIGHTS

• Steam explosion of copier paper reduces xylose and produces inhibitors.

• Steam explosion at SF 3.6 and 3.9 increased initial rates of saccharification.

• Steam explosion at moderate severities may reduce processing times.

• Co-steam explosion of waste paper and wheat straw reduces inhibitor production.

ARTICLE INFO

ABSTRACT

Article history:

Received 16 January 2015

Received in revised form 19 March 2015

Accepted 20 March 2015

Available online 25 March 2015

Keywords:

Steam explosion

Enzyme saccharification

Fermentation

Cellulosic biomass

This study evaluated steam (SE) explosion on the saccharification and simultaneous saccharification and fermentation (SSF) of waste copier paper. SE resulted in a colouration, a reduction in fibre thickness and increased water absorption. Changes in chemical composition were evident at severities greater than 4.24 resulting in a loss of xylose and the production of breakdown products known to inhibit fermentation (particularly formic acid and acetic acid). SE did not improve final yields of glucose or ethanol, and at severities 4.53 and 4.83 reduced yields probably due to the effect of breakdown products and fermentation inhibitors. However, at moderate severities of 3.6 and 3.9 there was an increase in initial rates of hydrolysis which may provide a basis for reducing processing times. Co-steam explosion of waste copier paper and wheat straw attenuated the production of breakdown products, and may also provide a basis for improving SSF of lignocellulose.

© 2015 Published by Elsevier Ltd.

1. Introduction

Pre-treatments comprise an important part of the lignocellulosic bioalcohol process, opening up the cellulosic fibre structure through hydrolysis of hemicelluloses and the partial removal of lig-nin (Trajano et al., 2013). This increases enzyme accessibility and cellulose digestibility. Enzymatic digestion of lignocellulosic substrates is limited in the absence of any pre-treatment (Kumar et al., 2009). There is a large array of pre-treatments including: strong and weak acid and alkali, ammonia fibre expansion (AFEX), hot water and steam explosion, all with their own potential benefits and drawbacks. Acid (Sun and Cheng, 2005) and alkali pre-treat-ments (Hu and Wen, 2008) require relatively lower processing temperatures but had the drawback of requiring a further step to

* Corresponding author. Tel.: +44 (0)1603 255385; fax: +44 (0)1603 507723. E-mail address: keith.waldron@ifr.ac.uk (K.W. Waldron).

neutralise the acid/alkali before moving on the further steps as well as having to deal with high salt concentrations, removal and disposal. Similarly AFEX (Holtzapple et al., 1991) required additional steps as it utilises heated ammonia. Hot water treatments (Mosier et al., 2005) require higher temperatures (around 200 °C) but no additional chemicals. Steam explosion (Kokta et al., 1992) is similar to hot water pretreatment in that it uses only water but it uses high pressure steam to break down the biomass, but it can cause the formation of fermentation inhibitors as part of the process.

Waste (shredded) copier paper comprises approximately 50% (w/w) glucan (Elliston et al., 2013) and also forms a significant component of municipal solid waste (23% UK; (Defra, 2008)). It is difficult to recycle because of the reduced fibre quality (Confederation of Paper Industries, 2011) and therefore inevitably goes to landfill. Such waste is, however, a potentially good source of cellulose for second generation biorefining because the paper manufacturing process has effectively removed the lignin

http://dx.doi.org/10.1016/j.biortech.2015.03.089 0960-8524/® 2015 Published by Elsevier Ltd.

component and the bulk of the hemicellulose. Chemical pulping during paper manufacture utilises processes similar to those used as pre-treatments in second generation biofuel production (Biermann, 1993; Roberts, 1996), therefore leaving copier paper as a relatively pure cellulose substrate. As a result, waste copier paper can be readily saccharified using cellulase preparations (Elliston et al., 2013, 2014) and converted to bioethanol or other fermentation products (Elliston et al., 2013). Such exploitation could help to both reduce landfill use, the cost of which is rapidly increasing, and help meet demands for renewable energy sources (European Commission, 2010).

Recently, Zhao et al. (2015) demonstrated that the ease of sac-charification of cellulose from duckweed (a low lignin dicotyledonous aquatic plant) was significantly improved by steam explosion. Furthermore, Jacquet et al. (2011) demonstrated that pure cellulose could be significantly degraded by steam explosion. The aim of this study was to evaluate whether or not thermophysi-cal pretreatment could improve the biorefining of waste copier paper. This involved assessing the effect of pretreatment on the ease of enzymatic saccharification, and secondly on the nature and extent of the production of fermentation inhibitors particularly from the small but significant levels of non cellulosic sugars present in copier paper. The results of the study demonstrated that the component inorganic filler (calcium carbonate) had a significant attenuating impact on inhibitor generation. This was explored further as a potential means to mitigate formation of inhibitors during pretreatment of other lignocellulosic biomass.

2. Methods

2.1. Materials

2.3. Dry weights

Moisture content was measured using a Mettler LP-16 Infrared Dryer Balance (Mettler-Toledo Ltd, Leicester, UK).

2.4. FT-IR

A Bio-Rad 175 C FTS spectrophotometer (Bio-Rad Laboratories, Hemel Hempstead, UK) was used for experimentation, equipped with an MCT detector and Golden Gate single reflection diamond ATR sampling accessory. Samples were measured in triplicate over a range of 800-4000 cm-1 with a resolution of 4 cm-1. Air was used as a background and 64 scans were taken for each spectrum. Triplicate spectra were averaged and the final spectra area normalised but no other manipulation was carried out.

2.5. Enzyme digestion

Steam exploded samples were weighed out to give 0.5 g dry weight in 20 g total, therefore 2.5% (w/v) substrate concentration. 200 iL Accellerase® 1500 (Genencor, Rochester, N.Y., USA) (20 FPU/g of substrate) and 40 iL peta-Glucosidase (pG); Novozyme 188 (Novozymes Corp, Bagsvaerd, Denmark) (25 U/g of substrate) were added for digestion. Accellerase® 1500 was chosen on the basis of high enzymatic efficacy and industrial suitability. Digestions were carried out at 50 °C while shaking at 120 rpm on an orbital shaker in sodium acetate buffer (pH 5.0) with added thiomersal (0.01% w/v) to prevent microbial contamination. Further experimentation using low enzyme additions evolved the reduction of enzyme addition by a factor of ten (2 FPU/g of substrate Accellerase® 1500 and 2.5 U/g of substrate pG)

M-Real copier paper (The Premier Group, Birmingham, UK), Whatman No. 1 filter paper (Fisher Scientific UK Ltd, Loughborough, UK) and dust extracted wheat straw (Dixon Brothers, Norfolk, UK) were used as the substrates for these experiments, the paper substrates were shredded using a PS-67Cs (Fellowes, Doncaster, UK) cross shredder to 3.9 x 50 mm particle size (Din Security Level 3), the straw was supplied pre-shredded into lengths of approximately 40 mm. Previous work by the authors (Elliston et al., 2013) has shown composition of the paper to be as follows; 4.0% (w/w) moisture, 4.1% (w/w) starch, 46% (w/w) cellulose, 11.9% (w/w) hemicellulose, 1% (w/w) lignin and 33% (w/w) kaolin/calcium carbonate, therefore a total glucan composition of 50.1% (w/w), comparable to other literature analyses (Wang et al., 2012). Also (Merali et al., 2013) details the composition of the wheat straw; 7.9% (w/w) moisture, 37.1% (w/w) cellulose, 23.5 (w/w) hemicellulose, 0.9% (w/w) phenolic acids, 15.8% (w/w) lignin, 8% (w/w) ash.

2.2. Steam explosion

Steam explosion was carried out using pilot scale steam explosion apparatus, Supplementary Fig. S1 (Cambi, Asker, Norway), ali-quots of 250 g were exploded over a range of severity factors achieved by altering residence time and temperature. Severity factors were calculated from Eq. (1) (Overend et al., 1987). The variables t being residence time (minutes) and T temperature (°C) respectively.

{T(°Q-W0)

SF = log10 t(mrn)- expV 1475 !

The steam explosion apparatus was equilibrated to required temperature prior to the addition of material in order to reduce temperature fluctuation during actual explosion. Furthermore the apparatus was pressurised and exploded several times to ensure the removal of all material before severity factors were altered.

2.6. Simultaneous saccharification and fermentation

0.25 g dry weight of each sample was weighed into 20 mL glass bottles and brought to 8.8 mL with Yeast Nitrogen Base (Formedium, Hunstanton, UK) in 0.1 mol/L NaOAc buffer (5.0 pH). NCYC 2826 Saccharomyces cerevisiae (National Collection of Yeast Cultures, Norwich, UK) was chosen as the fermenting organism for these experiments due to its high ethanol tolerance (1520% v/v; (CECT, 2013)). 1 mL NCYC 2826 grown in YM media (Fisher Scientific UK Ltd, Loughborough, UK), with a cell count of 6.45 x 107cells/mL was added along with 75 iL Accellerase® 1500 and 25 iL pG, 20 FPU/g of substrate and 25 U/g of substrate respectively, giving a total volume of 10 mL liquid. A substrate blank was used to account for any residual fermentable sugars and produced ethanol transferred in the YM inoculum and enzyme addition. Bottles were incubated at 25 °C whilst being shaken at 120 rpm for 24, 48 or 120 h, then 2 mL samples are taken into gas tight screw cap tubes which were boiled to stop further fer-mentation/saccharification.

2.7. HPLC analyses

2.7.1. Analysis of carbohydrate by HPLC

Sugars present in the residual solid were analysed by HPLC using the Nation Renewable Energy Laboratory (NREL) procedure (NREL, 2011). Samples were filtered through AcroPrep™ 0.2 im GHP Membrane 96 Well Filter Plates (VWR International Ltd, Lutterworth, UK) in a centrifuge (Eppendorf, UK) at 500 rpm for 10 min into a 96 deep well collection plate (Starlab, Milton Keynes, UK). The plate was sealed and loaded directly onto a Series 200 LC instrument (Perkin Elmer, Seer Green, UK) equipped with a refractive index detector. The analyses were carried out using an Aminex HPX-87P carbohydrate analysis column (BioRad Laboratories Ltd, Hemel Hempstead, UK) with matching guard

columns operating at 65 °C with ultrapure water as mobile phase at a flow rate of 0.6 mL/min.

2.7.2. Dissolved carbohydrate by HPLC

Concentration of dissolved carbohydrates were directly analysed using the HPLC method described above proceeding from the filtration step.

2.7.3. Organic acids/inhibitors by HPLC

Levels of organic acids were analysed by HPLC using the Series 200 LC instrument equipped with both a refractive index detector and photodiode array detector reading at 210 nm. An Aminex HPX-87H organic acid analysis column (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) and matching guard operating at 65 °C with 0.005 mol/L H2SO4 as mobile phase at a flow rate of 0.6 mL/min.

3. Results and discussion

3.1. Effect of steam explosion on moisture content and recovery

Copier paper was steam exploded for between 10 and 45 min over a range of temperatures from 170 to 230 °C (severities from SF 3.06 to 5.48). At the highest severity tested (5.48) the moisture content increased (Table 1) to a level where the sample became a slurry. At lower severities there was no clear trend. Higher temperatures and residence times involve higher pressures and larger quantities of steam therefore imbuing the paper with more moisture. At high severities the samples were darker brown in colour; this is most likely due to the formation of organic acid and furfural products attributed to the caramelisation of the monomeric sugars (Joseph, 1989).

Steam explosion also resulted in a loss of mass (Table 1) which was generally greater at higher severities. The recovered weight accounted for 88-97% (w/w) of the starting material. The Cambi™ steam explosion system, at very high intensity, may cause some material to be blown from the vortex into the exhaust port, accounting for some losses. In addition, some loss will have been due to the production and evaporation of inhibitory products (see below). Despite the explainable difference in moisture contents and change in colour, inspection of the paper fibre revealed only a marginal difference in consistency.

3.2. Microscopic examination of steam exploded copier paper

Optical microscopy of steam exploded samples was conducted using an Olympus BX60 brightfield microscope (Olympus, Japan). The results (Fig. 1), show that at higher severities, the fibres become less clearly defined and thinner. There was also some change in the general dispersion of the fibres but there appeared to be no reduction in fibre length for any of the samples.

Table 1

Steam explosion moisture contents, pH and recovered weights.

Temp (°C)/time Severity pH Moisture Recovered weight

(min) factor content (% w/w) (g) (% w/w)

170/10 3.06 7.8 82.05 220 92

180/10 3.36 7.8 80.00 214 90

190/10 3.65 7.6 83.12 219 92

200/10 3.94 7.8 80.06 231 97

210/10 4.24 7.7 78.84 222 93

220/10 4.53 7.1 87.85 213 89

230/10 4.83 7.0 80.95 211 89

230/45 5.48 6.4 93.40 209 88

3.3. Chemical analysis of steam exploded material

Samples of steam exploded copier paper were freeze dried and analysed for sugar composition. The results are shown in Fig. 1a. Two phases of the impact of severity can be identified with a transition at SF 4.24. At severities of SF 4.24 and below, the carbohydrate composition of the material remained quite constant with glucose remaining between 73.9 and 76.3 mol% and xylose between 17.9 and 19.5 mol%. This was in keeping with the similarity in visual appearance of the fibres seen by optical microscopy (Supplementary Fig. S2) where little to no change in fibres were observed at except at severity 5.48 where there was a marginal shortening of fibre length and thickness as compared to other samples. At severities above 4.24, the levels of xylose declined by up to 75% at a severity of 5.48 reducing to 5.6 mol% while glucose increased to 89.6 mol% (Fig. 1a). This change accompanied, and may partly explain, the visual reduction in fibre thickness. The expected loss of xylose and other non cellulosic sugars was also accompanied by an increase in the release of fermentation inhibiting components particularly formic and acetic acids (Fig. 1b). Acetic acid is most likely to have originated from the acetyl-substi-tuted xylan hemicelluloses as they were hydrolysed. Formic acid will have been produced from the degradation of 2-FA (2-furfural aldehyde) and 5-HMF (5-hydroxymethyl furfural) created by the degradation of both pentose and hexose sugars respectively (Rasmussen et al., 2013; Meyer and Pedersen, 2010).

Similar phases have been detected in studies on steam explosion on pure microcrystalline cellulose (Jacquet et al., 2011). They showed that low severity causes some thermal degradation of the pure cellulose and at severities above 4.0 this becomes depolymerisation. In the present study, the results for copier paper show that not even the non cellulosic xylose component was degraded until severity was greater than 4.24. This is probably due to the buffering effect of the calcium carbonate in the paper and reducing the effective severity of the steam explosion (Meyer and Pedersen, 2010). This is corroborated by the observation that the pH only begins to appreciably reduce above SF 4.24 (Table 1).

3.4. FT-IR analyses

FT-IR spectra were taken of the pretreated copier paper in order to provide further information on the chemical changes occurring during steam explosion. Severity had only a minor effect on the spectra with a majority of the peak heights remaining static as severity increased (results not shown). Peaks in the region 9861052 cm-1 showed the most turbulence over the change in severity, this is probably due to the formation of degradation products which disrupt the cellulose and hemicellulose peak contours.

Following the methods of Wistara et al. (1999), the spectra were used to evaluate changes in the crystallinity of cellulose and the influence of accompanying hemicellulose. The calculation of the crystallinity index was based on the relative intensities of infrared bands: Index A1433/897 cm-1 and Index B 1372/2900 cm-1. These indices are shown in Table 2. Whilst index B did not show much change as a result of the pretreatments, Index A showed significant increases at the higher severities. This is consistent with the removal of xylose from the copier paper, enabling any amorphous cellulose chains to associate more easily and thus become more crystalline (Wan et al., 2010). FTIR signature for calcium carbonate showed little change as a result of pretreatment (results not shown).

3.5. Effect of pretreatment on enzyme digestion

Enzyme hydrolysis of untreated and steam exploded copier paper was performed for up to 220 h at 2.5% (w/v) substrate levels

(a) 100%

80% 70% 60% 50% 40% 30% 20% 10% 0%

I Formic ■ Acetic 5-HMF 2-FA

Fig. 1. a) Sugar composition of the steam exploded residues in mol %; b) Inhibitors from steam exploded residues, mg of inhibitor per g of recovered solids.

and either 2 or 20 FPU/g cellulase (Accellerase 1500) and supplementary BG. Hydrolysis was complete after 48 h at which point the yields reached a plateau. With cellulase at 20 FPU/g, copier paper alone hydrolysed to give a 89.9% (of theoretical) yield of glucose (Fig. 2). Theoretical maximum glucose is calculated from the cellulose content of the material including a factor of 1.111 to take into account the water of hydrolysis as per Eq. (2), the yield is then calculated as a percentage of this.

Theoretical glucose maximum (mg) = mass of sample (mg)x percentage cellulose (%) x 1.111

This is similar to that reported by Elliston et al. (2014). Steam explosion resulted in no improvement to the final glucose yield (Fig. 2). Furthermore, at severities of 4.53 and 5.83 the yield was reduced to about 70% theoretical maximum. With cellulase at 2 FPU/g, the final yield of glucose for copier paper was about 19% theoretical maximum (in keeping with Elliston et al., 2014). However, steam explosion of the paper had a significant impact on the final yield which was increased to about 25% for severities of 3.65, 3.94 and 4.24, and was accompanied by an increase in the initial hydrolysis rates (Fig. 2). However, severities of 4.53 and 4.83 resulted in lower yields.

The mechanism of cellulase action is an area of considerable research and limitations to cellulase performance include

Table 2

Summary of normalised FT-IR data.

severity factor Wavenumber

Index A (1433 cm-1/897cm-1) Index B (1372 cm-'/2900cm-')

3.06 12.9 5.8

3.36 12.2 5.2

3.65 11.6 4.6

3.94 11.4 4.6

4.24 14.6 5.6

4.53 16.3 4.4

4.83 18.4 4.8

5.48 17.9 5.3

enzyme deactivation, the involvement of two-phase substrates (amorphous vs. crystalline cellulose), substrate reactivity (digestibility) and accessibility, and the non-uniformity of mixed reaction species resulting in fractal kinetics (Bansal et al., 2009).

Copier paper contains virtually no lignin or hemicelluloses as a result of the extensive processing during pulp and paper manufacture. However it does contain significant quantities of hemicellulosic moieties (Table 1). It is likely that the removal of xylose-containing hemicelluloses by the higher severity treatments is responsible for facilitating a high initial rate of hydrolysis at the lower enzyme concentration. The observation that at severities of 4.5-5.5 the yields of glucose are reduced is of significance not only to waste paper exploitation but to the pretreatment of lignocellulose generally. It is likely that the creation of saccharide/furfural complexes from the small quantities of residual xylans at severities that are not sufficient to facilitate their solubilisation into the surrounding liquor results in the creation of obstacles and blockage points along the microfibrils. This would enhance the fractal nature of the substrate, causing the cellulases to jam up and prevent them from completing their hydrolysis. At the highest severity (230 °C 10 min) such

100% -

components are likely to have been hydrolysed from the cellulose, thus facilitating enzymolysis.

3.6. Simultaneous saccharification and fermentation

Steam-exploded samples were subjected to simultaneous saccharification and fermentation (SSF) over a period of 48 h. Fig. 3 shows the percentage yields of ethanol and unfermented free glucose as a function of cellulosic material. The segregation into two severity-related phases can again be observed. At SF 4.24 the yield is generally similar to that of untreated copier paper. At SF 4.535.5 the yield is much reduced, consistent with the accompanying increase in fermentation inhibitors (Pienkos and Zhang, 2009). Several studies into the levels of inhibitors that will start to cause inhibitory effects in the yeast have been conducted (Ando et al., 1986; Clark and Mackie, 1984; Maiorella et al., 1983; Palmqvist et al., 1999; Phowchinda et al., 1995). This literature shows that the levels of these compounds present in the steam exploded samples are reaching concentrations that would be expected to cause inhibition during fermentation (Fig. 1b) furfural, 79% inhibition at 4 mg/mL (Palmqvist et al., 1999); 5HMF, 50% inhibition at 8 mg/ mL (Clark and Mackie, 1984); acetic acid, 74% inhibition at 6 mg/ mL (Phowchinda et al., 1995); formic acid, 80% inhibition at 2.7 mg/mL (Maiorella et al., 1983) were reported. None of the pre-treated samples achieve a greater yield than that of untreated paper (93% w/w). Hence in contrast to saccharification alone, pre-treatment of waste copier paper does not provide any clear advantage in relation to SSF performance.

3.7. Waste paper as an inhibitor-reducing agent

Wheat straw is one of the main substrates of interest to the second generation bioalcohols industry, but it is well known for its production of inhibitors when subjected to steam explosion pre- 100%

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о со

20% Ф £

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Fig. 2. Ten day enzyme digestion - % digestion based on total carbohydrate available. Initial rates of hydrolysis (after 30 minutes) - % digestion per hour. Low Enzyme Concentration 2 FPU/g substrate; High Enzyme concentration 20 FPU/g substrate. Theoretical digestion (%) measured on left hand axis, initial rates (%/hour) measured on right hand axis.

1-* M 00 o M U3 o NJ NJ N» Ni O N> U> O NJ

o O O O

p p p n p O p P

I-» o i-» o I-» o I-» o I-» o I-» O I-» o m

3 3 3 3 3 3 3 3

p p p p' p' p' p' p"

ia ~n ia n ia -n ia Tt ia -n </> -n ~n Tl

LU LU LU LU ■U ■fc» «k m

o m UJ CT> en U1 ÏD -Ê» N> -Ê» In W 00 U> 45» CO

Fig. 3. SSF % yields based on cellulose from steam exploded CP at a range of severities.

treatment (Bellido et al., 2011; Horn et al., 2011; Tomas-Pejo et al., 2008). Detoxification of pre-treated liquors to remove inhibitory compounds has been considered (Cantarella et al., 2004), one such methodology, over liming, uses compounds, such as calcium carbonate, to neutralise any undesired inhibitory components. As calcium carbonate is present in paper, we hypothesised that a combination of wheat straw and paper might yield smaller quantities of inhibitory breakdown products when compared to wheat straw alone when steam exploded.

Samples of wheat straw were therefore mixed with either calcium carbonate at a ratio of 3:1 or with copier paper in a 1:1 ratio to give comparable amounts of calcium carbonate in the mixtures. Steam explosion was conducted at a high severity (4.83; 230 °C 10 min) which would be expected to result in the production of inhibitory compounds. In addition samples of Whatman No. 1 filter paper were subjected to the same treatment with additions of calcium carbonate and copier paper to investigate and compare the effect on a "pure" cellulose substrate. The results (Fig. 4) show that the addition of calcium carbonate in both cases considerably reduces inhibitor concentrations in the final liquor, the maximum being a 94% (w/w) reduction in the case of the 5-Hydroxymethlyfurfural (5HMF) in filter paper, even taking into account the dilution effect of adding paper and filter paper together 1:1. Calcium carbonate appears to offer more protection against the formation of 5HMF and acetic acid, suggesting that cellulose (whose break down products these constitute) is afforded greater protection than hemicellulose, this is probably because hemicellulose is degraded more readily by thermal pretreatment whereas cellulose requires a greater reduction in pH to depoly-merise. The addition of waste copier paper also has a positive effect on the reduction in acetic acid (61% w/w reduction), formic acid (51% w/w reduction) and 2FA (2-furaldehyde) (59%) with wheat straw substrate. In the case of filter paper there was an increase in 2FA but this would be due to the addition of hemicellulose in the copier paper (CP). This incidence again can be explained by the inclusion of additional hemicellulose in the paper giving rise to its breakdown products of 2FA and formic acid (Meyer and Pedersen, 2010).

These results highlight the possibility of using one waste substrate (paper) in synergy with others (e.g., wheat straw) in order to produce peak ethanol yield for a material that would otherwise potentially produce too many inhibitory products for optimal fermentation.

3.8. SSF on mixed substrate pretreated samples

To establish the benefit of reducing fermentation inhibitors by using a mixed substrate a set of SSF experiments were carried out on straw, straw and paper, and straw and CaCO3. The samples were pretreated at 230 °C for 10 min as in Fig. 4. The SSFs were carried out at 25 °C for 24 h and 72 h, the shorter incubation period was selected in order to highlight the difference between the sample's levels of inhibitors, as 2FA and 5HMF have an effect of increasing the lag time of the yeast growth and therefore differences between samples would be most stark after a shorter incubation. Furthermore a short incubation period is also important industrially allowing for a higher throughput. The differences in levels of ethanol and glucose in the three samples at 24 and 72 h time points based on the theoretical maximum yield that could be achieved from the quantity of cellulose within the samples was calculated (Supplementary Fig. S3). The theoretical concentration of ethanol achievable can be calculated as in Eq. (3), the factor of 1.111 takes into account the water of hydrolysis and 51.1% of glucose is converted to ethanol during fermentation (with the remainder transformed to carbon dioxide). It should be noted that this does not take into account non-glucose carbohydrates which cannot be metabolised by the chosen yeast strain.

Theortical ethanol maximum (mg) = Mass of sample (mg)x percentage cellulose (% w/w)

x 0.511 x 1.111 (3)

After 24 h incubation, pretreated straw alone had a yield of only 3%, pretreated straw and paper 34% and pretreated straw and CaCO3 50%. This shows that there is a marked effect of the addition of CaCO3 either as a pure substance or indeed within the matrix of

■ Formic Acetic ■ 5HMF ■ 2FA

Fig. 4. Steam exploded filter paper (FP) and straw inhibitors at SF 4.83.

the waste paper. It was seen that after 72 h incubation there is much less difference in the yield of ethanol from the three samples (straw, 74%; straw + CP, 67% and straw + CaCO3, 80%), this suggested that the inhibitors are metabolised and the fermentation is able to continue, once the lag phase has been accounted for. However it is likely that the incorporation of pure or waste paper-associated CaCO3 will reduce the time taken for SSF to reach completion. Whilst the addition of pure CaCO3 directly has a larger effect it should be noted that the paper is a waste resource and would therefore be a useful addition to a system where there are large levels of inhibitors produced such as in straw.

4. Conclusion

Steam explosion (SE) at severities greater than 4.24 resulted in a loss of xylose and the production of breakdown products (particularly formic acid and acetic acid). SE did not improve final yields of glucose or ethanol and at severities 4.53 and 4.83 reduced yields probably due to the production of inhibitors. However, moderate severities of 3.6 and 3.9 increased initial rates of hydrolysis which may provide a basis for reducing processing times. Co-steam explosion of waste copier paper and wheat straw attenuated the production of breakdown products, and may also provide a basis for improving SSF of lignocellulose.

Acknowledgements

The Authors gratefully acknowledge the support of the UK Biotechnology and Biological Sciences Research Council in funding this work through a CASE studentship to AE and the Institute Strategic Programme 'Food and Health' (grant number BB/ J004545/1). They would also like to thank Miss Marie Julie Soucouri (visiting student from ESIROI, Université de la Réunion, Réunion, France) for her assistance with HPLC analyses of fermentation products.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.03. 089.

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