Sulfite post-treatment to simultaneously detoxify and improve the

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Sulfite post-treatment to simultaneously detoxify and improve the enzymatic hydrolysis and fermentation of a steam pretreated softwood lodgepole pine whole slurry Na Zhong, Richard P Chandra, and Jack (John) N Saddler ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06092 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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ACS Sustainable Chemistry & Engineering

Sulfite post-treatment to simultaneously detoxify and improve the enzymatic hydrolysis and fermentation of a steam pretreated softwood lodgepole pine whole slurry Na Zhong†, Richard Chandra† and Jack (John) N. Saddler† †

Forest Products Biotechnology and Bioenergy Group, Department of Wood Science, Faculty of

Forestry, The University of British Columbia, 2424 Main Mall, Vancouver BC, Canada. Corresponding author: Jack (John) N. Saddler

Email address: [email protected]

ABSTRACT Previous work has shown that sulfonation post-treatment employed at a temperature of 130-140 °C modifies the lignin in steam pre-treated softwood to improve the ease of hydrolysis of the resulting “post-treated” substrate. Sulfite has also been shown to detoxify water soluble fractions originating from the steam pretreatment of softwood to enhance fermentation. Consequently, lignin modification and detoxification could be combined in a single process step to simultaneously improve the ease of hydrolysis and fermentability of a high solids (25% w/v) substrate which contains the combined 5% (w/v) water soluble and 20% (w/v) water insoluble fractions (“high solids whole slurry”), originating from the steam pretreatment of softwood biomass. Unlike previous work on sulfite post-treatment applied to the water insoluble fractions of steam pretreated softwood, the sulfite treatment of the steam pretreated softwood high solids whole slurry was effective when performed at a reduced temperature (70 °C) when sodium carbonate was added as an alkali source. The alkaline sulfite

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treatment increased the enzymatic hydrolysis yield of the high solids whole slurry from 55% to 67% while simultaneously improving fermentability, resulting in an ethanol concentration of 56.4 g/L. Key words: Acid steam pretreatment, Sulfite post-treatment, Whole slurry, High solid loading hydrolysis, Fermentation, Detoxification

INTRODUCTION Conventional bioethanol production from sugar/starch feedstocks are mature industries where a high gravity fermentation approach is routinely used to obtain high ethanol concentrations of 10-15% (v/v) and yields of >90%, within a relatively short period of time (6-10 hours). 1 However, ethanol produced from woody lignocellulosic biomass is still largely at the research, development and demonstration stage of deployment. This is mostly a result of the high recalcitrance of woody biomass towards the pretreatment and enzymatic hydrolysis stages of biochemical conversion and the relative toxicity of the biomass derived sugar stream which compromises downstream fermentation. 1

Steam pretreatment has been widely utilized to overcome the recalcitrance of lignocellulosic biomass due to its simplicity, and its ability to liberate hemicellulose derived sugars into a water-soluble stream, while retaining the cellulose in the water insoluble fraction. However, for the biochemical conversion of woody biomass to obtain comparable sugar concentrations to first generation starch based ethanol, the water-soluble hemicellulose derived sugars solubilized during steam pretreatment must be combined with the water insoluble cellulose enriched fraction and treated as a high solids “whole slurry”. Despite the advantages of processing a high solids slurry, when processing woody biomass such as softwoods, there are several challenges that need to be overcome to achieve effective enzymatic hydrolysis and fermentation. First, due to the solubilization of hemicellulose, the lignin content of steam pretreated softwood typically approaches approximately 50% of the overall substrate composition. As well as being enriched in the water insoluble fraction, much of the biomass lignin 2 ACS Paragon Plus Environment

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typically undergoes condensation during acid catalyzed steam pretreatment.

2

The high residual

condensed lignin has been shown to inhibit enzymatic hydrolysis by physically impeding enzyme accessibility to cellulose and non-productively binding cellulases. This has been shown to be amplified when the biomass lignin is more condensed. 3 As well as the high recalcitrance of softwood towards enzymatic hydrolysis, water-soluble compounds such as acetic acid, furan aldehyde and phenolic compounds originating from the steam pretreatment of softwood are also inhibitory to fermentation. 4

Previous work has assessed the use of “post-treatments” such as sulfonation which could modify the lignin to improve its hydrophilicity. An increased hydrophilicity was shown to increase substrate swelling while decreasing the tendency of the lignin to non-productively bind with cellulases. For example, treating steam pretreated softwood using 16% sulfite (loaded based on solid pretreated biomass) at a temperature of 160 ℃, and a residence time of 80 mins, Kumar et al. achieved the complete hydrolysis of the cellulosic fraction at an enzyme loading of 15 FPU/g cellulose, despite the high lignin content (44%) of the substrate 5. However, it should be noted that the aforementioned post-treatment was applied to a well-washed steam pretreated softwood substrate that was washed again after the post-treatment. This removed the high concentrations of excess sulfite that could potentially inhibit the function of cellulases and yeasts. As well as modifying lignin to improve enzymatic hydrolysis of steam pretreated softwoods substrates, sulfite has also been shown to detoxify inhibitors that limit the fermentation of the sugars present in the water-soluble fractions originating from the steam pretreatment of softwood.

6,7,8

Although the underlying mechanisms

responsible for the detoxification are not fully understood, it is likely that reduction reactions predominate, as dithionite and reducing agents such as sodium borohydride have also been shown to detoxify the water-soluble streams from pretreated softwood. 6,7



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Although the addition of sulfite improves the enzymatic hydrolysis of the water insoluble fraction and the fermentability of the water insoluble fractions of steam pretreated softwoods, these separate approaches have yet to be combined into a single sulfite treatment. A one-step approach could potentially, simultaneously, improve the hydrolysis and fermentation of the whole slurry (water insoluble and water-soluble fractions) from steam pretreated softwood. However, the conditions that have been employed for sulfite treatment to improve fermentation vs enzymatic hydrolysis are quite different. The detoxification of the water-soluble streams has typically been achieved using low concentrations of sulfite (5-10 mM) at low temperatures while treatments to improve enzymatic hydrolysis of the water insoluble fractions have typically been applied at higher temperatures (>140 °C) and sulfite loadings (16 % based on the steam pretreated dry biomass). 5,7,9 Since the wholeslurry sulfite treatment would not involve washing of the substrate after the initial sulfite treatment, the presence of excess sulfite that is required to modify the substrate could also be inhibitory towards subsequent enzymatic hydrolysis and fermentation. 10,11 Therefore, in this study we assessed whether it was possible to improve both the hydrolysis and fermentation of a steam pretreated softwood whole slurry using a single step sulfite treatment. The work hoped to find “compromise” conditions where both the enhancement of hydrolysis of the water insoluble and good fermentation of all of the wood derived sugars, by decreasing inhibition, could be achieved.

MATERIAL AND METHODS Biomass The dissolving pulp (DsP) substrate (with 94% of cellulose) used in this research was kindly supplied by Neucel. Inc. Mountain beetle killed lodgepole pine wood chips were kindly provided by Canfor. Inc, with the moisture content of 7%. Pretreatment


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The lodgepole pine wood chips were screened to retain chips between 6 and 38 mm “Prior to pretreatment 8% SO2 (wt/wt of the dry substrate) was added to the wood chips, 200 grams (oven dry basis), for around 12 hours at room temperature. The amount of SO2 adsorbed was determined by weighing the total substrates weight before and after the addition of the sulfur dioxide gas. This resulted in the retention of 4% SO2 on the biomass.” Steam pretreatment was conducted in a 2 L StakeTech II batch steam gun (constructed by Stake Tech-Norvall, Ontario, Canada) at 200 °C for 5 mins. The acid steam pretreated lodgepole pine contained 48% glucan, 45% lignin, 3.7% mannan, 1.3% xylan, 0.1% arabinan, 0% galactan. Post-treatment Post-treatments were conducted in different equipment according to the temperatures required (160 °C in Parr high pressure batch reactor, 121 °C in the autoclave and 70 °C in a water bath) with the addition of the desired loading of Na2SO3 or Na2SO3 + Na2CO3 based on the dry weight of the substrate at the specified reaction time. “The post-treatments that were conducted in the autoclave at 121 °C, were performed at a 25% solid loading in a beaker sealed with aluminum foil.” For the washed substrates, after the reaction, the slurry was filtered using a Buchner funnel and the water insoluble fraction was washed extensively with water (using approximately 5 times the volume of water compared to the original reaction volume). Lower temperatures sulfite-treatment was conducted in a water bath at a 25% solid loading at 70 °C at the specified reaction time. For the hydrogen peroxide treatment, prior to the addition of enzymes and yeast to the hybrid hydrolysis and fermentation experiment, a 30% hydrogen peroxide solution was added in volumes ranging from (0.05-0.17 mL) per 10 mL of the sulfite post-treated whole slurry (25% solids loading).



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Hydrolysis The enzymatic hydrolysis was conducted in duplicate. Commercial Cellic Ctec3 preparation was used in this paper and it was obtained from Novozymes (Franklinton, NC). The Cellic Ctec3 is the stateof-the-art enzyme cocktail from Novozymes. It likely contains additional “cellulase components”, such as advanced AA9, improved β-glucosidases and various hemicellulases. 12 The enzyme activity of the Cellic Ctec3 was 71.7 FPU/mL using Whatman No. 1 filter paper as a substrate and the protein concentration was 277 mg/mL. The commercial Cellic Ctec3 enzyme preparation has been shown to contain a certain amount of electron donor for LPMO activity, 13 so we did not supply any additional electron donors to the hydrolysis experiments. The hydrolysis of dissolving pulp and the steam pretreated lodgepole pine substrate were conducted at 50 °C and pH 4.8 in a rotary shaker at 150 rpm. The high solid loading enzymatic hydrolysis was performed at 25% (w/v) dry matter, which contains 5% (w/v) water soluble and 20% (w/v) water insoluble fraction. After 2-3 days, hydrolysis was ceased and the liquid fractions obtained by centrifugation of the whole slurry were used for downstream fermentation. Fermentation The fermentation trials were carried out in 30 mL septa bottles with butyl-PFTE seals, at a working volume of 10 mL or 20 mL. The Saccharomyces cerevisiae T2 strain was provided by Tembec Limited (Temiscaming, Quebec, Canada). The strain had been previously adapted to grow on spent sulfite liquor and had been traditionally used by the sulfite mill to ferment the softwood derived, hemicellulose hydrolysate.14 Both separate hydrolysis and fermentation (SHF) as well as Hybrid hydrolysis and fermentation (HHF) configurations where performed in this work. SHF refers to the separation of the water soluble and water insoluble fraction after the enzymatic hydrolysis with the liquid fraction subsequently utilized for the downstream fermentation process. “Hybrid hydrolysis and fermentation (HHF) refers to the process where the substrate is “prehydrolyzed” at 50 °C using cellulases after which the temperature is decreased to 30 °C and the pH is adjusted to neutral with


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subsequent addition of the yeast to allow fermentation to proceed. This approach differs from simultaneous saccharification and fermentation where both the yeast and enzymes are added at the beginning of the reaction to allow for the simultaneous hydrolysis of the cellulose and utilization of the liberated sugars by the yeast.” Prior to fermentation, the pH of the lodgepole pine hydrolysate or dissolving pulp hydrolysate was adjusted to 5.5 with NH4OH. The reaction bottles loaded with cultures were incubated in an orbital shaker at 30 °C and 150 rpm. During the course of fermentation, 400 μL samples were taken at different sample hours. The samples were centrifuged at 5000 rpm for 5 mins and the supernatant was stored at −80 °C prior to further analysis. Ethanol yield were expressed as a percentage of the maximum theoretical yield obtained. The calculation was based on the total amount original fermentable sugars (including glucose and mannose) present in the liquor. By using a maximum stoichiometric ethanol yield of 0.51 g per gram of sugar, the percentage of ethanol yield was calculated as:

（1） where PEtOH is the total amount of ethanol formed in the fermentation and S0 is the initial amount of C6 sugars present in the original liquor. Analytical methods “Sugars were measured using a Dionex (DX-500, Dionex Corp., Sunnyvale, CA, USA) HPLC (ICS3000) equipped with an ion exchange CarboPac PA-1 column (4×250 mm) equilibrated with 1 M NaOH and eluted with deionized water at a flow rate of 1 mL/min (Dionex Corp.). An ED 40 electrochemical detector (gold electrode), an AD 20 absorbance detector and an auto sampler (Chromatographic Specialties, Brockville, Canada) were also used. Sodium hydroxide (0.2 M) was added post-column (for detection) at a flow rate of 0.6 mL/min. Prior to injection, samples were



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filtered through 0.45 μm HV filters (Millipore, MA, U.S.) and a volume of 20 μL sample was typically loaded.” Ethanol was determined using gas chromatography and a Hewlett Packard 5890 GC equipped with a HP-Innowax column (15mx0.53mm). Helium was used as the carrier gas at a flow rate of 20 mL/min. The temperatures of the injection unit and flame ionization detector (FID) were set at 175 and 250 °C respectively. The oven was heated to 45 °C for 2.5 mins and the temperature was raised to 110 °C at a rate of 20 °C/min and later held at 110 °C for 2 mins. Standards were prepared using ethanol (Sigma) and butanol (0.5 g/L) (Fisher) was used as an internal standard.

The sugar degradation products, furfural and 5-hydroxymethyl furfural (HMF) as well as acetic acid were analyzed using a HPLC (ICS-500) with Aminex HPX-87H column (Bio-Rad, Hercules, CA). The HPLC was fitted with an AS3500 auto sampler, a UV detector at a wavelength of 280 nm and a GP40 gradient pump (NREL, 2008). Standard concentration of HMF (Sigma) ranged from 0.1 - 4.0 g/L, while the concentration of furfural ranged from 0.1 - 2.0 g/L. All of the standards and samples were filtered through a 0.45 μm syringe filter (Chromatographic Specialties, Brockville, Canada). 5 mM H2SO4 was used as an eluent at a flow rate of 0.6 mL/min.

The concentration of total phenolics in the pretreated substrates were quantified using Folin– Ciocalteu reagent (Sigma), as proposed by Singleton and Rossi (1965). A 100 μL aliquot of the diluted sample was first mixed with 250 μL of the Folin–Ciocalteu reagent. After 5 mins, the reaction was stopped by adding 750 μL of 20% (w/v) Na2CO3 and the total volume was brought up to 5 mL using de-ionized purified water. The flasks were incubated for 2 hours at 22 °C with constant stirring on a magnetic stir plate. The absorbance of each reaction was measured spectrophotometrically at 760 nm. Reaction blanks with de-ionized purified water were also run in parallel. Calibration was done using


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vanillin as the standard. The reactions were performed in duplicate for each sample and the average values were reported.

Bulk acid groups in the pretreated and post-treated substrates were determined by conductometric titration according to standard methods. In brief, wet substrates containing 1 g dry matter were added to 300 mL of 0.1 N HCl and stirred for 1 hour. The pulp was then filtered and washed with 2000 mL of deionized water. The washed pulp was then treated with 0.001 M NaCl (250 mL) and 0.1 N HCl solutions (1.5 mL), stirred and conductometrically titrated with 0.05 N NaOH. The total acid groups were extrapolated from plots of the titration data (volume of NaOH vs. conductivity). Initially, conductometric titration curves indicate a rapid decrease in conductivity which represents the neutralization of strong acid groups. The first equivalence point (intersection of the graph) represents weaker carboxylic acids beginning to dissociate, and the second equivalence point (intersection) represents increases in conductivity due to excess NaOH.

RESULTS AND DISCUSSION The main goals of this work were to assess the ease of hydrolysis and fermentation of high solids (>25% w/v) whole slurries of steam pretreated softwood. “Initially, the enzymatic hydrolysis was performed at 25% solids concentration (5% (w/v) water soluble and 20% (w/v) water insoluble solids), and the fermentation was carried out after hydrolysis in the same flask.” We anticipated that the high lignin content of the steam pretreated softwood water insoluble substrate (solid fraction from steam pretreatment) might inhibit enzymatic hydrolysis, 15,16 while the inhibitors present in the hexose rich softwood derived water soluble fraction (liquid fraction from steam pretreatment) might inhibit fermentation.

17

Therefore, the initial work evaluated the challenges associated with the hydrolysis

and fermentation of the water insoluble, water soluble fractions and whole slurries from steam pretreated softwood while assessing the potential of sulfite treatments to overcome these challenges. 9 ACS Paragon Plus Environment


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As discussed earlier, effective enzymatic hydrolysis at high solids loadings is necessary in order to obtain the high sugar concentrations that are required for effective fermentation. However, increasing solids loadings typically requires higher enzyme loadings due to various factors such as, inefficient mass transfer, increased end-product inhibition and unproductive enzyme binding to lignin.

18

To

establish a baseline, the required enzyme loading for high solids loading (25%) steam pretreated softwood whole slurries was assessed using dissolving pulp as a control. This allowed us to assess the likely influence of lignin on the ease of hydrolysis of the steam pretreated softwood at high solids loadings. A range of enzyme loadings were used with the aim of achieving 70% cellulose hydrolysis after 48 hrs (Figure. 1). No more than 70% hydrolysis of the DsP substrate could be obtained over the range of enzyme loadings assessed in this study. In contrast, the steam pretreated substrate reached a hydrolysis yield of approximately 80%, but required an enzyme loading of 90 mg /g of glucan (Figure 1 A). It is likely that at these high substrate concentrations end product inhibition is a factor as no more than about 65% of the DsP substrate, which contained >94% cellulose, could be hydrolyzed at a 25% substrate concentration. It is worth noting that, although 80% of steam pretreated substrate (SPLP) could be hydrolyzed, albeit at a protein loading of 90 mg/g glucan, the resulting glucose concentration was only 137 g/L (Figure 1 B). It was also apparent, that the hydrolysis yield of the SPLP reached a maximum of close to 80% regardless of the increases in enzyme loading. This was similar to the results observed by Nakagame et al. where the lignin content of the steam pretreated softwood appeared to limit the accessibility of the cellulose regardless of increases in enzyme loading 19

. The result with the SPLP was quite different from that obtained with DsP where a sugar

concentration 167 g/L was obtained at a conversion of only 66% (Figure 1 A and B). This was likely due to the higher lignin content and lower cellulose content of the SPLP substrate.


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Figure 1 Enzymatic hydrolysis of dissolving pulp (DsP) and whole-slurry steam pretreated lodgepole pine (SPLP) with 25% (w/v) solid loading at different Cellic Ctec3 enzyme loadings (15-90 mg protein per g cellulose) for 48 hrs. (A) Percentage of cellulose to glucose concentration (g/L). (B) Final glucose concentration (g/L)

Hydrolysis and fermentation of the high solids SPLP whole slurry was assessed using an enzyme loading of 35 mg/g cellulose, 25% solids, in a HHF process configuration using a “pre” enzymatic hydrolysis of the substrate with subsequent commencement of the fermentation while the hydrolysis was allowed to continue. Interestingly, when the solid substrate was included in the hydrolysate (whole slurry hydrolysate) at the 25% solids loading, the sugars in the whole slurry hydrolysate could hardly be fermented (Figure 2). However, the water soluble fraction of the whole slurry hydrolysates which contained most of the water soluble inhibitors were readily fermented (Figure 2). The main difference between these two types of fermentation media (whole slurry hydrolysate vs. the liquid fraction of whole slurry hydrolysate) was that the whole slurry hydrolysate contained both the water soluble and water insoluble fractions, while the liquid stream of the whole slurry hydrolysate only contained the water soluble fraction. It was evident that the water insoluble fraction also contained some inhibitory material that was limiting fermentation of the wood derived sugars. 11 ACS Paragon Plus Environment


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Figure 2 The fermentation profile of strain T2 (with an Optical Density of 13) grown on steam pretreated lodgepole pine whole slurry hydrolysate with/without the removal of solid hydrolysis residues.

The higher solids loading at 25% presents several obstacles for both the fermentation and hydrolysis steps, including higher initial sugar concentrations, higher concentrations of residual recalcitrant solids and an increased concentration of fermentation and enzyme inhibitors. The use of a softwood substrate is also far more challenging due to their higher lignin content which approaches 50% after acid catalyzed steam pretreatment. 19 Recent work has shown that the simultaneous saccharification and fermentation (SSF) of corn stover at a solids loading of 15% using a pretreated whole slurry was feasible. 20 However, the liquid fraction from the steam pretreated corn stover contained only 2.7 g/L acetic acid, 0.7 g/L furfural and 1.0 g/L HMF, while the liquid fraction of the steam pretreated lodgepole pine used in this study contained 9.2 g/L acetic acid, 2.8 g/L furfural, 3.0 g/L HMF and 4.9 g/L phenolic compounds. Therefore, at these higher solids loadings, the inhibitory nature of the solid material towards fermentation, substrate


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recalcitrance and the higher amount of known fermentation inhibitors likely compromised the ability to ferment the whole slurry originating from the steam pretreated softwood. Thus, our subsequent work next assessed the use of sulfite treatments to simultaneously improve the hydrolysis and fermentation of high concentrations of steam pretreated softwood whole slurries.

Previous work had reported that sulfite treatment at temperatures of 160 °C could enhance the sulfonation of lignin, consequently improving the enzymatic hydrolysis of steam and mechanically treated softwood substrates.

5,21

The improvements in hydrolysis were attributed to an increase in

substrate swelling, which enhanced cellulose accessibility while also decreasing the non-productive binding of cellulase enzymes to lignin. 22 However, when temperatures as high as 160 °C are applied to a steam pretreated whole slurry, the solubilized hemicellulose derived sugars present in the watersoluble fraction can undergo degradation during the sulfonation reaction. Initially the sulfonation reaction was applied to the washed steam pretreated substrate to confirm earlier work

2,3,5

. Using a

10% (w/v) solid loading, 160 °C treatment for 70 mins with subsequent washing to remove excess sulfite, it was apparent that both an 8% (w/w) and 16% (w/w) sulfite loading (based on dry biomass) could boost the hydrolysis yield of the washed substrates from 62% (unsulfonated) to 88% (sulfonated) (Figure 3 B). When the extent of sulfonation was assessed by measuring the strong acid group content, after sulfonation at 160 °C for 70 mins, it was also apparent that the strong acid groups on the washed substrates were increased from 11 to 92 u mol / g substrates.

Even though it was clear that sulfonation of the washed substrates enhanced hydrolysis, sulfite posttreatment of the steam pretreated whole slurry did not appear to improve enzymatic hydrolysis compared to the control sample. It was likely that, although the higher temperature enhanced the sulfonation of lignin, the residual sulfite in the whole slurry system inhibited cellulase enzymes, offsetting the beneficial effects of sulfonation (Figure 3 A). Therefore, the effects of sulfite treatment 13 ACS Paragon Plus Environment


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of the whole slurry system on enzyme activity was next assessed using the dissolving pulp as a model substrate.

Figure 3 The effect of sulfite post-treatment (8% and 16% sulfite loading) on the enzymatic hydrolysis of 10% solid loading (A) unwashed and (B) washed steam pretreated lodgepole pine whole slurry at Cellic Ctec3 dosage of 15 mg protein per g cellulose for 48 hrs. The sulfite post-treatments were conducted at 160 °C for 70 mins in a Parr reactor.

To try to minimize the influence of substrate characteristics such as lignin content as well as watersoluble inhibitors on the activity of cellulase enzymes, a dissolving pulp was used as in this series of experiments, to assess the possible inhibition of sulfite addition on enzyme activity. A solids loading of 25%, an enzyme dosage of 35 mg protein/ g cellulose and a sulfite loading of 5% to the pulp was initially used. When compared to the hydrolysis yield of the dissolving pulp control, without the addition of sulfite, it was apparent that 0.13 mol/L of sulfite completely inhibited enzymatic hydrolysis (Figure 4). As mentioned earlier, the presence of sulfite during enzymatic hydrolysis may result in the denaturation of the enzyme through the reduction of disulfide bonds. 11


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When we assessed if the inhibition of enzyme activity was due to the ionic strength of the sodium sulfite, by adding sodium chloride to the enzyme mediated hydrolysis of the dissolving pulp (Figure 4) it was apparent that the addition of salt did not influence enzyme activity. Thus, it was likely that the sulfite was acting as a reducing agent which was denaturing the enzymes. To test this hypothesis, hydrogen peroxide was used to oxidize sulfite prior to the enzymatic hydrolysis, plus, the addition of sodium sulfate, which is the oxidization product of sodium sulfite, was also assessed. It was apparent that, with the addition of sulfate or peroxide oxidized sulfite, the hydrolysis efficiency was similar to that observed with the dissolving pulp control. Thus, although sulfite addition was able to modify the lignin present in the water insoluble substrate, while simultaneously detoxifying the water soluble fraction, when it was applied to whole slurries of steam pretreated softwood, the excess sulfite was acting as a reducing agent which inhibited enzyme activity (Figure 4).

Figure 4 The influence of mineral salts (NaCl, Na2SO3 and Na2SO4) on the enzymatic hydrolysis of 12% solid loading dissolving pulp (DsP) at Cellic Ctec3 dosage of 30 mg protein per g cellulose for 48 hrs. The mineral salts loading was 0.13 mol/L.



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As hydrogen peroxide was shown to decrease the inhibitory effects of the sulfite on the enzyme during the hydrolysis of dissolving pulp, it was subsequently used to quench the sulfite in the post-treated whole slurry, to decrease its detrimental effects on enzymatic hydrolysis. A 30% hydrogen peroxide solution was added in volumes ranging from 0.05 to 0.17 mL per 10 mL of the whole slurry (25% solids loading). However, unlike the case with the dissolving pulp substrate, the addition of hydrogen peroxide did not result in a boosting effect on enzymatic hydrolysis while, similar to the effects of sulfite, adding too much peroxide actually inhibited the enzyme (Figure 5 A). As well as inhibiting hydrolysis the hydrogen peroxide treatment of the whole slurry completely inhibited ethanol production during fermentation (Figure 5 B). This was likely because hydrogen peroxide also acts as a reactive oxygen species which is toxic to yeast cells.

23,24

This suggested that the peroxide also

reacted with the enzymes and yeast as well as the residual sulfite. It should be noted that it was challenging to obtain a homogeneous distribution of peroxide when adding to the high solids whole slurry. This may have compromised the ability of the peroxide to provide a targeted reaction with the sulfite separate from the other components such as the enzymes and yeast.


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Figure 5 (A) The effect of hydrogen peroxide treatment (0.05, 0.08 and 0.17 mL) on the enzymatic hydrolysis of 25% solid loading sulfite post-treated steam pretreated lodgepole pine whole slurry at a Cellic Ctec3 loading of 40 mg protein per g cellulose for 48 hrs. The sulfite post-treatments were conducted at 121 °C for 40 mins in an autoclave. (B) The effect of hydrogen peroxide treatment (0.05 and 0.08 mL) on the ethanol production profile of strain T2 grown on 25% solid loading sulfite posttreated steam pretreated lodgepole pine whole slurry hydrolysate. The sulfite post-treatments were conducted at 121 °C for 40 mins in an autoclave. The yeast concentrations (cell optical density: OD) were 13.

It was apparent that a sufficiently high temperature and sulfite loading was necessary to achieve sulfonation to improve hydrolysis of washed substrates. However, the conditions that improved the hydrolysis of the washed substrates were detrimental when applied to whole slurries as the excess sulfite inhibited fermentation and hydrolysis and the high temperatures compromised sugar recovery. To try to avoid sugar degradation, a milder temperature of 70 °C was next assessed, based on previous research. 9 However, in order to carry out effective sulfonation at such a mild temperature, a longer residence time was necessary, using a sulfite loading of 8% on the pretreated biomass for 12 hrs, based on previous research. 9 However, after post-treatment at 70 °C for 12 hrs using the whole slurry at a solids loading of 25%, the water soluble fraction still contained 20.7 g/L glucose and 12.5 g/L mannose. This was quite similar to the SO2 steam pretreated control. When increasing the sulfite loading to 16% at the 70 °C, the whole slurry also showed significant sugar degradation (13% and 24% of the glucose and mannose were degraded respectively). The hydrolysis of the post-treated slurry showed that the substrates treated with 16% sulfite suffered a yield loss of 12% (Figure 6 A), likely due to the excess sulfite inhibiting enzyme activity, as discussed above. However, the digestibility of the substrates post-treated using a sulfite loading of 8% at 70 °C was quite similar to that of the SO2 steam pretreated control. In order to maximize the sulfonation at this lower temperature, sodium carbonate was added to the post-treatment reaction, to assist the sulfite react with lignin. It was likely that, the addition of sodium carbonate as a source of



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alkali, deprotonates phenolic lignin moieties, causing the formation of quinone methides that are susceptible to nucleophilic attack by sulfite anions. The addition of alkali has been shown to enrich the amount of sulfonic acid groups during previous work detailing the sulfonation of poplar biomass. 22

It was apparent that the addition of carbonate along with sulfite to the whole slurry at a temperature of 70 °C increased the ease of hydrolysis of the steam pretreated substrate from 55% to 67% (Figure 6 A). Assessing the amount of sulfonic acid groups on the substrate after the post-treatment showed that the sulfonic acid groups increased from 85 µmol / g to 106 µmol / g upon the addition of carbonate during the post-treatment. Therefore, it was likely that the sodium carbonate enhanced sulfonation at the lower temperature, limiting sugar degradation while sulfonating the lignin, resulting in an increase in hydrolysis. The enhancement of sulfonation by sodium carbonate addition also decreased the amount of residual excess sulfite, by incorporating a greater amount of the sulfite into the substrate lignin. Consequently, this decreased the toxicity of the residual sulfite towards the yeast and the enzymes during HHF. Concentrations of 108 g/L glucose and 13.3 g/L mannose were obtained after 48 hours of hydrolysis of the whole slurry. An ethanol titer of 56.4 g/L was obtained from the whole slurry enzymatic hydrolysis and fermentation, as compared to the 15.3 g/L obtained from the processing of the untreated steam pretreated softwood whole slurry (Figure 6 B). Thus, it was apparent that this 70 °C “post-treatment” could simultaneously enhance enzymatic hydrolysis while also detoxifying the whole slurry such that the fermentation yield was enhanced substantially.


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Figure 6 (A) The effect of sulfite post-treatment (8% and 16% sulfite loading) with/without 2% carbonate on the enzymatic hydrolysis of 25% solid loading steam pretreated lodgepole pine whole slurry at a Cellic Ctec3 loading of 40 mg protein per g cellulose for 48 hrs. (B) The effect of sulfite (8% sulfite + 2% carbonate loading) post-treatment on the ethanol production profile of strain T2 grown on the 25% solid loading steam pretreated lodgepole pine whole slurry hydrolysate.

CONCLUSIONS This study indicated that the high solids hydrolysis and fermentation of steam pretreated softwoods remains challenging. The work reported here assessed the feasibility of using a simultaneous sulfite post-treatment and detoxification to improve both the hydrolysis and fermentation of this recalcitrant substrate. Although the potential of sulfite treatments to simultaneously improve enzymatic hydrolysis and fermentation of a steam pretreated softwood lodgepole pine whole slurry proved successful there were several challenges that had to be resolved. These included the conditions used for sulfite treatment to minimize sugar degradation and the toxicity of the unreacted sulfite towards enzymes and yeast during subsequent hydrolysis and fermentation of the post-treated whole slurry. Reducing the post-treatment temperature used resulted in a decrease in the amount of sulfonation occurring in the water insoluble fraction of the whole slurry. However, sulfonation at low 19 ACS Paragon Plus Environment


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temperatures (70 °C) could be enhanced through the addition of sodium carbonate. The use of a lower temperature limited sugar degradation, while the increased sulfonation improved enzymatic hydrolysis and fermentation. Now that this approach has been shown to have some potential, ongoing studies are focused on reducing chemical loadings with the objective of lowering the potential cost of this approach

ACKNOWLEDGEMENT We thank Novozymes (Sarah Teter) for their generous gift of cellulase enzyme mixtures, CSC for scholarship support of NZ, and NSERC for funding support.

REFERENCES

(1)

Walker, G. M. 125th Anniversary Review : Fuel Alcohol : Current Production and Future Challenges. J. Inst. Brew. 2011, 117 (1), 3–22, DOI: 10.1002/j.2050-0416.2011.tb00438.x.

(2)

Chandra, R. P.; Bura, R.; Mabee, W. E.; Berlin, A; Pan, X.; Saddler, J. N. Substrate Pretreatment: The Key to Effective Enzymatic Hydrolysis of Lignocellulosics? Adv. Biochem. Eng. Biotechnol. 2007, 108 (May), 67–93, DOI: 10.1007/10_2007_064.

(3)

Nakagame, S.; Chandra, R. P.; Saddler, J. N. The Effect of Isolated Lignins, Obtained from a Range of Pretreated Lignocellulosic Substrates, on Enzymatic Hydrolysis. Biotechnol. Bioeng. 20 ACS Paragon Plus Environment

Page 21 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2009, 105 (5), 871–879, DOI: 10.1002/bit.22626. (4)

Jönsson, L. J.; Martín, C. Pretreatment of Lignocellulose: Formation of Inhibitory by-Products and Strategies for Minimizing Their Effects. Bioresour. Technol. 2016, 199, 103–112, DOI: 10.1016/j.biortech.2015.10.009.

(5)

Kumar, L.; Chandra, R.; Saddler, J. Influence of Steam Pretreatment Severity on PostTreatments Used to Enhance the Enzymatic Hydrolysis of Pretreated Softwoods at Low Enzyme Loadings. 2011, 108 (10), 2300–2311, DOI: 10.1002/bit.23185.

(6)

Cavka, A.; Alriksson, B.; Ahnlund, M.; Jönsson, L. J. Effect of Sulfur Oxyanions on Lignocellulose-Derived Fermentation Inhibitors. Biotechnol. Bioeng. 2011, 108 (11), 2592– 2599, DOI: 10.1002/bit.23244.

(7)

Alriksson, B.; Cavka, A.; Jönsson, L. J. Improving the Fermentability of Enzymatic Hydrolysates of Lignocellulose through Chemical in-Situ Detoxification with Reducing Agents. Bioresour. Technol. 2011, 102 (2), 1254–1263, DOI: 10.1016/j.biortech.2010.08.037.

(8)

Guo, X.; Cavka, A.; Jönsson, L.; Hong, F. Comparison of Methods for Detoxification of Spruce Hydrolysate for Bacterial Cellulose Production. Microb. Cell Fact. 2013, 12 (1), 93, DOI: 10.1186/1475-2859-12-93.

(9)

Kumar, L. Steam Pretreatment as an Effective Front End for a Softwood Based Biorefinery. Ph.D. Dissertation, University of British Columbia, Vancouver, BC, Canada, 2013.

(10) Schimz, K. L. The Effect of Sulfite on the Yeast Saccharomyces Cerevisiae. Arch. Microbiol. 1980, 125 (1–2), 89–95. (11)

Bailey, J. L.; Cole, R. D. Studies on the Reaction of Sulfite with Proteins. J. Biol. Chem. 1959, 234 (7), 1733–1739.

(12)

Hu, J.; Arantes, V.; Pribowo, A.; Gourlay, K.; Saddler, J. N. Substrate Factors That Influence the Synergistic Interaction of AA9 and Cellulases during the Enzymatic Hydrolysis of Biomass. Energy Environ. Sci. 2014, 7 (7), 2308–2315, DOI: 10.1039/C4EE00891J.

(13)

Peciulyte, A.; Samuelsson, L.; Olsson, L.; McFarland, K. C.; Frickmann, J.; Østergård, L.;



Page 22 of 24

Halvorsen, R.; Scott, B. R.; Johansen, K. S. Redox Processes Acidify and Decarboxylate Steam-Pretreated Lignocellulosic Biomass and Are Modulated by LPMO and Catalase. Biotechnol. Biofuels 2018, 11 (1), 1–16, DOI: 10.1186/s13068-018-1159-z. (14)

Helle, S.; Cameron, D.; Lam, J.; White, B.; Duff, S. Effect of Inhibitory Compounds Found in Biomass Hydrolysates on Growth and Xylose Fermentation by a Genetically Engineered Strain of S. Cerevisiae. Enzyme Microb. Technol. 2003, 33 (6), 786–792, DOI: 10.1016/S01410229(03)00214-X.

(15)

Kumar, L.; Arantes, V.; Chandra, R.; Saddler, J. The Lignin Present in Steam Pretreated Softwood Binds Enzymes and Limits Cellulose Accessibility. Bioresour. Technol. 2012, 103 (1), 201–208, DOI: 10.1016/j.biortech.2011.09.091.

(16)

Weiss, N.; Börjesson, J.; Pedersen, L. S.; Meyer, A. S. Enzymatic Lignocellulose Hydrolysis: Improved Cellulase Productivity by Insoluble Solids Recycling. Biotechnol. Biofuels 2013, 6 (1), 5, DOI: 10.1186/1754-6834-6-5.

(17)

Jönsson, L. J.; Alriksson, B.; Nilvebrant, N.-O. Bioconversion of Lignocellulose: Inhibitors and Detoxification. Biotechnol. Biofuels 2013, 6 (1), 16, DOI: 10.1186/1754-6834-6-16.

(18)

Kristensen, J. B.; Felby, C.; Jørgensen, H. Yield-Determining Factors in High-Solids Enzymatic Hydrolysis of Lignocellulose. Biotechnol. Biofuels 2009, 2 (1), 11, DOI: 10.1186/1754-6834-2-11.

(19)

Nakagame, S.; Chandra, R. P.; Kadla, J. F.; Saddler, J. N. The Isolation, Characterization and Effect of Lignin Isolated from Steam Pretreated Douglas-Fir on the Enzymatic Hydrolysis of Cellulose.

Bioresour.

Technol.

2011,

102

(6),

4507–4517,

DOI:

10.1016/j.biortech.2010.12.082. (20)

Liu, Z.-H.; Qin, L.; Zhu, J.-Q.; Li, B.-Z.; Yuan, Y.-J. Simultaneous Saccharification and Fermentation of Steam-Exploded Corn Stover at High Glucan Loading and High Temperature. Biotechnol. Biofuels 2014, 7 (1), 167, DOI: 10.1186/s13068-014-0167-x.

(21)

Mooney, C.; Mansfield, S. D.; Touhy, M. G.; Saddler, J. N. The Effect of Initial Pore Volume


Page 23 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and Lignin Content on the Enzymatic Hydrolysis of Softwoods. 1998, 64 (2), 113–119, DOI: 10.1016/S0960-8524(97)00181-8. (22)

Chandra, R. P.; Chu, Q. L.; Hu, J.; Zhong, N.; Lin, M.; Lee, J. S.; Saddler, J. The Influence of Lignin on Steam Pretreatment and Mechanical Pulping of Poplar to Achieve High Sugar Recovery and Ease of Enzymatic Hydrolysis. Bioresour. Technol. 2016, 199, 135–141, DOI: 10.1016/j.biortech.2015.09.019.

(23)

Perrone, G. G.; Tan, S. X.; Dawes, I. W. Reactive Oxygen Species and Yeast Apoptosis. Biochim. Biophys. Acta - Mol. Cell Res. 2008, 1783 (7), 1354–1368, DOI: 10.1016/j.bbamcr.2008.01.023.

(24)

Izawa, S.; Inoue, Y.; Kimura, A. Oxidative Stress Response in Yeast: Effect of Glutathione on Adaptation to Hydrogen Peroxide Stress in Saccharomyces Cerevisiae. FEBS Lett. 1995, 368 (1), 73–76, DOI: 10.1016/0014-5793(95)00603-7.

TABLE OF CONTENTS (TOC) GRAPHIC



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One-step sulfite post-treatment based on the acid steam pre-treated softwood whole slurry to both enhance downstream enzymatic hydrolysis and fermentation.


Sulfite post-treatment to simultaneously detoxify and improve the

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