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Biofuels and Biomass
Effects of washing, autoclave and surfactants on enzymatic hydrolysis of negatively-valued paper mill sludge for sugar production Suiyi Zhu, Jun Sui, Ya Liu, S.F. Ye, Chuanxin Wang, Ming-xin Huo, and Yang Yu Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.8b03586 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on February 6, 2019
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Energy & Fuels
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Effects of Washing, Autoclave and Surfactants on Enzymatic
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Hydrolysis of Negatively-Valued Paper Mill Sludge for Sugar
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Production
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Suiyi Zhu1, 2, Jun Sui2, Ya Liu3, Shufeng Ye3, Chuanxin Wang2, Mingxin Huo1, Yang Yu1, 2, 4*
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1. Science and Technology Innovation Center for Municipal Wastewater Treatment and Water Quality Protection, Northeast Normal University, Changchun 130117, China 2. Guangdong Shouhui Lantian Engineering and Technology Co. Ltd., Guangzhou 510075, China 3. State Key Laboratory of Multiphase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, P. O. Box 353, Beijing 100190, China 4. Biorefining Research institute, Lakehead University, Thunder Bay, Ontario, P7B 5E1, Canada
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Abstract
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Paper mill sludge (PMS) is a waste of paper industry but can be a potential feedstock for
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cellulosic sugar production. In this study, washing, autoclave and surfactants were investigated
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for PMS pretreatment before enzymatic hydrolysis to produce cellulosic sugars. It was
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demonstrated that washing and autoclave had limited impact on improvement of enzymatic
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hydrolysis of PMS but washing reduced ash content, resulting less acid used in neutralization.
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Adding nonionic surfactants of Triton X-100, Tween 80 and PEG 8000 improved conversion of 1 ACS Paragon Plus Environment
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PMS and the highest rates were 56.3% and 55.4%, achieved by adding 1% Triton X-100 and 5%
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PEG 8000, respectively. Lowest conversion rates were showed by 1% and 5% Tween 80,
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probably because it had a hydrophobic alkyl chain. After optimizing concentrations of enzyme
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and PMS in hydrolysis with supplement of PEG 8000, the highest PMS conversion of 74.7% was
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achieved by 10% PMS and 3% enzymes. With addition of PEG 8000, the conversion of PMS
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was reduced at high concentrations of enzyme and PMS compared with the non-PEG control,
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which was more significant at the later stage of hydrolysis. We proposed that the combined
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negative effects of endproducts and surfactants were more significant on hydrolysis than the
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endproducts alone.
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Key words: paper mill sludge; washing; surfactants; enzymatic hydrolysis; inhibitory effect.
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1. Introduction
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Pulp and paper production is about 300 to 350 million ton annually worldwide1, of which about
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3~5% is lost in the form of paper mill sludge (PMS) because of the undesirable fine fibres
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produced in paper-making2-4. Currently, PMS is generally disposed in landfill, incineration and
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land application4. Disposal at landfills is expensive, which costs about US$20 ~ US$30 per wet
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ton1,5. Incineration is also unfeasible because less heat is generated from polysaccharides. For
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example, the heating value of cellulose is 3.85 kJ/g, significantly lower than 5.88 kJ/g of lignin
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and 8.12 kJ/g of wood extractives6. On the other hand, commercialization of cellulosic biofuels
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is impeded by the high costs of intensive disintegration of recalcitrant lignocellulose to extract
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cellulose7-8. It was estimated that pretreatment and fractionation accounted for 38.7% and 37.7%
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of the total cost of bioethanol production from hardwood and softwood, respectively9. In addition,
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products generated from plant biomass pretreatment, such as lignin and xylo-oligosaccharides,
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are inhibitors of enzymatic hydrolysis and the subsequent fermentation for bioethanol
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production10-16. Alternatively, when the negatively-valued PMS is used as a feedstock for sugar
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production, pretreatment is not required, which significantly lowers the overall cost of cellulosic
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bioproducts. It was estimated that the cost of enzymes and other chemicals was less than US$0.1
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to produce one pound of cellulosic sugars from PMS11. Compared to the conventional biomass
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feedstock, such as wood chips, PMS contains limited lignin11, which reduces its inhibitory
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effects on microorganisms in the subsequent biofuel production. The big surface area of fine
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fibers of PMS is much more amenable to enzymatic hydrolysis10. In addition, PMS is a reliable
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feedstock and the process of PMS enzymatic hydrolysis can be easily integrated into current
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pulping facilities17.
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However, PMS contains high contents of clays, fillers, solid rejects and other impurities4,11. In a
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typical PMS, about 1/3 of its dry content is ash, which may adsorb enzymes and negatively affect
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enzymatic hydrolysis of cellulose18-19. It was demonstrated that ash had higher affinity to
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enzymes than fibers and about 3-5 mg of enzymes was lost in one gram of acid insoluble ash19-20.
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Both acid soluble (CaCO3) and insoluble (clay) ash of PMS reacted with acids21, which increased
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cost in pH neutralization required by enzymatic hydrolysis. Thus, removal of ash is of
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significance for reducing acid and enzymes used in PMS hydrolysis. It was reported that sugar
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production increased by 12 to 27% after 82~98% of ash was removed, and much less acid was
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used in pH neutralization19. In another study, sugar conversion increased by 88% after ash was
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neutralized with HCl22. Software specifically designed for pulp and paper economic analysis
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demonstrated that cellulosic ethanol production was much more profitable from fractionated
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PMS23.
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The improvement of enzymatic hydrolysis of cellulosic biomass by nonionic surfactants has been
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showed in many studies24-26. It was reported that, by adding Berol 08, cellulose conversion
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increased by 70% in hydrolyzing acid-treated wheat straw27. In another study, the enzymatic
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conversion rate of steam-pretreated spruce (SPS) increased from 42% to 78% with addition of
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PEG 4000 but it was not improved when PSP was delignified28, suggesting the hydrolysis was
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improved by binding of PEG4000 to lignin. By adding 2.5g/L of Tween 20, the used enzymes
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were reduced to half while keeping the same yield of cellulosic ethanol in a process of
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Simultaneous Saccharification and Fermentation (SSF) using spruce chips as substrate29. It is
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believed that the non-specific binding of enzymes is reduced by surfactants, which results in the
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improvement of cellulase activities30, probably by changing the enzyme-substrate interactions25.
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A possible mechanism of improvement of enzymatic hydrolysis of lignocellulose with addition
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of an amphiphilic surfactant is that the hydrophobic sites of lignin are occupied by interactions
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with the hydrophobic part of surfactant, displacing the enzymes attached to it, while the
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hydrophilic part of surfactant forms a barrier, preventing the unproductive binding of enzymes25.
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The theory was supported by adding the nonionic surfactant Triton X-100 to enzymatic
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hydrolysis of waste newspaper, which contained 19.2% of lignin, and the conversion rate showed
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45% improvement. In contrast, when Triton X-100 was added to hydrolysis of waste office paper,
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which contained much less lignin, its effect on the enzymatic conversion rate was not
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significant31. Several studies have also showed that hydrolysis of cellulose is improved by adding
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nonionic surfactants. It was demonstrated that by adding 5% (w/w) of PEG4000, the hydrolysis
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of Avicel PH101 increased from 41.1% to 78.9%26. In another study, both nonionic surfactants
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Tween 80 and biosurfactants increased sugar production about six times in hydrolysis of
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Sigmacell 10032. However, it was found that high concentrations of nonionic surfactants had
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negative effects on cellulose hydrolysis33. The aim of the study is 1) Find practical ways to
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hydrolyze PMS for cellulosic sugar production; and 2) Understand the effects of nonionic
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surfactants in PMS hydrolysis. Enzymes costs significantly in production of cellulosic ethanol34,
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and it is of significance to study the pretreatment method to facilitate enzymatic hydrolysis of
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PMS.
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2. Materials and Methods
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2.1 Materials and chemicals
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PMS in this study was collected from the primary clarifier of wastewater treatment plant of a
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pulp mill in Northern Ontario, Canada, after pressing through a belt-filter press (Fig. 1A). The
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Kraft process was used for pulp-making and the used wood chips were largely softwood and
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only small proportion of hardwood was added.
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Fig. 1. Brief schematic diagrams of the pulping process, formation and conversion of PMS to
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bioethanol. (A), the process of pulp production. PMS was generated at the washing and
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dewatering steps of the process and collected at primary clarifier of the wastewater treatment
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plant; (B), the process of PMS conversion to bioethanol.
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Sulfuric acid, acetic acid, sodium acetate, the nonionic surfactants of Triton X-100, Tween 80
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and PEG 8000 were purchased from Fisher Scientific (Nepean, ON, Canada) and the molecular
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structure of Triton X-100, Tween 80 and PEG 8000 was demonstrated in Fig. 2, respectively.
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Glucose and xylose for the standard solution were purchased from Sigma-Aldrich (Oakville, ON,
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Canada). The cellulase cocktail Cellic CTec2 was kindly provided by Novozymes North
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America (Franklinton, North Carolina, USA).
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Fig. 2. Structures of nonionic surfactants for facilitating PMS hydrolysis. (A), Triton
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X-100; (B), Tween 80; and (C), PEG 8000.
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2.2 Preparation of PMS feedstock for enzymatic hydrolysis
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In a typical pulping process, pulp is produced after wood chips are treated with cooking, washing,
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bleaching and dewatering and PMS is mainly generated at the washing and dewatering steps,
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where the fine fibers are rejected and washed into the waste stream with fillers and other 6 ACS Paragon Plus Environment
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impurities from the pulping process (Fig. 1). After PMS was collected from primary clarifier of
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the wastewater treatment plant, it was allocated to small portions and stored at -20°C in 4L Zap
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plastic bags until use. Before PMS was used for hydrolysis, it was firstly thawed at 4°C in a
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refrigerator, then fully mixed with the decanted water in plastic bag before the pH was adjusted
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to 5.0 with 2 mol/L acetic acid except otherwise stated. After the pH was stabilized at pH5.0,
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PMS was used immediately or stored at 4°C for later use.
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2.3 Enzymatic hydrolysis
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After pH was adjusted to 5 from the initial pH of about 9.4 of the raw PMS, the consistency of
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PMS was about 30%, which was diluted to the desired concentrations with 50 mmol/L acetate
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buffer (acetic acid + sodium acetate), pH 5.0, based on the equivalent dry weight before and after
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dilution. In general, about 3 to 5g dry mass of PMS was added to 125mL flask, together with 10g
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of 4# glass beads (about 60 pieces) to facilitate mixing. The surfactant was added to the flask a
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day before hydrolysis and fully manually mixed with PMS before shaking overnight at 200rpm
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and 50°C in an incubator shaker (Excella E5, New Brunswick Scientific, Enfield, Connecticut,
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USA). Depending on requirement of experiments, 1% to 6% Cellic CTec2 of dry mass was
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added to PMS the next day and fully manually mixed before the flasks were incubated at 50°C
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and 200rpm for hydrolysis. To further facilitate mixing, glass beads were kept in the flask
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throughout the experiment. 1mL of sample was taken at 0h, 24h, 36h, 48h, 72h and 96h and
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stored at -20°C for sugar analysis. The sample for 0h was taken just before adding the enzymes.
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To better describe the exponential stage of the hydrolysis, two more samples were taken between
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0h and 24h, and 24h and 36h, respectively. CTec2 activity was determined following the
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protocol of Measurement of Cellulase Activities by NREL35 and the Filter Paper Activity of
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CTec2 was 114.2 FPU/g, slightly smaller than 120 FPU/g reported by Cannella and Jørgensen36.
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2.4 Determination of PMS components and acid hydrolysis
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Ash and organic content in PMS was determined following the protocol of Determination of Ash
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in Biomass developed by the National Renewable Energy Laboratory (NREL)37. The
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components of organic content in PMS was determined by acid hydrolysis with a modified
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protocol of Determination of Structural Carbohydrates and Lignin in Biomass developed by
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NREL38. In general, PMS was fully washed to completely remove ash before it was air-dried at
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room temperature. The washed-PMS was then disintegrated in a Wiley Mill (Thomas Scientific,
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Swedesboro, New Jersey, USA) and thus prepared ash-free PMS dust was collected through an
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80 mesh sieve. The acid hydrolysis was carried out by mixing 0.30g of the PMS dust with 4.92g
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of 72% sulfuric acid in a 12ⅹ100mm glass test tube. After incubating at 30 ± 3 °C in a water bath
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for 60 ± 5 min, the mix was added 84.00g of deionized water to dilute the sulfuric acid to 4% and
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autoclaved at 121°C for 1h. Followng compensation of water loss in autoclave, the hydrolysate
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was filtrated to determine carbohydrates and acid soluble lignin in the soluble fraction, and ash-
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free acid insoluble lignin in the solid fraction.
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2.5 PMS deashing
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The deashing of PMS was carried out by washing 100g of raw PMS 3 and 5 times (without
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neutralization of pH), respectively, with 100mL of deionized water (denoted as 3W and 5W).
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Another 100g of raw PMS was washed 15 times, each time with 1L of deionized water, which
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was used as the ash-free control (denoted as 15L). For each wash, PMS was fully mixed with 8 ACS Paragon Plus Environment
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deionized water, followed by filtration through a 40 mesh sieve and pressing until moisture was
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75% ~ 80%. After washing, the pH of PMS was adjusted to 5 using 2 mol/L acetic acid,
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followed by adjusting to desired concentrations with 50 mmol/L acetate buffer, pH5.0. For the
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unwashed control (denoted as Control), the pH of raw PMS was adjusted to 5 directly without
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washing, followed by adjusting to desired concentrations with 50mmol/L acetate buffer, pH5.0.
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2.6 Autoclave treatment of PMS
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The ash-free and unwashed PMS was treated as previously described. It was then separated into
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two fractions after the concentrations of PMS were adjusted to 10% by adding 50 mmol/L
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acetate buffer, pH5.0. One fraction was autoclaved at 121°C for 20min for the autoclaved trials
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and the other was kept at room temperature for the non-autoclaved trials. After the treatment, for
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trials for autoclaved and washed, autoclaved and unwashed, non-autoclaved and washed, and
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non-autoclaved and unwashed were prepared. A trial without pH adjustment of PMS was used as
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a control. The hydrolysis was carried out based on the steps described previously but with 6% of
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CTec2, and the samples were taken at 96h of hydrolysis for sugar analysis.
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2.7 Determination of sugar concentrations and conversion rates of PMS
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Sugar concentrations were determined following a modified protocol of Determination of
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Structural Carbohydrates and Lignin in Biomass developed by NREL36 and measured by HPLC
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(1260 Infinity, Agilent Technologies, Santa Clara, CA, USA) equipped with a refractive index
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detector (RID). Because the major sugars after enzymatic hydrolysis of PMS were glucose and
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xylose and glucose concentration was significantly higher than xylose’s11,17, an Aminex HPX-
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87H column (Bio-Rad, Hercules, California, USA) was used to determine sugar concentrations
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in experiments. The mobile phase of HPLC was 5 mmol/L H2SO4 at a flow rate of 0.5 mL/min.
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The temperature of the column and detector was maintained at 60°C and 35°C, respectively. A
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stock of 20.00g/L of glucose and xylose standard was made, allocated and stored at -20°C.
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Before analysis, the stock was thawed along with samples at room temperature and diluted to a
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series of 10.00 g/L, 5.00 g/L, 2.50 g/L, 1.25 g/L, 0.75 g/L and 0.375 g/L. The standard series and
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samples were filtrated through a 0.22µm nylon syringe filter before applying to HPLC.
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The conversion of PMS was determined by the modified equation of Chen and coworkers19:
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𝜂 = [Cg × V0 × 0.9 + Cx × V0 × 0.88] / (M0 –MA–ML )
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Where, 𝜂: conversion rate of PMS (%); Cg: glucose concentration at t hour (g/L); Cx: xylose
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concentration at t hour (g/L); V0: initial hydrolysate volume (L); M0: initial PMS dry mass (g);
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MA: initial ash dry mass (g); ML: initial lignin dry mass (g).
Eq. (2)
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The conversion factors between glucose and cellulose was 0.919; and between xylose and xylan
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was 0.8811.
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2.8 Statistic analysis
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The data was processed and statistically analyzed by using SPSS Statistics 21 (SPSS Co. Ltd,
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Chicago, USA) with One-way Analysis of Variance (ANOVA), followed by Tukey’s test.
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Differences were considered to be significant when P 0.05), indicating it had
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limited effects on improvement of hydrolysis. It was reported that Tween 20 had positive effect
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on hydrolysis of Avicel10126 but other results demonstrated that the effect of Tween 20 on
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hydrolysis of Avicel and delignified SPS was limited25. Our results were consistent with
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observation of Wang and coworkers, in which all Tweens with different structures slightly
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increased enzymatic hydrolysis of cellulose at a low concentration but inhibited the hydrolysis at
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a higher one44. The conversion rates of PMS were 53.2% for 1% and 55.4% for 5% of PEG 8000,
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7.7% and 9.9% over the control, respectively (p < 0.05). The same rate was observed with 1%
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and 5% PEG 8000 until 48h, when the rate of 5% PEG 8000 increased slightly higher (Fig. 6C).
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It was reported that the activity and stability of cellulase improved by adding PEG4000, which
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led to 91% increase of enzymatic hydrolysis of cellulose Avicel 101, the best among the selected
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additives26. Enzymatic hydrolysis of PMS improved by adding nonionic surfactants in a certain
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range of concentrations, In our study, the hydrolysis was enhanced by 1% and 5% Triton X-100
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and PEG8000, respectively, and by 1% Tween 80 (p 0.05),
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indicating two sides of the non-ionic surfactants, which may accelate the hydrolysis at a low
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concentration but inhibit at a higher one. The highest conversion rates were achieved by 1%
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Triton X-100 and 5% PEG 8000, which were 56.3% and 55.4%, respectively. The enzymatic
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hydrolysis of PMS was least effective by Tween 80 probably because it has an alkyl chain in one
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of its four hydrophilic moieties45, which might confer it more hydrophobic property. Compared
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to PEG 8000, which only has the EO repeat, Triton X100 has a hydrophobic head, which confers
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it hydrophobic property, leading to the inhibitory effects of Triton X100 at a higher
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concentration. The results indicated that nonionic surfactants in general improved enzymatic
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hydrolysis of PMS in a certain range of concentrations. Their different structures might
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determine their effects on hydrolysis and their hydrophobic property might have inhibitory
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impact on it. Our results were consistent with the observation of Zhou and coworkers with the
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same types of surfactants (Triton, Tween and PEG), in which Tween 20 and 80 showed the most
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significant inhibition on hydrolysis and Triton X100 was only demonstrated the inhibitory effect
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with a higher concentration33. It was reported that nonionic surfactants with only hydrophilic
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fraction and long EO repeats increased efficiencies of enzymatic hydrolysis of softwood
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lignocelluloses28. PEG 8000 only contained a long EO repeat without hydrophobic moiety,
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which was selected as the surfactant for further studies.
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(A) 60
(B) 60
PMS conversion (%)
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PMS conversion (%)
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0
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100
50 40 30 20 10 0
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Time (h)
20
40
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Time (h)
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(C) 60
PMS conversion (%)
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0
20
40
60
80
100
Time (h)
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Fig. 6. Effect of nonionic surfactants on hydrolysis of PMS. (A), Triton X-100, (B),
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Tween 80 and (C), PEG8000. (■), 1% and (▲), 5% of surfactants was demonstrated. (○)
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and dash-line: negative control without surfactant. 5% PMS and 1% CTec2 (1.1FPU/g
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glucans) were added
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3.5 Effect of PEG 8000 on enzymatic hydrolysis of PMS with different enzyme doses
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To investigate the effect of PEG 8000 on enzymatic hydrolysis of PMS, the trials with different
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CTec2 enzyme dosage of 1%, 3% and 6% (w/w) were supplemented with PEG 8000 and
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compared with the hydrolysis without it. It was demonstrated that the hydrolysis of PMS
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improved by increasing the addition of CTec2, and no significant differences were observed for
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both PEG and non-PEG trials in the first 10h (Fig. 7). After 10h, the conversion rates were
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higher with PEG 8000 than those without it in the 1% and 3% CTec2 trials. At 96h, the
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conversion rates of PMS were 46.8% and 60% with PEG 8000 for the 1% and 3% CTec2 trials,
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respectively, which were 3.7% and 3.3% higher than the non-PEG counterparts (Fig. 7). Tukey’s
387
test demonstrated that the differences of hydrolysis between the PEG 8000 trials with 1% and 3%
388
CTec2, respectively, were significant with their non-PEG controls (p
