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Factors affecting the enzymatic hydrolysis of primary clarifier sludges Thomas, Paul E. 1998

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FACTORS AFFECTING THE ENZYMATIC HYDROLYSIS OF PRIMARY CLARIFIER SLUDGES By Paul E . Thomas B. A . Sc. (Chemical Engineering) Queen's University, Kingston A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMICAL ENGINEERING We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1998 © Paul E . Thomas, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her represen-tatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Chemical Engineering The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V 6 T 1Z1 Date: Abstract Bioconversion of cellulose to ethanol, by means of enzymatic hydrolysis, has been, and still is, the focus of much research interest since the early 1980s. The enzymatic hydrolysis process is well developed and offers an environmentally attractive option. However, many obstacles prevent the process from becoming commercially attractive. These obstacles include kinetic inhibitions (competitive reactions with lignin, intermediate and end-product inhibitions), dif-ficulties in enzyme recycling, and economics. These hurdles have resulted in intense research over the last several decades. Despite the economic disadvantages of the enzymatic hydrolysis process, niche opportu-nities, such as the operations at the Tembec mill in Temiscaming, Quebec, still exist. The ethanol produced from its spent sulphite liquor is distilled to food-grade ethanol, which de-mands higher prices than fuel ethanol. Additional ethanol produced from Tembec's waste primary clarifier sludge (PCS), using enzymatic hydrolysis, could also be sold for similar use. This work explored several aspects of enzymatic hydrolysis to optimise the production of ethanol from Tembec's sulphite PCS. Batch hydrolysis results indicated that up to 50% of the PCS was hydrolysable by Trichoderma reesei enzymes. Adsorption experiments revealed that a significant fraction of enzyme activity was recoverable in the liquid hydrolysate. Oxygen delignification, as a pretreatment option for the PCS, was also examined. It was found that on a mass basis, a 68% increase in sugar yield from delignified PCS was possible over untreated PCS. A semi-continuous laboratory-scale hydrolysis reactor (22 L working volume) was run on both untreated and oxygen delignified PCS. In both cases, the yields represented 90% of the levels attained by batch hydrolysis. ii Table of Contents Abstract i i List of Tables vi List of Figures vi i Acknowledgements ix 1 Introduction 1 2 Background 5 2.1 Substrate characteristics 5 2.1.1 PCS (Primary Clarifier Sludge) production 7 2.1.2 PCS as a feedstock 7 2.2 Pretreatment 8 2.2.1 Chemical pretreatment 8 2.2.2 Mechanical pretreatment 10 2.3 Enzymatic hydrolysis 10 2.3.1 Scope & limitations 12 2.3.2 The enzymes and their mechanism . . . 13 2.3.3 Enzyme recycle 15 2.4 Economics 18 3 Research Objectives 21 iii 4 Experimental 22 4.1 Materials 22 4.1.1 Substrates 22 4.1.2 Enzymes 23 4.1.3 Small-scale hydrolyses 24 4.1.4 Reactor apparatus 24 4.2 Methods 24 4.2.1 Hydrolysis in buffered solution 24 4.2.2 Adsorption 25 4.2.3 Oxygen delignification 26 4.2.4 Reactor operation 26 4.3 Analyses 29 4.3.1 Sampling protocol, all experiments 29 4.3.2 Reducing sugars assay 29 4.3.3 Glucose assay 30 4.3.4 Filter paper assay 30 4.3.5 Protein in solution 30 4.3.6 Lignin content (Kappa number, TAPPI T236 cm-85 and CPPA G.18) 31 4.3.7 Statistics 31 5 Results and Discussion 33 5.1 Hydrolysis experiments 33 5.1.1 Maximum sugar conversion 33 5.1.2 Effect of acetate buffer on hydrolysis 35 5.1.3 Effect of enzyme loading and substrate concentration on hydrolysis . . 35 5.2 Protein adsorption experiments 43 5.2.1 Adsorption during hydrolysis 43 iv 5.2.2 Adsorption without hydrolysis 49 5.3 Pretreatment experiments 51 5.3.1 Comparison of pulp from three mills 51 5.3.2 Oxygen delignification of PCS 56 5.4 Laboratory-scale reactor experiments 62 5.4.1 PCS run 63 5.4.2 02-delignified PCS run 64 6 Conclusion 66 7 Recommendations 68 References 70 A Acronyms 77 v List o f Tables 4.1 List of substrates and how they were used 23 4.2 Comparison of the operating conditions of the laboratory 02-delignification and a typical medium consistency commercial processes 27 4.3 Operating conditions of the reactor. 27 5.4 Percentage sugar yields for the enzymatic hydrolysis of different substrates (based on total reducing sugars) 34 5.5 Summary of solka floe hydrolysis' data 37 5.6 Summary of PCS hydrolysis' data 40 5.7 Summary of pre- and post-oxygen delignified pulp hydrolyses [33]. A constant enzyme loading of 2 FPU/g pulp was used throughout 52 5.8 Summary of PCS hydrolysis' data at 25 g/L 60 vi List of Figures 2.1 Cellulose structure [62] 5 2.2 Two-dimensional representation of the repeating unit of spruce lignin [62]. . . 6 2.3 Simplified hydrolysis process 12 2.4 Cellulose to ethanol by enzymatic hydrolysis and fermentation with interme-diate and end-product inhibitions 14 4.5 Schematic of the reactor setup 25 5.6 Maximum sugars conversions for the enzymatic hydrolysis of different sub-strates. The hydrolysis time was 72 hr for 50 g/L substrate, on a dry basis, at an enzyme loading of 50 FPU/g 34 5.7 Effect of acetate buffer concentration on the enzymatic hydrolysis of solka floe. 36 5.8 Enzymatic hydrolysis of different solka floe concentrations and enzyme load-ings. Measured by total reducing sugars 38 5.9 Enzymatic hydrolysis of different solka floe concentrations and enzyme load-ings. Measured by glucose concentrations 39 5.10 Enzymatic hydrolysis of PCS for 25 g/L at different enzyme loadings 41 5.11 Enzymatic hydrolysis of PCS for 50 g/L at different enzyme loadings [45]. . . 42 5.12 Hydrolysis of solka floe and Sigma Cell 50 at 10 g/L and 10 FPU/g substrate. 43 5.13 Total protein concentrations throughout the enzymatic hydrolysis of Sigma Cell 50 and solka floe for 10 g/L substrate at an enzyme loading of 10 FPU/g. 44 5.14 Time-dependent enzyme activity for the hydrolysis of 10 g/L solka floe at an enzyme loading of 10 FPU/g 46 vii 5.15 Protein levels in solution for the hydrolysis of solka floe 47 5.16 Protein levels in solution for the hydrolysis of Sigma Cell 50 47 5.17 Protein levels in solution for the hydrolysis of Tembec PCS 48 5.18 Protein adsorption on solka floe (30 g/L) without hydrolysis. Total protein measured using the B i o - R A D assay. 49 5.19 Protein adsorption on solka floe (30 g/L) without hydrolysis. Total protein measured by absorbance at 280 nm 50 5.20 Hydrolysis of Howe Sound pulp for 10, 25 and 50 g/L concentrations [33]. . 53 5.21 Hydrolysis of Intercontinental pulp for 10, 25 and 50 g/L concentrations [33]. 54 5.22 Hydrolysis of Northwood pulp for 10, 25 and 50 g/L concentrations [33]. . . 55 5.23 Effect of oxygen delignification on the hydrolysis of 50 g/L PCS at an enzyme loading of 50 FPU/g 57 5.24 Effect of oxygen delignification on the hydrolysis of 10 g/L PCS at an enzyme loading of 10 FPU/g 58 5.25 Enzymatic hydrolysis of oxygen delignified PCS for 25 g/L at different enzyme loadings 59 5.26 Determination of enzyme fraction & activity after a 48 hr hydrolysis [33]. . . 61 5.27 Time run of the semi-continuous reactor on untreated PCS. A n enzyme loading of 10 FPU/g PCS for 25 g/L substrate was used. . . 63 5.28 Time run of the semi-continuous reactor on oxygen delignified PCS. A n en-zyme loading of 10 FPU/g PCS for 25 g/L substrate was used 65 viii Acknowledgements I wish to express my sincere appreciation for the assistance provided by my thesis supervisor, Dr. Sheldon Duff. I would like to thank Chris Kurniawan for his invaluable help in the laboratory. This work was funded by a N S E R C Strategic Grant. I gratefully acknowledge the donation of enzyme samples by Novo Nordisk, PCS and pulp samples from all the respective mills. Finally, sincere gratitude is extended to Dr. Dick Kerekes and the staff of the U B C Pulp and Paper Centre for their generous support, in particular to Rita Penco for her outstanding assistance. ix Chapter 1 Introduction Ethanol has been an important product in western societies for centuries. Before the develop-ment of the chemical industry in the nineteenth century, it was used for basically one purpose: human consumption. However, ethanol has characteristics that make it useful for a variety of applications, such as a chemical feedstock, an extractive agent, a disinfectant, or even a fuel. A s early as the 1890s, German and French institutions have exhibited an interest in ethanol as an automobile fuel [52]. In 1923 an English engineer, Ricardo, showed that 10% ethanol and 90% gasoline blends increased fuel octane rating by 9 points compared to gasoline alone. Despite this, the development of tetraethyllead as a cheap and efficient octane improver, and the 1933 "Dieterich Report" to the U.S. Congress marked a sharp decline in interest of ethanol as a fuel additive. Various disadvantages of ethanol were cited in the report, such as: 1) increased wear and corrosion, 2) lower mileage than gasoline on a volume basis, 3) higher cost and greater price variability than gasoline, and 4) most importantly at the time, the fact that ethanol was not as cost efficient as an octane booster as tetraethyllead [52]. Over the past two decades, renewed interest in ethanol was prompted by several factors, chief among them the oil crisis in 1973, and the recognition that tetraethyllead combustion products are toxic. Ethanol represented, and still represents, a renewable, clean source of energy to fuel automobiles. Ethanol gives no net contribution to global warming since the carbon dioxide produced by the combustion of ethanol is consumed by the growing raw material. 1 Chapter I. Introduction 2 Lignocellulosic materials are an attractive feedstock because they are available in large quantities at a relatively low cost. Many countries have invested considerable resources into researching several types of lignocellulosic biomasses, such as aspen wood, tropical hard-woods, sugar cane bagasse, wheat straw and willow [69]. However, the choice of feedstock has been primarily dictated by what was locally available. The implementation of fuel ethanol on a large scale has been limited to a handful of countries. A s of 1984, Brazil had 1.5 million cars running on pure ethanol derived from corn crops. In the U S A and Canada, ethanol is used as an additive and octane enhancer. The fuel mixture is composed of 5-10% ethanol, the remainder being gasoline. A s of March 1997, pilot studies involving fleet vehicles in Illinois and Wisconsin have been conducted to test and promote Ford vehicles modified to run on either gasoline or E-85 (85% ethanol and 15% gasoline blends) [49]. Currently, raw materials for ethanol production come primarily from dedicated agricultural crops, as opposed to agricultural or industrial waste products. The option of using waste lignocellulosic residues as the feedstock for ethanol production has been explored by many researchers [13, 14, 15, 16, 17, 25, 28, 31, 35, 37, 38, 40, 41, 45, 46, 59, 61, 68, 74, 76] and government agencies [10, 42, 47, 63] to address the high cost of using dedicated crops. Although the process has been experimented on a large-scale [6], and planned on an industrial scale [3], commercial acceptance has not been widespread. Among the available saccharification technologies, enzymatic hydrolysis is recognised as an environmentally benign cellulose hydrolysis option which reduces the discharge of harsh chemicals. The enzymatic hydrolysis process is well developed. However, many obstacles, such as kinetic inhibitions (competitive reactions with lignin, intermediate and end-product inhibitions), difficulties in enzyme recycling, and economics prevent the process from becoming commercially attractive. These hurdles have resulted in intense research over Chapter 1. Introduction 3 the last several decades. Today, there is growing interest in enzymatic hydrolysis as a result of increased envi-ronmental concerns, and the introduction of new enzymes and genetically altered enzyme-producing microbes [53]. Organisms which ferment cellulose-derived glucose are well known, and organisms capable of direct conversion of cellulose to ethanol have been developed. This is not the case for the degradation of hemicellulose. Given that the average composition of lignocellulosics is 50% cellulose, 25% hemicellulose and 25% lignin, use of the hemicellulose fraction is very significant for the yields of alcohol from biomass processes. For example, ge-netic engineering has resulted in recombinant bacteria incorporating the Zymomonas mobilis genes allowing the direct conversion of xylose and arabinose to ethanol [28]. This accounts for two of the five sugars that make up hemicellulose. Other researchers have taken similar approaches [31, 36, 53]. The development of microbes capable of the direct conversion of both cellulose and hemicellulose fractions of lignocellulosics to alcohol is foreseeable in the near future. To put these promising technologies into perspective, over 70% of the materials placed in U S landfills are lignocellulosic, consisting of paper, cardboard, yard trash, wood products, etc. This is sufficient to produce over 10 billion gallons of fuel ethanol per year [28]. Together with other industrial residues, a large part of the 100 billion gallons of liquid fuel burned each year could be supplied as ethanol. In the US pulp and paper industry alone, roughly 12 million tonnes of combined primary and secondary sludge are produced annually [17] from operations such as liquid effluent treatment systems and milling operations. Traditional means of disposal include either in-cineration, i.e. burning the sludge as a fuel, or landfilling. The burning of sludge incurs cost because of dewatering requirements, and also represents an environmental concern due to gaseous emissions. Landfilling is becoming a less attractive option due to rising costs [2] Chapter 1. Introduction 4 and, perhaps more importantly, increasingly limited space and concerns over the environmen-tal impact [17]. Given that the handling and disposal of waste sludge can represent upwards of 40% of the cost of operating a wastewater treatment system [2] , a need for alternative means of dealing with these wastes, such as hydrolysis into a saleable product, exists. The objective of this work is to investigate the production of sugar from sulphite primary clarifier sludge (PCS) by focusing on the hydrolysis step. Several aspects of enzymatic hy-drolysis were considered, with the ultimate goal of constructing a continuous saccharification system. O f particular interest is the PCS from Tembec (a mill in Temiscaming, Quebec). The Tembec operation produces large quantities of PCS (40 tonnes/day, on a dry basis), primar-ily from its sulphite pulping process, and also from chemithermomechanical pulp ( C T M P ) production. In addition to using the PCS as a dewatering agent for activated sludge and as a fuel source in hog fuel boilers, a fraction of the PCS is landfilled. The Tembec operation also produces ethanol from its spent sulphite liquor by fermentation. The possibility of im-plementing a PCS-to-ethanol conversion process could reduce the amount of PCS requiring disposal, and increase ethanol productivity. Based on enzymatic hydrolysis technology, such a process could be implemented at a modest cost by using existing Tembec equipment and personnel. Chapter 2 Background 2.1 Substrate characteristics The principal fraction of biomass is made up of lignocellulosics. Although the ratios vary between species, wood is made up of approximately 50% cellulose, 25% lignin, and 25% hemicellulose on a mass basis. Cellulose is a linear polysaccharide, composed of repeating sugar units of glucose ( C 6 H i 0 O 5 ) which are joined by 13-1,4 glycosidic bonds. The recurring unit is actually two consecutive glucose anhydride molecules, known as cellobiose [62]. The structure of cellulose is shown in Figure 2.1. Due to the large number of hydroxyl groups on the cellulose molecule, hydrogen bond-ing occurs between adjacent strands. These strands group to form fibrils high enough in molecular weight to render them insoluble in water. The fibrils themselves are enclosed in a hemicellulose-lignin matrix [45]. Hemicellulose is a polymer similar to cellulose, except that it is composed of five different —Primary hydroxyl Secondary hydroxyl— Cellobiose units Figure 2.1: Cellulose structure [62]. 5 Chapter 2. Background 6 Figure 2.2: Two-dimensional representation of the repeating unit of spruce lignin [62]. sugars: • Hexoses: Glucose, mannose and galactose. • Pentoses: Xylose and arabinose. Pulp which has undergone chemical treatment always has a lower fraction of hemicelluloses, as they are more easily degraded and dissolved than cellulose. Lignin is an amorphous, highly-polymerised substance of mostly aromatic alcohols, which is difficult to degrade. The chemistry of lignin is very complex, as can be seen by the three-dimensional structure shown in cross-section in Figure 2.2. Chapter 2. Background 7 2.1.1 PCS (Primary Clarifier Sludge) production PCS is a mixture of waste cellulose fibres, undigested wood chips, sand and grit [45]. The waste cellulose (nearly 100% carbohydrates) is derived from fibres which are mostly due to screen inefficiencies. The PCS from Tembec also contains C T M P fibres, which have retained the majority of their original hemicellulose and lignin content. Waste streams, which include sand, grit and other foreign matter (such as bark), entering the mill with the wood chips are removed from the pulp and usually end up as part of the PCS. 2.1.2 PCS as a feedstock There are numerous advantages for using PCS as a feedstock instead of dedicated cellulosic energy crops for bioconversion to ethanol, and they represent a niche opportunity for the pulp and paper industry. First, PCS has a very low or negative cost, as compared to biomass which is grown specifically as an energy crop [39]. Second, solids captured in the primary clarifier have been through some pretreatment to remove lignin and hemicellulose from the fibre fraction, resulting in a sludge which has a much higher proportion of cellulose and is far more amenable to enzymatic hydrolysis than the wood from which is was derived [45]. Third, the PCS is already in the form of an aqueous slurry; the medium in which hydrolysis is carried out. Therefore, no dewatering is necessary. Fourth, running such a hydrolysis process in association with a pulp mill can reduce operating and handling costs because of the pre-existence of most required facilities (i.e. lower capital costs) and the sharing of personnel. In addition, environmental benefits ensue from the reduction of solid wastes, and a saleable product in the form of a clean-burning fuel. O n the other hand, in addition to the technical challenges of efficiently hydrolysing the cellulose contained in the PCS, PCS is limited in its availability, and there are competing Chapter 2. Background 8 uses, such as use as an aid in dewatering secondary sludges. 2.2 Pretreatment In general, enzymatic hydrolysis requires an efficient pretreatment to make the raw material more susceptible to attack by the cellulase enzymes. Although a large fraction the main substrate (PCS) has already undergone some pretreatment, further pretreatment could be ben-eficial to sugar yields. Due to the importance of pretreatment in enzymatic saccharification, significant factors affecting the efficiency of pretreatments to promote hydrolysis will be discussed. Literature data indicate that less than 20% of the carbohydrate content of lignocellulosic materials, which have not been pretreated, is convertible to monomeric sugars (see [69] for a summary). Several forms of pretreatment have evolved to render lignocellulosics more suscep-tible to hydrolysis, primarily as a result of lignin removal. The more common pretreatments are either chemical and/or mechanical in nature. 2.2.1 Chemical pretreatment Chemical pretreatments can be very effective in rendering a cellulosic substrate susceptible to enzymatic hydrolysis. The principle chemical pretreatments with potential for industrial application are steam explosion, organosolv processes and dilute-acid prehydrolysis [17, 77]. Chapter 2. Background 9 Steam explosion (autohydrolysis) Steam-explosion pretreatment involves heating wood chips with high-pressure steam. The steam, which permeates the wood, initiates an auto catalyzed hydrolysis reaction, or autohy-drolysis. The organic acids that are initially formed from the acetyl groups present in wood catalyze the hydrolysis of most of the hemicellulose [17]. After a specific reaction time, the wood chips undergo explosive decompression by release of a ball valve. The combined effect of the autohydrolysis and explosive decompression results in a feed with a more open structure containing less hemicellulose, and lignin which is partially degraded and restructured. This facilitates the penetration and action of the cellulolytic enzymes [69]. Steam explosion is generally regarded as one of the most cost-effective pretreatment pro-cesses for hardwoods and agricultural residues. For example, hydrolysis yields as high as 90% are possible from pretreated poplar chips or bagasse, up from 15% without pretreat-ment [20, 24, 55]. Although initially restricted to hardwoods and to chemically similar types of biomass (with high acetylated xylan content), steam explosion can be made equally effective on softwood by impregnation with acidic compounds, such as SO2, prior to pretreatment [69]. Dilute acid prehydrolysis Dilute acid prehydrolysis is similar to steam explosion with the major differences being that the substrate is first ground to a coarse powder (particle diameters of 0.25-1 mm), and that the substrate is fed into a continuous plug flow reactor as a 5-10% particle slurry in 1% sulphuric acid (as a catalyst) [17]. In terms of economics, steam explosion pretreatment is more practical, as particle sizes can range as high as 32 mm in diameter, and the water requirements are significantly less [58]. Chapter 2. Background 10 Furthermore, steam explosion can also make use of acid catalysts to improve yields. Organosolv pretreatment Organosolv processes, as the name implies, rely on various solvents (organic or aqueous-organic) to degrade the lignin and hemicellulose in lignocellulosic feedstocks [66]. Organosolv processes have found limited use as an environmentally friendly alternative to Kraft pulping in the pulp and paper industry. When applied to pretreatment, an Organosolv process yields three separate fractions: dry lignin, an aqueous hemicellulose stream, and a relatively pure cellulose fraction that is quite susceptible to enzymatic hydrolysis [17]. The principle disadvantage of such processes is cost. A l l solvents must be recovered and all the product steams must be used to make the Organosolv technology economically viable. 2.2.2 Mechanical pretreatment Size reduction by mechanical means such as hammer, roll or ball milling increases surface area of the substrate. This would account for the moderate improvements in hydrolysability. However, these treatments are energy-intensive and expensive, and generally do not perform as well as steam explosion or chemically-based treatments [69]. 2.3 Enzymatic hydrolysis It is not within the scope of this work to give a detailed review of the state of the art biochemical technologies for the conversion of cellulose to ethanol. A general overview, drawing attention to key areas, will be provided along with pertinent references to which the reader can refer for more detailed information. Chapter 2. Background 11 Synthesising ethanol from lignocellulosic material centers around two main reactions: 1. Hydrolysis of polysaccharides to monosaccharides and 2. Fermentation of the monosaccharides to ethanol. There are two primary methods of performing the saccharification step: acid hydrolysis and enzymatic hydrolysis. Much research has focused on acid hydrolysis, as it was the first process to appear and it has had an economic advantage over enzymes. Two types of acid hydrolysis are employed, dilute acid hydrolysis and concentrated acid hydrolysis, which use on the order of 1 and >5% acid, respectively. Research interest has shifted to enzymatic hydrolysis in recent decades for several rea-sons. Fungal and bacterial strains with increased capacity to synthesize high concentrations of cellulolytic enzymes have been developed [51]. Recombinant bacteria capable of converting cellulose and hemicellulose (both 6-carbon and 5-carbon sugars) directly to ethanol are seri-ously being considered beyond pilot scale [31, 53]. Enzymes with higher specific activities are also appearing. Lastly, and perhaps most importantly, increased environmental concerns heavily favour the enzymatic route. Acid hydrolysis involves a harsh chemical environment, whereas enzymes are environmentally benign. The study of enzymatic hydrolysis may have gained momentum from the perceived limi-tations and drawbacks of acid hydrolysis. Sugar yield from dilute acid hydrolysis is relatively small compared to yields from enzymatic hydrolysis. Using acid, only about 50% of the avail-able polysaccharides can be converted to monomeric sugar, compared to >90% by enzymes, depending on the substrate. Although concentrated acid hydrolysis can give yields approach-ing 100%, environmental and corrosion problems, and the high cost of acid consumption and recovery attribute to its lack of widespread adoption [77]. Chapter 2. Background 12 Raw Material Pretreatment ][ Modified lignocellulosics Hydrolysis (enzyme or acid) ][ Monomeric sugars Fermentation ][ Ethanol, H,0 Distillation V Ethanol Figure 2.3: Simplified hydrolysis process. The problems, or rather the challenges facing enzymatic hydrolysis, are still significant. Pretreatment of the raw material is necessary to make it sufficiently accessible to enzymatic attack. The cost of enzyme is high compared to acid. The saccharification process is con-siderably slower when using enzymes (on the order of days versus minutes or hours for acid hydrolysis), thereby requiring larger equipment. Furthermore, end-product inhibitions and enzyme denaturation limit the efficiency of the hydrolysis. 2.3.1 Scope & limitations The general process for biomass-to-ethanol conversion is outlined in Figure 2.3. A s discussed above, the hydrolysis step can be accomplished by means of cellulolytic enzymes or acid. In this research, all experiments used enzymes; acid hydrolysis was not investigated. The final two steps in the process (fermentation and distillation) were not considered in the experimental Chapter 2. Background 13 work. 2.3.2 The enzymes and their mechanism It is beyond the scope of this work to explore the detailed structures, functions and synergistic activities of the various cellulase enzymes that have been studied. Rather, a brief overview of current knowledge of the enzymes and their mechanisms, in addition to recent advances in understanding, shall be covered. Cellulases are a combination of several enzymes which act in concert to hydrolyse cel-lulose. The two principle sources in nature are bacterial and fungal cellulases. Cellulolytic bacteria generally produce cellulases with high specific activity as compared to fungal cel-lulases. However, bacterial cellulases are not easily produced in high titres and the bacteria themselves generally have a low growth rate. As a result, most applied research has focused on fungal-derived cellulases [17]. Fungal cellulases are a combination of three major classes of enzymes [12, 17, 73]: • endocellulases or endoglucanases (EG, endo-1,4-D-glucan glucanohydrolase, E C, • exoglucanases or cellobiohydrolases ( C B H , 1,4-fl-D-glucan cellobiohydrolase, E C, and • fi-glucosidase (EC Endocellulases attack randomly along the cellulose fibre, cleaving the internal 15-1,4-glycosidic bonds, resulting in a rapid decrease in chain length and yielding glucose, cellobiose and other water-soluble oligosaccharides. Exoglucanases attack both the reducing and non-reducing ends of these intermediates yielding cellobiose. Finally, B-glucosidase hydrolyses the cellobiose to produce glucose. Chapter 2. Background 14 © cellulose cellulase cellobiose (J-glucosidase glucose veast -> ethanol Generally, < > > © > © > © Figure 2.4: Cellulose to ethanol by enzymatic hydrolysis and fermentation with intermediate and end-product inhibitions. Figure 2.4 is a simplified illustration of the steps involved in the enzymatic hydrolysis of cellulose to glucose. The further step for the fermentation of glucose to ethanol is included, as would apply to a simultaneous saccharification and fermentation (SSF) operation. In this work, the last step in Figure 2.4 (inhibition 3) does not come into play. The reaction rates of the enzymatic hydrolysis are severely affected by intermediate and end-product inhibitions as illustrated in Figure 2.4. The endo- and exocellulases' activities are significantly reduced by an accumulation of cellobiose in the bulk aqueous phase. The activity of B-glucosidase is inhibited to a lesser degree by glucose. For a detailed review of the kinetic inhibitions, the reader is referred to Esterbauer et al. [18] and Penner et al. [50]. Generally, enzymatic hydrolysis is carried out in a cellulase broth to which is added a slurry of water-washed pretreated cellulosic material, such as the PCS used in this work. Augmenting the broth with B-glucosidase is commonly done to keep cellobiose levels low, and reduce cellulase inhibition. Lignocellulosic substrate concentrations usually do not exceed 5-10% (w/v) due to mixing difficulties. Chapter 2. Background 15 Although the structure of cellulose is quite well understood, much remains to be discovered of the mechanism of its enzymatic degradation [4, 17]. O f the numerous models that recently have been proposed to describe the mechanism of the enzymes [4, 11, 65, 71], the simplest is that of Walker et al. [73]: 1. transfer of enzymes from the bulk aqueous phase to the surface of the sold cellulose, 2. adsorption of the enzymes and formation of enzyme-substrate complexes, 3. hydrolysis of cellulose, 4. transfer of the intermediate hydrolysis products (water-soluble oligosaccharides includ-ing cellobiose, and glucose) from the surface back to the bulk aqueous phase, and 5. hydrolysis of the intermediates into glucose in the aqueous phase. From the above steps, several factors can be identified as affecting the hydrolysis process including the structural features of the cellulose substrate, the interaction between the cellu-lases and the cellulose fibre (i.e. adsorption, denaturation, etc.), the nature of the cellulases used, and the enzymes' susceptibility to product inhibition [73]. As a result, when investigat-ing new substrates and enzymes, it is difficult and often misleading to estimate results from previous work on similar systems [1]. 2.3.3 Enzyme recycle U p to 80% of the total cost for enzymatic hydrolysis is attributed to the cost of the enzymes themselves [21]. This necessitates optimal use and re-use of the enzymes to make the process commercially viable. Several factors which affect enzymatic hydrolysis, as mentioned above, also hamper efforts to efficiently reuse the cellulase enzymes [9]. O f primary concern is the Chapter 2. Background 16 adsorption of the cellulase complex to the solid residues. The mode of action of adsorbed cellulases on the surface of the substrate is still not well understood, due mainly to the multiplicity of the cellulase enzyme complex [4]. Cellulase adsorption isotherms have been used in an attempt to estimate the recovery of enzyme from the hydrolysate [21]. Preliminary results from Galbe et al. [21] have shown that after hydrolysis of steam-pretreated willow by Trichoderma reesei cellulases, only 20% of the original CMC-ase activity1 remained in the hydrolysate. Elution of the unhydrolysed solid residues by several washings with tap water recovered an additional 11% of the original activity. Work examining the reuse of the bound cellulases has shown that fresh batches of Eu-calyptus could be hydrolysed with little loss of enzyme activity [54]. Although the results are encouraging, as they indicate the enzymes may be efficiently reused several cycles, the methodology employed by Ramos et al. [54] is not easily tranferrable to an industrial process. The presence of lignin in the lignocellulosic substrate, which can range anywhere from 15 to 36%, can be of particular concern [8, 67]. Due to its nature—an "encrusting" substance of the plant cell wall—lignin induces hindrances for the enzymatic attack of cellulases on cellulose by binding with the cellulose. Therefore, the degree of delignification can play an important role in the hydrolysability of lignocellulosic substrates. Furthermore, lignin can adsorb and inactivate cellulolytic enzymes, thereby eliminating them from the reaction zone. This effect can be particularly problematic for continuous hydrolysis reactors, since the elimination from the reactor of the unhydrolysed residue, mostly consisting of lignin with adsorbed cellulases, will increase the throughput of enzymes compared to the continuous hydrolysis of delignified cellulose [8]. Yu et al. [79] found that after a 12 h hydrolysis of steam-exploded birch using T. reesei 'The carboxymethyl cellulose (CMC) assay is a measure of endoglucanase activity (see [23]). Chapter 2. Background 17 cellulases, only 40-50% of the total initial activity could be recovered. The remainder of the cellulase activity was associated with the Hgnin residue. However, the effect lignin may have is highly dependant on the substrate itself and the nature of the enzymes. Ishihara et al. [29] observed no appreciable adsorption of specific cellulase components onto lignin during the prolonged hydrolysis of steamed shirakamba wood. A n d although the presence of lignin tended to slow down the enzyme adsorption to the cellulose, it did not appear to restrict the extent of hydrolysis of the carbohydrate moiety. The study concluded that lignin removal prior to hydrolysis of steamed hardwood is not always necessary for the recycling of cellulolytic enzymes. In addition to other studies examining the adsorption of cellulases onto various forms of cellulose [22, 32], there is evidence pointing towards interaction between the cellulase components influencing the degree of adsorption [34, 57, 79]. Researchers have modeled enzyme adsorption accounting for slow deactivation of the adsorbed enzyme [11], and also enzyme recycle [4]. The mathematical models agreed well with the experimental results, although the identification of the model parameters required considerable substrate- and enzyme-specific data. A n d despite the accuracy of the models, the mechanism of enzyme deactivation is still not understood. The most practical and simplest means of enzyme recycle is by recirculating the spent hydrolysis solids, thereby contacting the adsorbed enzymes with fresh substrate [70]. Reported gains in sugar yields were 7 and 14% for steam-exploded aspen wood and wheat straw, respectively [70]. A s an additional process improvement, further use of the spent substrate as the growth medium for cellulolytic enzyme-producing fungi was found to increase cellulase yields [43]. Chapter 2. Background 18 2.4 Economics Cellulose is the single most abundant renewable resource. It has been estimated that through photosynthetic fixation of CO2 as much as 2 2 x l 0 9 tons of cellulose are produced annually worldwide, and that as much as 4x 109 tons/year could be available for processing [52]. For lignocellulosic sources, standing timber has been estimated to cost approximately US$36/ton [19]. Tree plantations using new strains, either genetically enhanced or selectively bred, could supply biomass at a cost of US$20.5/ton [19]. But the most promising source is waste cellulosic materials which are currently being landfilled or burned. The cost of such wastes may be very low, or even negative. A report for the Ontario Ministry of Environment & Energy [75] found that a municipal solid waste (MSW) to ethanol operation, by enzymatic hydrolysis, could realise a 20% ROI by requiring a tipping fee of about C$62/ton, assuming an ethanol selling price of 25 cents per litre. A n ROI of 25 and 30% could be attained by requiring tipping fees of C$86 and 109/ton, respectively. Such fees are significantly lower than municipal tipping fees, which may reach as high as C$175/ton for Metropolitan Toronto [75]. It is worth noting that only a 6.4% ROI is realised if the waste paper is accepted at zero cost, which is not enough to attract private investors. These price estimates include all plant construction costs (fixed capital, working capital, engineering and management during construction and financing) and annual operating costs (including maintenance and depreciation). A significant reduction in capital and operating costs for a waste paper to ethanol plant could be realised by partnering with a nearby plant which has available excess low pressure steam and/or electricity, such as a pulp and paper mill [75]. O n the process side, lignocellulosic substrates generally require pretreatment to make Chapter 2. Background 19 the cellulosic fraction susceptible to the cellulase enzymes. Lignocellulosic wastes, such as PCS, have the advantage of having undergone pretreatment for removal of the lignin and hemicellulose portions of the wood [17]. The residual cellulose fraction of PCS has been shown to be highly amenable to enzymatic hydrolysis [15, 45]. Several general factors affect the production costs of ethanol, such as the assumptions made in the technical and economic calculations, the nature of the raw materials used (MSW, softwood, hardwood, etc.), the type of process utilised (enzymatic, dilute/concentrated acid or A C O S 2 ) , and the design of the process (separate hydrolysis and fermentation, S S F 3 , pen-tose fermentation, etc.). The difficulty in economic comparisons of enzymatic hydrolysis to competing technologies is that assumptions are, for the most part, unavoidable as there are no full-scale plants from which information on yields and other crucial data are available [72]. For a full review of the economics of ethanol production from lignocellulosics the reader is referred to von Sivers et al. [72]. The study reviewed enzymatic hydrolysis, dilute and concentrated acid hydrolysis processes. The review found that ethanol production costs (in 1994 dollars) were in the range of US$0.40-0.80/L. Not surprisingly, ethanol production costs for enzymatic hydrolysis decreased with increased plant capacity and higher yields. A n d yet, work conducted by Clesceri et al. [9] on enzyme recycle in batch systems suggests that there may be an economic tradeoff in a slight sacrifice in yield if enzyme recovery is improved. Cost projections from the US Department of Energy [48] indicate the selling price of wood-derived ethanol at about US$0.32/L, based on a simultaneous saccharification and fermentation process which is capable of fermenting xylose at 70% efficiency [48]. In order to be competitive with gasoline as a neat fuel, ethanol would have to sell for US$0.18/L at an oil price of US$25/bbl [17]. Nevertheless, substantial opportunities still exist to lower the projected selling price [78]. 2Acid catalyzed organosolv saccharification. 3 Simultaneous saccharification and fermentation. Chapter 2. Background 20 Niche opportunities also exist, such as the operations at the Tembec mill in Temiscaming, Quebec. The ethanol produced from its spent sulphite liquor is distilled to food-grade ethanol, which sells for up to C$0.50/L. Additional ethanol produced from Tembec's waste PCS using enzymatic hydrolysis could also be sold for similar use. It is promising that enzymatic hydrolysis, as compared to dilute or concentrated acid hydrolysis, has been and still is the focus of most research interest since the early 1980s [72]. Chapter 3 Research Objectives The goal of the research in this thesis was to investigate the production of sugar from sulphite primary clarifier sludge. Several aspects of enzymatic hydrolysis were considered to determine practical ways to reuse enzyme in a continuous system. The effort can be subdivided into three stages. First, the nature of the substrate is investigated by batch hydrolysis and adsorption exper-iments. Issues such as maximum sugar yield, maximum enzyme loading for enzyme binding site saturation, and increasing sugar yield through pretreatment were examined. Second, the question of whether enzyme activity loss is responsible for limiting hydrolysis was addressed. Third, the results gathered from the operation of a continuous, laboratory-scale hydrolysis reactor were compared to those measured in batch experiments under similar conditions. 21 Chapter 4 Experimental 4.1 Materials 4.1.1 Substrates Primary clarifier sludge (PCS) from sulphite pulp was supplied by Tembec Inc. (Temiscaming, Quebec) as approximately 10% solids, by mass. Solka floe (James River Corporation, Berlin, N H ) was used as a source of pure, amorphous cellulose. Sigmacell 50 (Sigma, St. Louis, M O ) was used as a source of pure, crystalline cellulose. Pure celluloses were used for experiments that required a substrate without impurities. Additional PCS samples were obtained from three B . C . mills: Kraft and T M P from Fletcher Challenge Canada Ltd. (Elk Falls M i l l , Campbell River), C T M P from Fiberco Pulp Inc. (Taylor), and Kraft from Western Pulp Ltd. Partnership (Squamish mill). Integrated PCS was made from a 70/30% mixture of TMP/Kraft PCS from Elk Falls. The above PCS samples were used to compare the amenability to hydrolysis of Sulphite PCS to that of sludges produced from different pulp processes. Pulp samples were obtained from three B . C . mills: Howe Sound Pulp & Paper Ltd. (Port Mellon), Canadian Forest Products Ltd. Intercontinental M i l l (Prince George) and Northwood Pulp & Timber Ltd. (Prince George). Samples from both upstream and downstream of the oxygen-delignification process were used to determine the effect of commercial pretreatment 22 Chapter 4. Experimental 2 3 Table 4.1: List of substrates and how they were used. Experiment Substrates Maximum hydrolysis Solka floe Sigmacell 50 Sulphite PCS Kraft PCS T M P PCS C T M P PCS Adsorption Solka floe Sigmacell 50 Pretreatment Howe Sound Pulp Intercontinental Pulp Northwood Pulp Sulphite PCS Reactor operation Sulphite PCS on enzymatic hydrolysis. Table 4.1 lists the substrates and in which experiments they were used. 4.1.2 Enzymes Cellulase enzymes derived from Trichoderma reesei were obtained from Novo-Nordisk (Den-mark) as Celluclast C C N 1.5L (78 FPU/mL glucanase activity). Cellobiase enzyme de-rived from Aspergillus Niger were also obtained from Novo-Nordisk as Novozym T N 188 (780 IU/mL fi-glucosidase activity). Chapter 4. Experimental 24 4.1.3 Small-scale hydrolyses The small-scale hydrolyses were performed in 250 m L Erlenmeyer flasks, at 125 m L working volume. The flasks were placed in a shaker/incubator (model 4628SHOW, Lab-Line Instru-ments Inc., IL), in which the hydrolyses were carried out under controlled temperature and agitated conditions. 4.1.4 Reactor apparatus The laboratory-scale reactor consisted of a 5 L-capacity C S T R with a sealed water jacket (Figure 4.5). The feed tank was a 20 L STR, also with a sealed water jacket. A l l reactor flow lines were 3/4" flex-hose tubing. A Jabsco model 30510-0001 flexible impeller pump was used in conjunction with two three-way actuated ball valves for both reactor recycle and introduction of fresh feed. Solids separation was accomplished using a 20 L gravity clarifier. Both supernatant and solids streams were split and either diverted back to the reactor or to waste. Although the main reactor was operated at a 5 L volume, the total operating volume was approximately 22 L . This is the summation of the reactor, tubing ( « 1 . 5 L), and clarifier ( « 1 5 . 5 L) volumes. 4.2 Methods 4.2.1 Hydrolysis in buffered solution Reactions were performed in a 0.2 M sodium acetate buffer solution (pH 4.8), at 5 0 ° C and agitated at 150 R P M . These conditions have been found to be optimal for cellulolytic enzymes [17]. Prior to enzyme addition, the substrate and buffer solutions were combined Chapter 4. Experimental 25 2 0 L P C S F e e d t a n k ( s t i r r e d & t e m p e r a t u r e c o n t r o l l e d ) E n z y m e s 5 L C S T R ( t e m p e r a t u r e c o n t r o l l e d ) ( - 3 % s o l i d s i n 0 . 2 M a c e t a t e b u f f e r , p H 4 . 8 ) Figure 4.5: Schematic of the reactor setup. and allowed to reach reaction temperature conditions for 1 hr. Hydrolyses were initiated by the addition of enzymes (6-glucosidase followed by glucanase). The substrate levels in the flasks were varied from 5-50 g/L. Enzyme loadings varied between 5-50 F P U of glucanase and 50-500 IU of B-glucosidase activities per gram of substrate. PCS and enzymes were stored at 4 ° C until use. Duplicate flasks were run in hydrolysis experiments (and in all other experiments, except where noted). 4.2.2 Adsorption Experiments were carried out in 35 m L test-tubes (30 m L liquid volume), in a 0.2 M sodium acetate buffer solution (pH 4.8). The substrate and buffer solution were combined in the Chapter 4. Experimental 26 tubes and were placed in a refrigerator (4°C) to allow them to reach reaction temperature for 1 hr. At this temperature, cellulotic enzymes are mostly inactive and thereby hydrolysis is prevented. Enzyme (glucanase) was added, the tubes were agitated by inversion, and then incubated for 5 minutes. The tubes were centrifuged (model CU-5000, IEC Ltd., M A ) and the supernatant was sampled. The process was repeated several times (addition of more enzyme, shaking and incubation, centrifugation and sampling), for enzyme loadings of approximately 5-35 IU/g. 4.2.3 Oxygen delignification The PCS was delignified by sparging it with oxygen (1 L/min) in a solution of 0.8 M N a O H at 2% consistency. Reactions were carried out at 8 0 ° C and atmospheric pressure under vigorous agitation. The oxygen delignification was terminated by neutralizing the PCS solution with 2 M H2SO4. After washing, the residual PCS was hydrolysed and tested for lignin content. Washed, untreated PCS samples were used as standards for the hydrolysis experiments. A second set of delignifications were carried out using a BioFlo IV 20 L reactor (New Brunswick Scientific Co., NJ). The reactions were carried out at 8 0 ° C and 30 psi in a solution of 0.8 M N a O H at 2% consistency, under vigorous agitation while sparging with oxgygen. Magnesium sulphate (MgS0 2 ) was added to supply Mg""" at 0.5 g/kg PCS, to prevent cellulose degradation. A comparison the operating conditions of a typical commercial oxygen delignification process with the lab-scale operation is shown in Table 4.2. 4.2.4 Reactor operation Operation parameters of the semi-continuous reactor are listed in Table 4.3 on the next page. Chapter 4. Experimental 27 Table 4.2: Comparison of the operating conditions of the laboratory 02-delignification and a typical medium consistency commercial processes. Units Commercial! Lab-scale Pulp consistency % 10-14 2 Delignification % 40-45 30 Rentention time min 20-30 60 Temperature °C 85-95 80 Alkali consumption kg/t 18-21 20 Oxygen consumption kg/t 20-24 20 M g + + kg/t 0.5 0.5 Reactor pressure psig 80-97 30 f Source: [26] Table 4.3: Operating conditions of the reactor. Parameter Value Total reactor volume Initial PCS consistency Enzyme loading Residence time Recirculation rate Recycle ratios: solids liquid 22 L 2.5 % 10 FPU/g PCS ^22 hr 60 L/min 0.9 0.1 1 \ Chapter 4. Experimental 28 Reactor startup The steps for starting up the hydrolysis reactor were as follows: 1. Fill feed tank (20 L) and reactor vessel (5 L) with a PCS/acetate buffer mixture. The maximum initial sludge consistency should be no higher than 3%, otherwise the 3 /4" tubing and/or the valves will plug. 2. Activate stirrers and water bath/circulator and allow the PCS mixture to reach reaction temperature ( 5 0 ° C ) . 3. Close all valves contolling the recycle and drainage from the settling tanks. Before starting the pump, ensure the lines to the pump are primed with some water (do not run the pump dry). 4. Activate the valve diverting flow from the feed tank to the settling tank. Activate the pump, gradually raising the pump speed up to no higher than 30% of maximum. 5. Once the settling tank over-flow line begins to flow, stop the pump. Activate the valve diverting flow from the reactor tank to the settling tank. 6. To begin reactor operation, add initial load of enzymes ((B-glucosidase followed by glucanase). Start the pump, gradually raising the pump speed to no higher than 50%. 7. Open the valves contolling the recycle from the settling tanks. Adjust the pump speed and recycle valves to maintain a constant fluid level (at the 5 L mark) in the reactor, and to control the solid/liquid recycle flow ratio. 8. Every hour, harvest 1 L from solid and/or liquid recycle streams, and introduce fresh feed from the feed tank, and add fresh enzymes. Chapter 4. Experimental 29 Reactor shutdown The steps for shutting down the hydrolysis reactor were as follows: 1. Shut off the main pump. 2. Close the settling tank recycle valves. 3. Open the drain valves to empty the settling tank. The reactor contents can be pumped out via the settling tank. 4. Thoroughly wash all tanks and flow lines with water to avoid any build-up of dry pulp. 4.3 Analyses 4.3.1 Sampling protocol, all experiments Representative 1.4 m L samples were withdrawn and centrifuged (13,000 g, 10 min) in a Fisher Micro Centrifuge (model 235C, Fisher Scientific, Fair Lawn, NJ). The supernatant liquids were then pipetted into capped 1.5 m L vials and frozen (at - 2 0 ° C ) for later analysis. 4.3.2 Reducing sugars assay The concentration of total reducing sugars was determined colorimetrically using a dinitros-alicylic acid (DNS) reagent [44]. Three millilitres of an appropriately diluted sample was combined with 3 m L of D N S reagent in a sealed 10 m L Hach test tube and placed in boiling water for 15 min. Chapter 4. Experimental 30 A Hach single-beam, direct reading spectrophotometer (model DR/2000) was used to read the absorbances of the coloured solutions. Calibration using glucose as a standard by this method revealed a linear range of 0.1-0.3 g/L at a wavelength of 575 nm. 4.3.3 Glucose assay The concentration of 13-D-glucose was determined by analysis in an enzyme-based glucose analyzer (Glucose Analyzer 2, Beckman Instruments Inc., C A ) . Ten microlitres of an appro-priately diluted sample was injected into the analyzer. The detection range of the analyzer is 0.10-4.50 g/L, with a measurement error of approximately ± 0 . 0 3 g/L. 4.3.4 Filter paper assay The enzyme activity was determined by the filter paper assay, as described by the IUPAC procedure [23], measured in filter paper units (FPU), which is an estimate of the enzyme concentration necessary to release exactly 2.0 mg of glucose from hydrolysis of a Whatman No. 1 filter paper strip. A Polystat #12050-00 (Cole-Parmer Instruments Co., IL) circulating bath/shaker was used. 4.3.5 Protein in solution Bio-RAD assay Total protein was determined by the Bio-Rad assay, based on the method of Bradford [7]. One hundred microlitres of an appropriately diluted sample was pipetted into a test tube, then 5.0 m L of diluted dye reagent was added to each tube. After vortexing and incubation at room temperature for 15 min, the absorbance of the samples was measured at 595 nm with Chapter 4. Experimental 31 the Hach DR/2000 spectrophotometer. Calibration using bovine serum albumin as a standard revealed a dynamic linear range of 0.2-1.0 g/L. A280 absorbance A s the detection range of the B i o R A D assay was limited, total protein was also determined by measuring absorbance at 280 nm using a HP Scanning U V Diode Array Spectrophotometer (model HP8452A). Calibration using bovine serum albumin and Celluclast/Novozym enzyme as standards revealed a dynamic linear range of 0.01-0.16 g/L. 4.3.6 Lignin content (Kappa number, TAPPI T236 cm-85 and CPPA G.18) The percentage of lignin in an unbleached sulphite softwood pulp may be estimated as about one-seventh of its Kappa number1. TAPPI and CPPA standards stipulate that Kappa number tests use 1 gram of oven dry pulp oxidized in a 1 litre solution of 0.1 N potassium perman-ganate (100 mL) and 4 N sulphuric acid (100 mL) for 10 minutes at 2 5 ° C . The volume of potassium permanganate consumed is determined by thiosulphate titration and corrected to 50% consumption of the permanganate added. 4.3.7 Statistics A l l error bars in graphs represent 95% confidence intervals based on the average of dupli-cate runs sampled three times each, unless otherwise noted. Best fit curves for hydrolysis experiments used the Michaelis-Menten model for enzyme kinetics (a hyperbolic function). Goodness of fit was tested using the chi-squared distrubition at a 95% confidence level. 1 Lignin content (%) = Kappa number x 0.187 Chapter 4. Experimental 32 Comparison of population means was done using the t test at a 95% confidence level. Analysis of the factorial design was done using Jass version 2.1 computer software to deter-mine the significance of parameter and interaction effects. Chapter 5 Results and Discussion 5.1 Hydrolysis experiments A number of small scale batch hydrolysis experiments were performed on various substrates in addition to PCS. These experiments were designed to study the nature of the substrate and how variables such as buffer concentrations, enzyme loading, and substrate concentrations affect the degree of hydrolysis. Besides providing useful general information on enzymatic hydrolysis, the effect of these variables will assist in the operation of the continuous hydrolysis reactor. 5.1.1 Maximum sugar conversion To determine the hydrolysable fraction of cellulose and hemicellulose, extreme conditions were applied to various PCS substrates. A n enzyme loading of 50 FPU/g substrate was used. A n initial substrate concentration of 50 g/L was chosen, as from a commercial standpoint, economically viable processes require concentrated sugar product streams. The hydrolysis results are illustrated in Figure 5.6. As expected, the pure forms of cellulose (solka floe and Sigma Cell 50) yielded the highest sugar levels. O f the sludges, those from Elk Falls and Fibreco have one half as much a hydrolysable fraction as the Tembec PCS. The Kraft sludge from Western Pulp was largely grit or dirt, and few cellulose fibres could be discerned visually. This would explain the very low sugar conversion. 33 Chapter 5. Results and Discussion 34 Solka Floe Sigmacell Kraft CTMP TMP Sulphite Kraft (WP) Integrated* 50 Figure 5.6: Maximum sugars conversions for the enzymatic hydrolysis of different substrates. The hydrol-ysis time was 72 hr for 50 g/L substrate, on a dry basis, at an enzyme loading of 50 FPU/g. Table 5.4: Percentage sugar yields for the enzymatic hydrol-ysis of different substrates (based on total reduc-ing sugars). Substrate Percentage Conversion (%) Solka floe 79 Sigma Cell 50 62 Sulphite 50 Kraft (Elk Falls) 31 T M P (Elk Falls) 28 C T M P (Fibreco) 17 Integrated 13 Kraft (Western Pulp) 2.4 Chapter 5. Results and Discussion 35 A s can be seen by the percentage conversions in Table 5.4, the amenability to hydrolysis of the Tembec PCS compares favourably to the pure forms of cellulose. A relatively high fraction of the PCS is hydrolysable cellulose. Considering that the conversions of the pure forms of cellulose average to 70%, the 50% conversion from the sulphite PCS suggests a relatively high fraction is cellulose (on the order of ). The remaining unhydrolysed fraction was likely made up of cellulose and hemicellulose inaccessible to the enzymes, lignin, extractives, sand and other inorganic matter [45]. 5.1.2 Effect of acetate buffer on hydrolysis The sodium acetate/acetic acid buffer is necessary to maintain a p H of 4.8, which is the optimum for Trichoderma reesei enzymes, both in terms of thermal stability and peak ac-tivity [5, 56]. Figure 5.7 illustrates the effect of acetate buffer strength on hydrolysis over a range of 0.02 M to 1.0 M . The trend is dilute buffer solutions have little effect on the hydrolysis, after which point, increasing buffer strengths significantly inhibit enzyme activity, thereby reducing sugar conversions. The results indicate that the maximum buffer strength, without having a significant impact on the enzymatic hydrolysis, is 0.2 M . 5.1.3 Effect of enzyme loading and substrate concentration on hydrolysis Both the rate and the degree of enzymatic hydrolysis of lignocellulosics is proportional to the extent to which the cellulose fraction of the material is accessible to the enzymes. For the degradation of the cellulose by cleavage of the glycosidic bond to occur, a cellulase molecule must be in contact with an active site of the cellulose surface. Such contact is achieved in a solid/liquid mixture, by maintaining a homogeneous slurry through continuous agitation. Chapter 5. Results and Discussion 36 25 h 0 4 8 12 16 20 24 Time, hr Figure 5.7: Effect of acetate buffer concentration on the en-zymatic hydrolysis of solka floe. In the hydrolysis system under study, mass transfer effects, such as diffusion rates of enzymes to the active sites on the substrate, and reaction products from the substrate/liquid interface to the bulk liquid, were not studied as it was beyond the scope of this project. Several substrates were hydrolysed to observe the effect of different enzyme loadings and substrate concentrations on the hydrolysis reaction. Solka floe A two-way factorial hydrolysis experiment was conducted using solka floe (see Table 5.5 for the conditions), and all runs were conducted in parallel. A s solka floe is a pure and finely-ground amorphous cellulose, rheology problems associated with PCS are avoided, and therefore substrate accessibility effects are minimized. The effects of enzyme loading and substrate concentration on hydrolysis can be determined without the presence of additional Chapter 5. Results and Discussion Table 5.5: Summary of solka floe hydrolysis' data. 37 Solka floe Enzyme Percentage Initial hydrolysis Specific initial concentration loading conversion rate hydrolysis rate (g/L) (FPU/g PCS) (% after 24 hrs) (g/(L h)) (g/(L h FPU)) 10 10 60.9 1.31 0.131 10 5 21.5 0.88 0.176 1 10 16.2 0.27 0.027 1 5 9.0 0.18 0.036 impurities introduced by PCS. Figure 5.8 and Figure 5.9 illustrate the hydrolysis of solka floe, measured by total reducing sugars (TRS) and glucose concentrations, respectively. At the low substrate concentration of 1 g/L, it can be seen that a doubling of the enzyme loading corresponded to almost double the final glucose conversion. This indicates that the system is not diffusion limited, but enzyme limited. At the low enzyme loading, the number of active sites available on the substrate surface exceed the number of active cellulase molecules. Whether the active sites have been saturated at the higher enzyme loading is not apparent. Results from Hogan et al. [27] suggest that saturation is reached at 25 FPU/g solka floe BW300, based on initial adsorption, actual hydrolysis and/or combined hydrolysis and fermentation data. Although the solka floe substrate was similar, the enzyme preparation was derived from Trichoderma harzianum E58. Analysis of the factorial experiment at 24 hr revealed that the most significant factor was the enzyme loading, whose effect on average was to increase T R S by 2.7 g/L at the high level (10 g/L solka floe). The next significant factor was substrate concentration which on average raised T R S by 2.4 g/L at the high solka floe level. Finally, there was a positive interaction effect between solka floe concentration and enzyme loading (a 1.8 g/L increase on average). A l l effects were found to be statistically significant. Chapter 5. Results and Discussion 38 • 10 g/L , 10 F P U / g So lka Floe • 10 g/L , 5 F P U / g Time, hr Figure 5.8: Enzymatic hydrolysis of different solka floe concentrations and enzyme loadings. Measured by total reducing sugars. For the two substrate concentrations, a doubling of the enzyme loading resulted in a 50% increase in the initial hydrolysis rate. This translates to increases of 33 and 34% in the specific initial hydrolysis rates for the 1 and 10 g/L substrate concentrations, respectively. The number of active sites does not increase if the substrate concentration remains con-stant. Therefore, the kinetics of the enzyme complex are somehow affected by the doubling of the enzyme concentration. The mechanism of cellulase adsorption and cellulose hydrolysis is still not completely understood due to the multiplicity of the cellulase enzyme complex. A possible explanation for the observed trend could be as follows: i f a complex of enzymes must act at a "common locus" of an active site on the solid cellulose substrate, whether it be simultaneously or sequentially, the probability of the complex acting correctly in tandem is increased with a rise in enzyme concentration in the bulk solution. A ten-fold increase in the solka floe concentration (from 1 to 10 g/L) results in only Chapter 5. Results and Discussion 39 • 10 g/L, 10 FPU/g Solka Hoc • 10 g/L, 5 FPU/g • 1 g/L, 10 FPU/g 0 4 8 12 16 20 24 T i m e , hr Figure 5.9: Enzymatic hydrolysis of different solka floe concentrations and enzyme loadings. Measured by glucose concentrations. a five-fold increase in both initial hydrolysis rates. It has been suggested by Penner and Liaw [50] that a form of substrate inhibition at higher concentrations is responsible. Pen-ner and Liaw's experimental work showed an asymptotic decrease in T. reesei activity with increasing substrate concentration. It was postulated that the competitive adsorption of incom-plete components of the cellulase complex at physically distinct active sites acted to inhibit overall enzyme activity. At higher enzyme concentrations, a relatively higher activity could be due to a synergistic effect, yielding a kinetic advantage over enzymes acting independently. At the high substrate level, a three-fold increase in the 24 hr glucose conversion is ob-served. Although the initial glucose levels are approximately one half of the 10 F P U enzyme loading for the first four hours, further hydrolysis results in disproportionately higher conver-sions at the higher enzyme level. Such behaviour can likely be a result of a combination of substrate inhibition [50], irreversible enzyme adsorption and/or enzyme deactivation, which becomes progressively more significant as the hydrolysis proceeds. Chapter 5. Results and Discussion Table 5.6: Summary of PCS hydrolysis' data. 40 PCS Enzyme Percentage Initial hydrolysis Specific initial concentration loading conversion rate hydrolysis rate (g/L) (FPU/g PCS) (% after 48 hrs) (g/(L h)) (g/(L h FPU)) 5 22.2 1.75 0.35 25 7 35.0 2.50 0.36 10 43.8 3.35 0.34 15 49.0 4.23 0.28 2 18.6 2.1 1.07 50t 5 27.7 4.8 0.95 7 30.3 6.6 0.94 10 31.5 7.9 0.79 fSource: [45] A s can be seen by comparing Figures 5.8 and 5.9, the total reducing sugars results are an average of 24% higher than the measured glucose levels. Although some complex sugars may not have been fully converted to the glucose end-product, it is more likely that the T R S values are high [1, 60]. PCS Similar hydrolysis experiments were conducted on the Tembec PCS. One substrate concen-tration (25 g/L) was investigated at several enzyme loadings, as tabulated in Table 5.6 and illustrated in Figure 5.10. Results from Moritz's work are included for comparison, and are illustrated in Figure 5.11 [45]. The most significant trend observed in Figure 5.10 is that the maximum sugar conversion is a direct function of the enzyme loading. This indicates that the enzyme activity is being deactivated by the PCS, probably from a combination of irreversible adsorption, thermal denaturation, and substrate inhibition described in the previous section. Unfortunately, little Chapter 5. Results and Discussion 41 14 r Time, hr Figure 5.10: Enzymatic hydrolysis of PCS for 25 g/L at dif-ferent enzyme loadings. of these processes is understood, and it is difficult to separate their individual contributions. A s compared to the higher substrate level in Figure 5.11 on the following page, the 25 g/L hydrolyses reach equilibrium more rapidly. This may be due, in part, to the higher enzyme to substrate ratio, and also in part to rheology. At 50 g/L, mixing is very poor during the first few hours of hydrolysis, due to the higher PCS consistency. A s a result, accessibility of active sites on the PCS surface to the enzyme is hampered. At 25 g/L, the reaction mixture does not suffer the same mixing-related problems. Maximum sugars conversions for the 25 g/L hydrolyses appear to be reached at 24 hours. The decline in the sugar levels after 24 hours is probably due to microbial growth in the reaction mixture. From a process standpoint, however, it is noteworthy that by 7 hours, on average, 72% of the 24-hour maximum conversion has already been reached. After 12 hours of hydrolysis have elapsed, 87% of the maximum sugar conversion has been achieved. Insofar as the design of a continuous system is concerned, reactor solid residence times far in excess Chapter 5. Results and Discussion 42 3 5 30 2 5 ; J 2 0 o> co 10 5 0 0 8 16 2 4 32 4 0 4 8 Time, hr Figure 5.11: Enzymatic hydrolysis of PCS for 50 g/L at dif-ferent enzyme loadings [45]. of 12 hours might not be economically justified for the modest increase in conversions. However, longer residence times would be necessary for the hydrolysis of a higher con-sistency PCS slurry. The 50 g P C S / L hydrolysis takes far longer to reach a maximum. Nevertheless, at 24 hours an average of 82% of the 48-hour sugar conversion is reached, and at 16 hours, 74%. The one obvious advantage of the higher consistency is the higher final concentrations (for 10 FPU/g PCS loading, 25 g/L consistency gives a maximum conversion of 11 g T R S / L , whereas 50 g P C S / L yields 31 g TRS/L) , which greatly reduce production costs associated with downstream purification of the resulting alcohol from fermentation of the sugars. From these hydrolysis results, it is clear that there is a compromise between production rate (conversion efficiency and product concentration) and efficiency of enzyme use. Chapter 5. Results and Discussion 43 6 h 0 8 16 24 32 40 48 Time, hr Figure 5.12: Hydrolysis of solka floe and Sigma Cell 50 at 10 g/L and 10 FPU/g substrate. 5.2 Protein adsorption experiments Several experiments were conducted to determine the extent to which the cellulase enzymes can be recycled. Estimates of the enzyme levels were measured by total protein in solution, and enzyme activities were measured by conducting either filter paper assays or further hydrolyses. 5.2.1 Adsorption during hydrolysis The hydrolysis experiment in Section 5.1.3 was further examined for protein content and enzyme activity over time. Included is the hydrolysis of Sigma Cell 50, so that both amorphous and crystalline forms of pure cellulose can be compared. The hydrolyses are illustrated in Figure 5.12. A s can be seen in the figure, the solka floe is hydrolysed a little more easily than the Sigma Cell 50 substrate over the first 16 hours. This is most likely due to the difference in the crystallinity of the cellulose, the solka floe being the amorphous of the two. The transfer Chapter 5. Results and Discussion 4 4 Figure 5.13: Total protein concentrations throughout the en-zymatic hydrolysis of Sigma Cell 50 and solka floe for 10 g/L substrate at an enzyme loading of 10 FPU/g. of enzymes from the bulk solution to the surface of the solid cellulose is facilitated by the amorphous structure of the solka floe. A s can be seen in Figure 5.13, the protein concentration in solution shows a steady increase over the first 8 hours of the hydrolyses, which is assumed to be attributable to the liberation of enzymes as they complete the hydrolysis of the substrate to which they were adsorbed. Baseline solutions of the buffer and substrate, in the absence of enzyme, were subtracted from the measured protein values from the hydrolysis. The increase of protein, from 1 hr to the highest levels, is 16.3% and 21.7% for the solka floe and Sigma Cell 50, respectively. Although this increase is statistically significant, i f the 95% confidence intervals are compared at 1 hr and 8 hr, it cannot be said with certainty that the released protein is, in fact, composed of cellulases. Efforts were made to obtain the exact Chapter 5. Results and Discussion 45 composition of the glucanase and B-glucosidase preparations. However, only rough estimates of the Celluclast and Novozym preparations were available, mainly that they are composed of 11% and 13% protein by weight, respectively. O f the protein percentages, it is uncertain whether they represent pure enzyme content. The remainder of the preparations is presumed to be impurities left over from the fermentation process used in their production. Given the high affinity of cellulases for cellulose [8], it is very likely that a significant fraction of the protein remaining in solution is made up of these impurities. This is a shortcoming of the research, even though one of the main purposes of this work is to examine the hydrolysis properties using an industrial enzyme preparation. A s would be expected, there is more protein in solution for the hydrolysis of the Sigma Cell 50. B y inference, less protein is adsorbed onto its crystalline surface, which indicates fewer accessible active binding sites are available compared to the amorphous solka floe. However, the difference in the initial hydrolysis rates (see Figure 5.12) is not very large. Perhaps the adsorption of other impurities onto the solka floe accounts for some of the difference in the protein concentrations. A measure of the enzyme activity in the residual supernatant of the solka floe hydrolysis was determined using the filter paper assay (see Section 4.3.4). The results are illustrated in Figure 5.14 on the following page, including a plot of the specific enzyme activity. It can be seen in Figure 5.14 that corresponding to the initial increase of protein over the first 12 hrs of the hydrolysis, the enzyme activity shows a significant increase (53% at 12 hr, and 57% at 24 hr). This trend indicates that the liberated protein observed in Figure 5.13 is mostly liberated cellulases. Moreover, the specific enzyme activity also shows a significant increase (37% at 12 hr, and 38% at 24 hr). Such an increase in the relative enzyme activity for the total protein in solution is likely explained by the release of cellulases from the hydrolysed cellulose surface Chapter 5. Results and Discussion 46 Figure 5.14: Time-dependent enzyme activity for the hy-drolysis of 10 g/L solka floe at an enzyme loading of 10 FPU/g. back to the bulk aqueous phase. These trends are of strong interest in the design of a continuous system in which enzyme recycle is a desired component: a significant amount of enzyme activity is reclaimed in the aqueous phase after the first 12 hours of hydrolysis. Adsorption over different enzyme loadings Similar hydrolyses were conducted on solka floe, Sigma Cell 50, and Tembec PCS for three different enzyme loadings. Baseline solutions (substrate and buffer) were used as controls to subtract from the hydrolysis data. The results are illustrated in Figures 5.15, 5.16 and 5.17. A similar trend for all the substrates can be seen over the first 8 hr of the hydrolysis, where there is a small increase in the protein levels. Chapter 5. Results and Discussion Al co ~ 0.25 2 0.20 £ 0.15 K 0.00 _ l . 1_ 12 16 T i m e , hr - 5 lU/g -10IU/g -20IU/g Figure 5.15: Protein levels in solution for the hydrolysis of solka floe. 0.40 0 .35 0 .30 _ l B> ^ 0 .25 c ' f f l 2 0 .20 h 0. S O 0 .15 0 .10 0.05 0 .00 - • - 5 I U / g - • — 1 0 I U / g -A— 20 lU/g _ l , I . I . I . L. 12 16 T i m e , h r 20 Figure 5.16: Protein levels in solution for the hydrolysis of Sigma Cell 50. Chapter 5. Results and Discussion 48 Figure 5.17: Protein levels in solution for the hydrolysis of Tembec PCS. Comparing the profiles of Figures 5.15 and 5.16, the increase in the protein levels directly correspond with the enzyme loadings, as would be expected. For the PCS in Figure 5.17, however, the protein levels in solution are significantly higher than for the pure celluloses at all enzyme loadings. The most likely explanation is due to the impurities in PCS, such as sand, grit and bark, that translate to less cellulose to which the cellulases can adsorb. No effort was made to equalize the cellulose loading of the PCS hydrolyses with the cellulose levels of the solka floe and Sigma Cell 50 hydrolyses. At the lowest enzyme loading (5 FPU/g), the release of protein is the most gradual, which is not unexpected. A s the substrate levels are constant for all the hydrolyses, at the lowest enzyme loading the cellulases will be degrading the cellulose longer, and therefore remain adsorbed for a longer period. Increasing the enzyme loading to 10 FPU/g increases the release of cellulases over the first four hours of the hydrolyses. Chapter 5. Results and Discussion 49 0 10 20 30 40 Enzyme Loading, FPU/g Figure 5.18: Protein adsorption on solka floe (30 g/L) with-out hydrolysis. Total protein measured using the B i o - R A D assay. A n interesting trend is visible for the highest enzyme loading (20 FPU/g), where there is a sharp initial decline of protein in solution, then it increases again. A possible explanation is that this same trend is happening at all enzyme loadings, only the equilibrium is reached slower at the higher loadings. 5.2.2 Adsorption without hydrolysis A series of adsorption experiments were conducted to determine whether the "saturation" point could be found—the point at which all available binding sites on the cellulose are covered by adsorbed enzyme. Solka floe was chosen because it lacks the impurities that are present in PCS. The results are illustrated in Figure 5.18. The B i o - R A D assay proved not to be sensitive enough to detect the protein in solution at the lowest loading (5 FPU/g). The total protein measurements were taken again using a Chapter 5. Results and Discussion 50 0 10 20 30 40 Enzyme Loading, FPU/g Figure 5.19: Protein adsorption on solka floe (30 g/L) with-out hydrolysis. Total protein measured by ab-sorbance at 280 nm. spectrophotometer to read the absorbance at 280 nm. The results are illustrated in Figure 5.19. The almost-uniformly increasing solka floe plot represents non-adsorbed proteins remain-ing in solution. These proteins are likely a combination of cellulases and impurities (i.e other proteins) from the cellulase preparation. The "shift" between the standard and the solka floe plots likely represents the adsorbed cellulases, and perhaps any other adsorbed impurities. There are three possibilities that Figures 5.18 and 5.19 illustrate. First, one would expect the solka floe plot to approach that of the standard curve (demarcated by a sharp decrease in the difference) to indicate the point at which binding-site saturation has been reached. Both figures show a gradual increase in the difference at about 20 FPU/g, but both increases are within the experimental error of the assays. Second, the expected sharp decrease in the difference has not yet been reached, and therefore the point of binding-site saturation has not been reached. Third, the substrate is saturated at all the enzyme loadings tested, and the Chapter 5. Results and Discussion 51 "shift" of approximately 6-8 FPU/g represents the point of saturation. A s mentioned earlier, results from Hogan et al. [27] suggest that saturation is reached at 25 FPU/g for solka floe. However, the enzyme preparation was derived from a different strain (Trichoderma harzianum E58). It is difficult to draw definitive conclusions from these results without further study. Given that desirable enzyme loadings are as low as possible, it appears that the enzyme loading ranges of greatest interest (2-10 FPU/g) are probably not saturating the substrate surface. Even if the point of saturation is reached at these loadings, other considerations, such as kinetics may be of greater importance. 5.3 Pretreatment experiments Although a large fraction of the PCS already undergoes pretreatment, a significant fraction of the sludge is waste (such as bark and debris) from other stages of the pulping process which is not readily hydrolysed. To make this fraction more amenable to hydrolysis, the effect of further pretreatment on the PCS was examined. Oxygen delignification was chosen to pretreat the PCS, as it is a common method applied in pulp and paper mills to degrade lignin. Using pulps from three B . C . pulps mills, the effect of the mills' own oxygen delignification processes on the amenability of enzymatic hydrolysis was examined. Lab-scale atmospheric pressure and elevated pressure oxygen delignifications were then applied to PCS. 5.3.1 Comparison of pulp from three mills Both non-delignified pulp and delignified pulp samples from three different mills were hydrol-ysed at different substrate concentrations (10, 25 and 50 g/L). A summary of the conditions Chapter 5. Results and Discussion 52 Table 5.7: Summary of pre- and post-oxygen delignified pulp hydrolyses [33]. A constant enzyme load-ing of 2 FPU/g pulp was used throughout. Pulp Average Maximum Initial Specific Pulp Concen- lignin percentage hydrolysis initial tration content conversion rate hydrolysis rate (g/L) (%) (% after 48 hrs) (g/(L h)) (g/(L h FPU)) 10 . 40.7 0.60 0.30 pre 25 12.2 17.4 0.36 0.18 Howe 50 17.4 0.72 0.36 Sound 10 60.2 0.61 0.30 post 25 7.0 47.6 0.76 0.38 50 47.5 1.51 0.76 10 40.0 0.55 0.28 pre 25 11.8 18.0 0.65 0.33 Inter- 50 15.4 1.29 0.65 continental 10 51.2 0.50 0.25 post 25 4.8 57.1 1.05 0.53 50 57.1 2.11 1.05 10 28.2 0.53 0.26 pre 25 6.0 34.2 0.81 0.41 Northwood 50 34.2 2.24 1.12 10 49.5 0.38 0.19 post 25 5.4 49.4 0.92 0.46 50 49.4 1.84 0.92 and results is presented in Table 5.7. The hydrolysis of the Howe Sound pulp is illustrated in Figure 5.20. A s can be seen for both pulps, an increase in substrate concentration results in an increase in sugar conversions. This is to be expected, for as long as the cellulases are not deactivated or irreversibly bound to non-hydrolysable substrate, the fraction of readily-hydrolysed cellulose will be degraded first. The interesting feature is the percentage conversions for 25 and 50 g/L—for both the Chapter 5. Results and Discussion 53 2 5 10 g /L pu lp 10 g/L de l ign i f led pu lp 25 g/L 25 g/L de l ign i f ied 50 g/L 50 g/L de l ign i f ied - Pu lp best- f i t De l ign i f ied pu lp best- f i t I "5) 15 h rr t-10 h 2 4 3 2 Time, hr Figure 5.20: Hydrolysis of Howe Sound pulp for 10, 25 and 50 g/L concentrations [33]. untreated and delignified pulps, their maximum percentage conversions are identical. This translates to double the efficiency in enzyme use, which is important for cost savings on an industrial scale. The trend also indicates that close to the maximum easily-hydrolysed fraction of cellulose in the pulp is being converted. To consolidate this trend with the 10 g/L run, it would appear that at that enzyme/substrate ratio, the easily-hydrolysed fraction has already been degraded by 12 hr (judging by the slopes over the first 12 hr), and that the small increase in conversions result from the degradation of less easily-hydrolysed cellulose. With a decrease in lignin content, there is a dramatic increase in conversions, particularly for the higher pulp concentrations. A combination of factors could account for the trend: less enzyme deactivation due to lignin and hemicellulose, and the delignification process renders the structure of the cellulose more accessible (i.e. more binding sites) to the cellulase enzymes. This is further discussed in the next section (see page 56). The hydrolysis of the Intercontinental and Northwood pulps are illustrated in Figures 5.21 Chapter 5. Results and Discussion 54 10 g/L pu lp 10 g/L del igni f ied pulp 25 g /L 25 g /L del igni f ied 5 0 g /L 5 0 g /L del igni f ied Pulp best-f i t Del igni f ied pulp best- f i t 0 8 16 2 4 3 2 4 0 4 8 Time, hr Figure 5.21: Hydrolysis of Intercontinental pulp for 10, 25 and 50 g/L concentrations [33]. and 5.22 on the following page. The Intercontinental pulp hydrolysis results are very similar to the Howe Sound pulp. Except for the untreated Howe Sound pulp, the specific initial hydrolysis rate increases as a function of the pulp concentration. That is to be expected, for similar reasons discussed earlier on the trend in percentage conversions. When comparing the three pulps, it appears that the larger the difference in lignin content, the larger the difference is for the conversions. For example, the Howe Sound pulp is reduced from 12 to 7% lignin, and shows an increase from 17 to 48% conversion for the 25 and 50 g/L runs. The Intercontinental pulp's lignin content is reduced from 12 to 5%, and results in an average conversion increase from 17 to 57%. The difference in conversions between the two Northwood pulps is far less dramatic, due in large part to the low lignin content of the untreated pulp. However, even the small drop Chapter 5. Results and Discussion 55 Figure 5.22: Hydrolysis of Northwood pulp for 10, 25 and 50 g/L concentrations [33]. of 6 to 5.4% lignin is sufficient to result in a significant increase in conversion. Again, it is likely that the reduction in lignin content is not the only factor affecting final sugar conversions. It is quite likely that both the degradation of hemicelluloses and the increase in available active sites on the cellulose structure are significant variables. Another possibility is that the 0*2 delignification step results in a washing of the pulp and removal of soluble, cellulase-inhibiting compounds. From the hydrolysis profiles and the relative hydrolysis rates of the untreated and treated pulps, it appears the characteristics of the substrate have a minor influence on the kinetics. Initial hydrolysis rates are comparable, and only at higher substrate levels (50 g/L) is there a noticeable increase in the rate of hydrolysis. It is important to note that in the case of 50 g/L substrate levels, to obtain high conversions requires a very high residence time, which translates to larger reactor vessels and increased capital costs. Furthermore, the pulp or PCS has to be a relatively "clean" substrate. Both Chapter 5. Results and Discussion 56 Howe Sound and Intercontinental have very low conversions prior to the oxygen delignifi-cation. Only the Northwood pulp (which already had low lignin before the delignification process) gave respectable conversions on the untreated sample. Although there is no obvious correlation of enzyme behaviour for different, yet similar substrates, the trend of increased substrate susceptibility to enzymatic action with decreasing lignin is unmistakable. Nevertheless, the results for the pulp after undergoing oxygen deligni-fication are very promising. The technology appears to be very well-suited as a pretreatment option for lignocellulosic substrates. 5.3.2 Oxygen delignification of PCS A series of oxygen delignification experiments were conducted to determine whether its effect on hydrolysis conversion justifies further pretreatment of the Tembec PCS. Atmospheric 02-delignification A sample of the Tembec PCS was delignified for 24 hrs to compensate for the low pressure (see Section 4.2.3 on page 26). Figure 5.23 shows the effect oxygen delignification has on the hydrolysis of the PCS. For the 24 and 72 hr hydrolyses, there is a 47% and 49% increase in sugar conversion, respectively. The difference is statistically significant. O n a mass basis, the conversion from the PCS jumps from 50% to 74%. Although this is a very significant improvement in conversion, there is a caveat. The oxygen delignification itself resulted in about a 40% loss of total solids in the PCS. This increase in conversion is likely due to a combination of factors. First, there is likely to be less enzyme deactivation due to lignin and hemicellulose [8, 67]. Second, the delignification process renders the structure of the cellulose more accessible to the cellulase Chapter 5. Results and Discussion 57 24 72 Hydrolysis Time, hr Figure 5.23: Effect of oxygen delignification on the hydrol-ysis of 50 g/L PCS at an enzyme loading of 50 FPU/g. enzymes by cleaving the cellulose chains. This creates more active binding sites for the cellulases to act on. Upon a visual inspection of the PCS before and after treatment, it is clear that the treated PCS has a significantly lower viscosity, and that the fibres are visibly shorter. Another oxygen delignification was conducted, and several PCS samples were taken at various stages of treatment. These samples were hydrolysed and sampled at 2 and 4 hr, as illustrated in Figure 5.24 on the following page. The results indicate that a lengthy delignification does not have a very large effect on short hydrolysis times. After 2 and 4 hours of hydrolysis, the overall effect of the delignification is not very pronounced—a 23% and 13%o difference, respectively. This minor trend is explained by the nature of the first few hours of enzymatic hydrolysis, where the most readily-attacked cellulose is degraded first. The untreated PCS already has a large fraction of readily-degradable cellulose, which Chapter 5. Results and Discussion 58 3.5 3.0 2.5 ; J 2 .0 cn Lo" t 1 5 1.0 0.5 0.0 0 4 8 12 16 2 0 2 4 Time of Delignification, hr Figure 5.24: Effect of oxygen delignification on the hydrol-ysis of 10 g/L PCS at an enzyme loading of 10 FPU/g. results in only the small differences seen in Figure 5.24. B y contrast, after a longer period of hydrolysis, such as 24 hr, as illustrated in Figure 5.23, delignification has a far more significant effect on the maximum hydrolysable fraction of the PCS. Although a shorter oxygen delignification time, such as four hours, is sufficient to improve the conversion, further work should be conducted to determine whether shorter pretreatments are equally effective on final hydrolysis conversions (i.e. after 24 or 48 hr hydrolyses). Pressurized 0 2-delignification Typical commercial oxygen delignification processes operate at elevated pressure. Efforts were made to approach similar operating conditions on the laboratory scale (see Section 4.2.3 on page 26). The hydrolysis of PCS, which underwent oxygen delignification at elevated 4 hr hydro lys is - • — 2 hr hydro lys is Chapter 5. Results and Discussion 59 A 10 F P U / g 0 8 16 2 4 3 2 4 0 4 8 Time, hr Figure 5.25: Enzymatic hydrolysis of oxygen delignified PCS for 25 g/L at different enzyme loadings. pressure, is illustrated in Figure 5.25. The results are summarized in Table 5.8, including the data from the hydrolysis of untreated PCS, for comparison. Two significant trends are apparent from the hydrolysis of the delignified P C S . First, the maximum conversions after 48 hr are far greater at the same enzyme loadings than for the untreated PCS. For the 5 and 10 FPU/g runs, almost the same conversions are attained (42.3 and 43.8 %) for the delignified and untreated PCS, respectively. Second, the specific initial hydrolysis rates are not significantly higher for either substrate. Both of these observations indicate that oxygen delignification reduces enzyme inhibition due to lower lignin levels in the PCS and/or the structure of the cellulose is made more accessible to enzymatic attack. A s was seen in Section 5.1.3, the majority of the cellulose conversion is complete after 24 hr (on average, 88% of the 48 hr conversions). After 12 hr, 73% of the 48 hr maximum conversion is reached. From a process standpoint, prolonged residence times beyond 24 hr may not be economically justified for the modest increase in conversions. Chapter 5. Results and Discussion Table 5.8: Sumrnary of PCS hydrolysis' data at 25 g/L. 60 Enzyme Percentage Initial hydrolysis Specific initial PCS loading conversion rate hydrolysis rate (FPU/g PCS) (% after 48 hrs) (g/(L h)) (g/(L h FPU)) 2 14.1 0.70 0.35 Delignified 5 42.3 2.13 0.43 10 53.2 3.06 0.31 , 5 22.2 1.75 0.35 Untreated 10 43.8 3.35 0.34 15 49.0 4.23 0.28 From the results of the oxygen delignification, it is clear that the process allows for higher sugar conversions from treated PCS compared to untreated PCS at a given enzyme loading. More importantly, comparable conversions can be attained for almost one half the enzyme, depending on the enzyme loading in question. A s mentioned earlier, there is one caveat: solids loss. For the atmospheric oxygen delig-nification, upwards of 40% of the PCS solids were lost. With the pressurized process, and the addition of MgS02, the solids loss was reduced to 12%. If a comparison is made on a mass basis, the efficacy of the O2-delignification process can be properly judged. For 1 kg of untreated PCS, the hydrolysis at 10 FPU/g PCS and 25 g P C S / L yields 0.438 kg of T R S after 48 hr. Treating 1 kg of PCS gives 0.883 kg of 0 2-delignified PCS, and hydrolysis at similar conditions yields 0.470 kg T R S , which represents only a 7.3% increase in conversion. However, i f the above calculations are repeated for an enzyme loading of 5 F P U / g PCS, the hydrolysis of 1 kg of untreated PCS yields 0.222 kg T R S . The hydrolysis of 0.883 kg of 02-delignified PCS yields 0.374 kg T R S , which represents a 68% increase in conversion. This result is extremely encouraging, as it indicates that at lower enzyme loadings, further delignification of the PCS can translate to large gains in overall conversion. Chapter 5. Results and Discussion 61 8 DC 4 2 • L iqu id El L iqu id Base l ine 1 1 1 Howe (Pre) Howe (Post) Figure 5.26: Deterrnination of enzyme fraction & activity after a 48 hr hydrolysis [33]. Enzyme recycle To determine the amenability of the cellulases to recycling, hydrolyses of the Howe Sound pulps were carried out for 48 hours (enzyme loading of 10 FPU/g pulp, 10 g/L). At that point, the solid and liquid fractions were separated by vacuum filtration. New buffer solution and fresh substrate (10 g/L) was mixed with both the solid residue and liquid fraction, then both were incubated for an additional 5 hours. The final sugar concentrations of the liquid fraction is illustrated in Figure 5.26. For comparison, the maximum conversion obtained from the previous section (Howe Sound, 2 FPU/g) is also plotted. Little can be said about the liquid fraction because the sugar levels after the 48 hr hy-drolysis was not performed. The final sugar levels (6.5 and 9.5 g/L for untreated and treated Chapter 5. Results and Discussion 62 pulp, respectively) are significantly higher than the maximum values obtained for the 2 FPU/g (4.1 and 6.0 g/L). It is very likely that the majority of the difference in the results is due to the enzyme loadings. Nevertheless, the very high sugar result (9.5 g/L) for the treated pulp is high enough that it is not unreasonable to assume a contribution from the additional 5 hr hydrolysis. Therefore, further work should be conducted to confirm the enzyme activity in the liquid fraction. Depending on the outcome of the work, continuous hydrolysis incorporating some form of sugar extraction could prove advantageous from an economic perspective. The solid residue was not washed so that any adsorbed enzymes would not be eluted. The total reducing sugars levels resulting from the supernatants after the 5 hour hydrolysis were too low to be measurable using the D N S assay. A s no new sugars were produced, it can be concluded that the adsorbed enzymes on the undigested PCS are either inactive or irreversibly bound to the residue. 5.4 Laboratory-scale reactor experiments A s was found in the small scale experiments, the majority of the hydrolysis (over 80%) is complete within 24 hrs. On this basis, the residence time for the solids in the reactor was chosen at 22 hrs, which translates to a feed and product stream of 1 L per hour. The original design intention was to run a fully continuous reactor, however, it was very difficult to achieve such operation due to the nature of the PCS substrate. Even at low consistency (2.5%), clogging problems were frequently encountered while running the reactor. Moreover, while reaching steady-state, the flow properties of the reactor contents changed as the cellulose fibres were degraded, requiring frequent adjustment to the pump speed to maintain a constant recycle flow rate. As a compromise, the reactor was fed and Chapter 5. Results and Discussion 63 12 r Time, hr Figure 5.27: Time run of the semi-continuous reactor on un-treated PCS. A n enzyme loading of 10 FPU/g PCS for 25 g/L substrate was used. harvested on a semi-continuous basis every hour, and sampled every half hour. 5.4.1 P C S run The results of the run on untreated PCS are illustrated in Figure 5.27. The striking feature is the rapid approach to steady-state, which was reached at about 6 hr after startup. At steady-state, the reactor sugar concentration is approximately 10 g/L, which represents 90% of the levels attained (11 g/L) in the shake flask experiments at the same conditions after 24 hr. This is very promising considering that the maximum hydrolysability of the PCS substrate is about 50% (or about 12.5 g/L T R S conversion). These results are encouraging, insofar as the semi-continuous operation can achieve con-version levels almost comparable to those in batch operations. This behaviour is most likely a Chapter 5. Results and Discussion 64 result of the increased mixing that resulted from the recycle flow. Enzyme-substrate contact was greatly facilitated in the reactor. Sound and Lynd [64] found that substrate cellulose conversion in a 1.6 L continuous upflow reactor was 80% higher than that of the batch reaction at the same reaction time (14.0 vs 7.8%). The reactor residence time was 7.7 hr. However, similar difficulties associated with the rheology (mixing, channeling, dead zones, etc.) were experienced while operating the reactor. Ballerini et al. [6] designed and implemented a large-scale ethanol production plant (25,000 L hydrolysis reactor). At similar conditions to the work presented here (temper-ature, p H , enzymes), the hydrolysis of pretreated poplar wood was run in batch mode for 72 hr at 16 FPU/g for a conversion of 96%. This is compared to an 80% conversion running semi-continuously at 10 FPU/g with a residency time of about 22 hr. 5.4.2 0 2 -delignified P C S run The results of the run employing oxygen-delignified PCS are illustrated in Figure 5.28. The same conditions were used as in Figure 5.27. The approach to steady-state was quite rapid, and was also reached at about 6 hr after startup. At steady-state, the reactor sugar concentration was approximately 11.7 g/L, which represents about 91% of the levels attained (12.8 g/L) in the shake flask experiments at the same conditions after 24 hr. As compared to the results from using untreated PCS in the reactor, the 16% improvement in conversion is comparable to the 21% improvement observed in the batch studies at similar conditions (see p. 58). One point of interest is that the viscosity of the 02-delignified PCS appeared lower as a result of the treatment, and fewer clogging problems were encountered while running the reactor. A s a result, higher consistencies could be more easily used with the treated PCS, as Chapter 5. Results and Discussion 65 Time, hr Figure 5.28: Time run of the semi-continuous reactor on oxygen delignified PCS. A n enzyme loading of 10 FPU/g PCS for 25 g/L substrate was used. compared to using untreated PCS. In summary, oxygen delignification is beneficial to hydrolysis conversions in both batch and continuous systems, resulting in 16-68% improvements in final conversions. In addition to increased hydrolabi l i ty of the substrate and improved rheology of the system, it is likely the benefits accrued from decreased enzyme inhibition due to decreased lignin content improve the prospects of enzyme reuse. Chapter 6 Conclusion The hydrolysis of several cellulose substrates has shown that primary clarifier sludges are readily degraded by cellulase enzymes. The hydrolysis of PCS from Tembec's low yield sulphite pulping operation resulted in a 50% conversion (by total mass) when an initial enzyme loading of 50 FPU/g PCS was applied to 50 g P C S / L . The effect that acetate buffer concentration had on hydrolysis rates was appreciable. The maximum buffer strength, which could be used without a significant impact on the enzymatic hydrolysis, was found to be 0.2 M . Higher levels of enzyme loading resulted in higher rates of hydrolysis. However, increasing enzyme loadings resulted in proportionately lower increases in hydrolysis rates. For the hydrolysis of solka floe at both 1 and 10 g/L, doubling the enzyme loading from 5 to 10 F P U / g resulted in 33 and 34% increases in the specific hydrolysis rates, respectively. This trend does not follow for final hydrolysis conversions—other factors, such as substrate concentration and enzyme deactivation affect the relationship between enzyme loading and final sugar conversion. Over the first 12 hr of hydrolysis of solka floe, an increase in protein concentrations in the supernatant was observed. There was a 53% increase in enzyme activity of the supernatant over the corresponding time period. Although this indicates that a large fraction of enzyme activity can be recovered in the supernatant, means of liberating the cellulase enzymes from the unhydrolysed solids is equally important for enzyme recycling. 66 Chapter 6. Conclusion 67 The experiments on adsorption in the absence of hydrolysis were inconclusive, as the point of maximum binding-site saturation was not clearly established. However, given that desirable enzyme loadings are as low as possible, and the goal of this work was to maximize enzyme use and minimize waste, it appears that the enzyme loading ranges of greatest interest (2-10 FPU/g) were probably not saturating the substrate surface. Even i f the point of saturation was reached at these enzyme loadings, other considerations, such as hydrolysis kinetics and final sugar concentrations were deemed to be of greater importance. The trend of increased substrate susceptibility to enzymatic action with decreasing lignin was clearly shown. The hydrolysis data for both pulps and PCS after undergoing oxygen delignification are very promising and result in significant increases in sugar concentrations. Concern over cellulose loss can be addressed by high pressure, low retention time delig-nification in the presence of MgS02. Depending on the enzyme loadings and substrate concentrations, increases in conversion as a result of CVdelignification were as high as 68% (5 FPU/g PCS; 25 g PCS/L) . The technology appears to be very well-suited as a pretreatment option for lignocellulosic substrates. Operation of a semi-continuous hydrolysis reactor on both untreated PCS and oxygen delignified PCS yielded approximately 90% of the conversions attained in batch conditions. While the results are encouraging, similar operating conditions could represent a problem upon scale-up because of the high energy input required to provide a comparable degree of mixing. This setup may be impractical on a larger scale, and similar results may not be attainable upon scale-up. Chapter 7 Recommendations To build on the research presented in this work, a number of different topics merit further consideration: 1. Given the discrepancy between glucose measurements taken with a glucose analyser, and the results obtained using the D N S assay, a better means of measurement should be employed in future work. Unfortunately, the alternatives are either very costly (HPLC) and/or lend themselves to other problems (e.g. methylated sugars & G C ) . See [1, 60] 2. The decline in sugar concentrations over prolonged hydrolyses is most likely due to microbial growth. Repeat hydrolyses using an anti-microbial agent, such as azide should be performed to confirm whether this is, in fact, the case. 3. A more careful study of the protein adsorption experiments should be undertaken using a more accurate means of protein measurement, such as by H P L C . Furthermore, the composition of the Celluclast/Novozym preparations should be ascertained. 4. Comparisons of hydrolyses of thoroughly washed PCS vs PCS straight from the mill should be made to determine whether there are other chemical agents which might significantly inhibit the cellulases. 5. The results for the PCS after undergoing oxygen delignification are very promising. The technology appears to be very well-suited as a pretreatment option for lignocellulosic 68 Chapter 7. Recommendations 69 substrates. Further work is warranted in determining how significant the individual contributions of the CVdelignification process are (i.e. reduced lignin, reduced hemi-cellulose, increased cellulose binding sites, etc.). 6. There are several reactor operation and design issues: (a) More work is justified on finding alternative practical means of enzyme recycle in a continuous system, such as the implementation of an ultrafiltration separator (see [30]). (b) The effect of the vigorous mixing on the enzyme kinetics should be further explored so that it could be determined whether high energy input in the form of mixing would be cost effective upon scale-up. (c) The next step is to make the process fully continuous. This is a very difficult challenge to do on a bench-scale reactor due to the rheological problems associated with the PCS slurry. A large pilot-scale plant with adequate tubing (>2") would be necessary. This would also allow the operation at higher consistencies (5% or more). 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Biotechnology and Applied Biochemistry, 21(2):203-216, April 1995. Appendix A Acronyms A C O S A c i d catalyzed organosolv saccharification C M C Carboxymethyl cellulose C S T R Continuous stirred tank reactor C T M P Chemithermomechanical pulp F P U Filter paper unit IU International unit M S W Municipal solid waste PCS Primary clarifier sludge ROI Return on investment (% annual return on capital investment) SSF Simultaneous saccharification and fermentation T M P Thermomechanical pulp T R S Total reducing sugars 77 


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