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Heatand Mass Transport in Processing of Lignocellulosic Biomass for Fuels and Chemicals

Heatand Mass Transport in Processing of Lignocellulosic Biomass for Fuels and Chemicals

Abstract Lignocellulosic biomass, a major feedstock for renewable biofuels and chemicals, is processed by various thermochemical and/or biochemical means. This multi-step processing often involves reactive transformations limited by heat and mass transport. These limitations are dictated by restrictions including (1) plant anatomy, (2) complex ultra-structure and chemical composition of plant cell walls, (3) process engineering requirements or, (4) a combination of these factors. The plant macro- and micro-structural features impose limitations on chemical and enzyme accessibility to carbohydrate containing polymers (cellulose and hemicellulose) which can limit conversion rates and extents. Multiphase systems containing insoluble substrates, soluble catalysts and, in some cases, gaseous steam can pose additional heat and mass transfer restrictions leading to non-uniform reactions. In this chapter, some of these transport challenges relevant to biochemical conversion are discussed in order to underscore the importance of a fundamental understanding of these processes for development of robust and cost-effective routes to fuels and products from lignocellulosic biomass. Keywords Lignocellulose · Biomass · Biofuels · Heat transport · Mass transport

1 Introduction
The biochemical conversion of lignocellulosic biomass requires several processing stepsdesigned to convert structural carbohydrates, such as cellulose and hemicellulose, to monomeric sugars, which include glucose, xylose, arabinose, and mannose. These sugars can be fermented to ethanol and other products, to varying degrees of effectiveness, by wild type and modified microbial strains. The front end of the process includes feedstock size reduction followed by a thermal chemical treatment, called pretreatment. In practice, this unit operation usually involves the exposure of
S. Viamajala (B) Department of Chemical and Environmental Engineering, The University of Toledo, Toledo, OH 43606-3390 e-mail: O.V. Singh, S.P. Harvey (eds.), Sustainable Biotechnology, DOI 10.1007/978-90-481-3295-9_1, C Springer Science+Business Media B.V. 2010 1

biomass to acid or alkaline catalysts at temperatures ranging from 120 to 200a—¦ C. Pretreated slurries (the hydrolysate liquor containing soluble sugars, oligosaccharides, and other released solubles plus the residual solids) are then enzymatically digested at 40–60a—¦ C to release sugars from the polysaccharides and oligomers remaining after pretreatment [1–9]. In both of these steps, adequate heat, mass, and momentum transfer is required to achieve uniform reactions and desirable kinetics. Plant cell walls, which make up almost all of the mass in lignocellulosic biomass, are highly variable both across and within plant tissue types. At the macroscopic scale, such as within a stem or leaf, uneven distribution of catalyst (chemical or enzyme) due to the differentproperties of different tissues results in heterogeneous treatment, with only a fraction of the plant material exposed to optimal conditions [10–13]. Tissues that do not get exposed to sufficient amounts of catalyst during pretreatment are incompletely processed, resulting in decreased overall enzymatic digestibility of pretreated biomass [6]. When pretreatment severity is increased, by increasing temperature, catalyst concentration, or time of reaction, areas of biomass readily exposed to catalyst undergo excessive treatment leading to sugar degradation and formation of toxic by-products (furfural, hydroxymethyl furfural, and levulinic acid) that inhibit downstream sugar fermentation and decrease conversion yields [1]. This problem continues at a microscopic scale due to the compositional and structural differences between middle lamella, primary cell wall, and secondary cell wall. At even smaller scales, intermeshed polymers of cellulose, hemicellulose, lignin, and other polysaccharides present another layer of heterogeneity that must be addressed during bioconversion of plant cell walls to sugars. Milling to fine particle sizes improves some of these mass transfer limitations, but can add significant costs [14, 15]. Size reduction, however, may not overcome heat transfer limitations associated with short time-scale pretreatments that employ hot water/steam and/or dilute acids. When such pretreatments are carried out at high solids loading (>30% w/w) to improve process efficiency and increase product concentrations, heat cannot penetrate quickly and uniformly into theseunsaturated and viscous slurries. It is thought that steam added to high-solids pretreatments can condense on particle surfaces impeding convective heat transfer. Depending on particle and slurry properties, the condensed steam can form temperature gradients within biomass aggregates, resulting in non-uniform pretreatment. Besides limiting heat transfer rates, biomass slurries can pose other processing challenges. At high solids concentrations, slurries become thick, paste-like, and unsaturated. Limited mass transfer within these slurries can cause localized accumulation of sugars during enzymatic hydrolysis, decreasing cellulase and hemicellulase activity through product inhibition [16–23]. In addition, slurry transport through process unit operations is challenging at full scale. As solid concentrations increase, hydrodynamic interactions between particles and the surrounding fluid as well as interactions among particles increase. At high solids concentrations “dense suspensions” are formed and the resulting multiple-body collisional or frictional interactions and entanglement between particles creates a complex slurry rheology [24–26]. A further complicating aspect is water absorption by biomass, causing the bulk to become unsaturated at fairly low insoluble solids concentrations (~ 30–40%

w/w) and behave as a wet granular material [27]. This material is highly compressible and the wet particles easily “stick” to each other and agglomerate. With no free water in the system, the material becomesdifficult to shear or uniformly mix. At the ultrastructural scale of plant cell walls, catalysts must penetrate through the nano-pore structure of the cell wall matrix to access the “buried” and intermeshed carbohydrate polymers. Based on reported average cell wall pore sizes of 5–25 nm [28–31], small chemical catalysts (1 cm) is often overlooked; however, milling biomass to reduce this problem can incur large energy and equipment costs [1, 14, 15]. This problem is compounded by the widespread use of process irrelevant biomass sizes for laboratory experiments. Most laboratory studies on biomass to ethanol conversion processes use finely milled materials (20–80 mesh is standard) where the effects of macroscopic transport processes are not easily observed or are masked altogether [43–45]. In larger pilot studies using compression screw feeders, these transport effects can be further masked by the high-shear feeder causing biomass size reduction [6, 8]. Often this size reduction occurs after catalyst impregnation, limiting catalyst effectiveness on pretreatment. A further complication is that compression of the feed stock may cause biomass pore structure collapse, leading to uneven heat and mass transfer during pretreatment [10, 13] as well as limitation of catalyst access to the interior of the biomass. Before larger biomass particles containing intact tissues are used in processing, it is essential to understand the catalyst transport processes and pathways and the limitations associated with them (Fig. 1). In living plants, vascular tissues such as xylem and phloem are the primaryroutes for transport of water and nutrients along the length of the plant stem and leaves. Additional transport within tissues and between adjacent cells is carried out through (1) the pits, areas of thin primary cell wall devoid of secondary cell wall between adjacent cells and (2) the apoplast, the contiguous intercellular space exterior to the cell membranes [46]. In dry senesced plants, studies with dyes to visualize fluid movement through tissues showed that the apoplastic space is the major catalyst carrier route, with limited fluid movement

occurring through the vascular tissue [11]. In untreated biomass, the pits do not appear to support significant transport. It is probable that these pits disintegrate and open up during pretreatment allowing fluid to flow through [40]. Thus, new pathways for catalyst penetration are formed either during the drying process that creates fractures in plant tissues or after some degree of biomass degradation. The primary major barrier to fluid transport into native dry plant tissue appears to be air entrained in the cell lumen. Simple exposure of tissues to high temperature fluids is insufficient to achieve catalyst distribution to all parts of the biomass [11]. The primary escape route for the intracellular air is most likely through pits. However, the small pit openings (approx 20 nm) could be blocked due to cell wall drying and water surface tension may prevent movement through these narrow openings. Forced air removal by vacuum provides additional drivingforce for the bulk fluid mobility necessary to enhance liquid and catalyst penetration into tissues as demonstrated by Viamajala and coworkers [11]. Heating dry biomass can minimize the amount of entrained air (due to expansion of air by heat) and assist in drawing liquid into the cells by contraction of the entrained air when cooled by immersion in catalyst-carrying liquid. Thus, bulk transport, rather than diffusive penetration, is the dominant mass transfer mechanism into dry biomass. Although movement of fluids is associated with catalyst transport, the primary goal of catalyst distribution is to deliver the catalyst to cell wall surfaces containing fuel-yielding carbohydrates, rather than to empty cytoplasmic space in dry tissues. In fact, entrainment of fluids in the biomass bulk can be detrimental to small time-scale dilute acid or hot water pretreatments, as the presence of excess water increases the net heat capacity of the material, increasing the heating time needed to achieve desired pretreatment temperatures. Data shown in Fig. 2 support this hypothesis. In this set of experiments, un-milled sections of corn stems

(~ 1 inch long) were saturated to various degrees with dilute sulfuric acid (2% w/w) and pretreated in 15 mL of the same acid solution at 150a—¦ C for 20 min. Milled corn stems (–20 mesh) pretreated under identical conditions served as controls. Allpretreatments were performed in 22 mL gold coated Swage-Lok (Cleveland, OH) pipe-reactors, heated in an air-fluidized sand bath [42]. After pretreatment, whole stem sections were air-dried, milled and enzymatically digested for 120 h with a 25 mg/g of cellulose loading of a commercial T. reesei cellulase preparation (Spezyme CP, Genencor International, Copenhagen, Denmark) supplemented with an excess loading (90 mg/g of cellulose) of commercial Aspergillus niger cellobiase preparation (Novo 188, Novozymes Ltd., Bagsvaerd, Denmark) using procedures described previously [47]. Milled stover pretreated as controls in this experiment was dried and digested similarly, but without any further comminution. In Fig. 2a, dry internodes pretreated without pre-impregnation of catalyst were poorly pretreated as evidenced by the high amounts of xylan remaining in the biomass after reaction. Stem sections pre-impregnated to achieve 20% saturation showed better reactivity and xylan removal and this trend continued when stem sections pre-impregnated to 50% saturation were pretreated. However, when completely saturated (100%) stem sections were pretreated, xylan conversion was observed to be lower. Milled materials with and without pre-impregnation of catalyst – conditions that would have lowest mass transfer limitations, showed comparable pretreatment performance with each other as well as with the 50% saturated stem sections. These results confirm that only limited catalyst penetration and pretreatment is achieved when air remains entrapped in cytoplasmic spaces such as in dry internodes. Enhancedcatalyst distribution and transport dramatically enhances pretreatability up to a certain point, after which excess fluid impedes pretreatment. Similar conclusions on the negative impacts of poor bulk transfer on biomass pretreatability can be inferred from other reported studies also. Tucker and coworkers [10] observed poor pretreatability of biomass during steam explosion of corn stover when materials were not pre-wetted with dilute acid and ascribed their results to mass transport limitations. In another study Kim and coworkers [13] observed poor pretreatment of biomass when the biomass was pressed prior to pretreatment and hypothesized that the mechanical compression of biomass caused pore structure collapse resulting in formation of material that was relatively impervious to heat and mass transfer. Enzymatic digestion results corresponding to pretreatments shown in Fig. 2a, are presented in Fig. 2b. As expected, release of monomeric sugars from pretreated whole stem sections was proportional to the degree of pretreatment they experienced. Unmilled biomass that was 50% saturated with acid before pretreatment showed better digestibility than the sections that were pre-saturated to lower or higher levels. Milled biomass, however, digested best, demonstrating the importance of enhanced enzyme transport – an outcome of the more thorough and uniform pretreatment of milled materials. With woody feedstocks, milling to fine particle sizes may be impractical and pre-impregnation of biomass with catalyst, as practiced in the pulp and paper industry [48], might need to beutilized to improve conversion efficiencies.

3 Microscopic Transport Through Plant Cell Walls
Enzyme penetration into plant cell wall is widely acknowledged to be a key barrier to economical and effective biochemical conversion of lignocellulosic biomass [5, 49]. In fact, the primary function of pretreatment of lignocellulosic biomass is to assist subsequent enzymatic digestibility by making cell walls more accessible to saccharifying enzymes [1, 4, ]. However, an accurate description of the methods by which enzymes penetrate cell walls and accomplish cellulose degradation has been lacking. A recent study by Donohoe and coworkers provided, for the first time, direct visual evidence of loosening of plant cell wall structure due to dilute acid pretreatment and the subsequently improved access by cellulases [49]. Figure 3c–f further demonstrate the penetration of cellulases into pretreated cell walls as detected by nano-gold labeled antibodies to Cel7A and other cellulases. This study shows that penetration of enzymes into mildly pretreated cell walls is minimal and that cells stay largely intact even after prolonged exposure to cellulases (Fig. 3a, b). In moderately pretreated cell walls, cellulases are able to partially penetrate and disintegrate the inner secondary layers (S3) only (Fig. 3c, d); whereas the outer layers (S1 and S2) remain impervious to enzymes. In severely pretreated cell walls, enzymes penetrate throughout (Fig. 3e, f). These data suggest that enzymatic digestibility of biomass isrestricted by transport of enzymes into cell walls. While not directly evidenced by this study, these results also suggest that thermal pretreatments (and possibly others) “loosen” cell walls in layers providing enzymes access only to these structurally compromised zones of the cell walls. Kinetic data on thermal pretreatments by several research groups also suggests likely mass transfer limited xylan removal that can be modeled as parallel fast and slow reactions [44, 50, ] and the fundamental observations made by Donohoe and coworkers [49] support this hypothesis.

4 Lignin Mobility and Impact on Biochemical Conversion
Lignin is a polymeric material composed of phenylpropanoid units derived primarily from three cinnamyl alcohols (monolignols): ρ-coumaryl, coniferyl, and sinapyl alcohols. Polymer formation is thought to occur via oxidative (radical-mediated) coupling between monolignols and the growing oligomer/polymer [52, 53] and is commonly believed to occur in a near-random fashion [54], although some recent studies suggest an ordered and protein-regulated lignin synthesis [55]. In any case, the resulting polymer is complex, heterogeneous, and recalcitrant to biological degradation. Although lignin loss is minimal during thermal-acidic/neutral pretreatments, it can undergo structural and chemical changes [56] that significantly influence downstream enzymatic conversion. Although enzymes thoroughly penetrate cell walls after high severity pretreatments [49], incomplete cellulose conversion by cellulases suggests additional barriers exist at the ultrastructurallevel. One potential barrier is occlusion of the cellulose microfibrils by residual lignin or hemicellulose that would sterically prevent

Fig. 1.3 Immuno-labeled electron micrographs of pretreated, digested corn stover cell walls. Gold particles (visible as dark dots especially in d and f) mark the location of Cel7A enzymes digesting through cell walls following dilute acid pretreatment of varying severity (120a—¦ C c, d; 150a—¦ C e, f). CL, cell lumen; ML, middle lamella; P, pit; 1a—¦ CW, primary cell wall; 2a—¦ CW, secondary cell wall. Scale bars = 1 μm a, c, e; 500 nm b, d, f

cellulases from binding to cellulose [42]. Other indirect mechanisms that impede complete cellulose hydrolysis are also possible such as non-productive binding of cellulases to lignin [34–36], however reports that contradict this theory also exist [57]. Enzymatic hydrolysis of biomass pretreated under alkaline conditions, which hydrolyzes less xylan than acidic pretreatments, supports the steric hindrance concept. Elevated cellulolytic activity is observed on alkaline pretreated biomass when cellulases are supplemented with xylanases and other hemicellulose degrading enzymes, likely a function of removing additional barriers to cellulose accessibility [58, 59]. A study in pretreatment variability by Selig and co-workers suggested that cellulose digestibility is improved directly by xylan removal, but only indirectly by lignin removal [47]. Removal of lignin by pretreatment appeared to increaseenzymatic removal of xylan, which in turn increased cellulose digestibility. Lignin removal alone had little impact on cellulose digestion. Lignin modifying enzymes, however, have been shown to synergistically work with cellulases during digestion of steam-pretreated biomass, improving sugar yields through at least partial removal of the lignin barrier [60]. In spite of a general consensus in the scientific community about the significance of the lignin barrier to cellulose digestibility, only limited attention has been given to the fate of lignin during widely used high temperature dilute acid, hot water, and steam pretreatments which only partially remove lignin [1, 8]. A recent study investigated the fate of lignin during high temperature acid and neutral pretreatments using electron microscopy and spectroscopy techniques [40]. This study revealed that lignin could be mobilized within the cell wall matrix at temperatures as low at 120a—¦ C during both neutral and low pH pretreatments, and appears to be, at least in part, dependent on pretreatment severity. On a relatively macro scale, part of the mobilized lignin deposits back on to biomass surfaces as spherical bodies, suggesting that lignin undergoes the following sequence of events during these pretreatments – phase-transition or melting, mobilization into bulk solution, coalescence, and deposition onto solid surfaces. Scanning- and transmission electron microscopy (SEM and TEM) of pretreated cell walls shows that the lignin droplets (stained with KMnO4 ) take a wide range of sizes (15% solids). Each data point wasgenerated as a single measurement from triplicate reactors after 5 days of digestion. As can be seen from Fig. 6a, conversion of cellulose to glucose decreases steadily as solids concentrations increase suggesting inhibition of enzymes, possibly due to poor mass transfer resulting in localized accumulation of sugars as suggested by Hodge and coworkers [22]. Clearly, slurry properties will play a major role in determining these transport parameters that are crucial to determine optimal process performance across multiple scales. As another example, Fig. 7 shows experimental data from tests performed to evaluate heating time in a closed reactor containing biomass slurries of varying concentrations. These data show significant retardation of heat transfer, even with the moderate density slurries containing 10% solids (w/w). Simple heat transfer simulation models have been developed for biomass slurries assuming conductive heat transfer and a one-dimensional system; however, their validity has not been verified with experimental data [64, 65]. In unsaturated biomass slurries containing discrete aggregates, the accurate determination and prediction of transport properties might be a challenging exercise.

6 Outlook for Challenges Associated with Transport Processes in Biochemical Conversion of Lignocellulosic Biomass
Significantly greater research and development effort in the conversion of lignocellulosic biomass, spurred by economic, national security and climate change concerns over the past few years have led to significant strides in development of a fundamental understanding oftransport processes that could appreciably

Fig. 1.6 5-day enzymatic digestibility data for pretreated corn stover showing (a) decrease in conversion with increasing solids concentration and (b) Plateau in glucose release after a solids concentration of 30%

improve overall performance and make renewable liquid transportation fuels sustainable and affordable. A thorough understanding of fundamental issues related to transport processes and the development of predictive models that integrate heat, mass and momentum transport are essential to the design, development and implementation of scale-independent processes. Continued synergism between science and engineering disciplines along with participation by industry is crucial to the development of cost-effective alternative motor fuels by 2012 and the significant displacement of fossil-derived fuels specified by the DOE (Energy Independence and Security Act of 2007) EISA for 2022. Improvements in process equipment,

Fig. 1.7 Effect of solid concentrations on heat up time of pretreatment reactor containing biomass slurries

enzymes and microbial systems, as well as improved understanding of the basis for biomass recalcitrance are critical determinants of the successful implementation of biorefineries.
Acknowledgements This work was funded by the US DOE Office of the Biomass Program. The authors also acknowledge the valuable intellectual insights provided by Dr. James McMillan, National Bioenergy Center, NationalRenewable Energy Laboratory, on issues related to transport processes in biochemical conversion of lignocellulosic biomass.

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