Although the biochemical conversion of lignocellulosic biomass to biofuels and bio- products requires the use of water as a carrier fluid and reaction solvent, there are substantial economic and environmental benefits to reducing the amount of water used throughout the conversion process. Performing the conversion process with less water results in relatively smaller process volumes, enabling smaller equipment sizes, improved fermentation yields due to higher sugar concentrations, and a reduction in the energy needed to separate water from the product (Humbird et al., 2010). How- ever, operating at low water conditions, and consequently at high solids concentrations
(≳10%insoluble solids), gives rise to several chemical and physical phenomena that can limit product yields and increase some operating costs. For the unit operation of enzymatic hydrolysis, these phenomena include inhibition of the enzyme cata- lysts, inefficient slurry transport and mixing, and ineffective heat and mass transfer.
Implementation of appropriate process technologies can reduce the impact of some of these phenomena, but ultimately there is an economic trade-off between reducing water content and obtaining high product yields (Humbird et al., 2010).
4.3.1 Conversion Yield Calculations
Scientific investigation and development of conversion processes with slurries at high solids concentrations requires careful definition and mathematical treatment of quan- tities of interest. Several investigators have addressed the need to use detailed mass- balance relationships when calculating the conversion yield of enzymatic hydrolysis at high solids loading (Hodge et al., 2009; Kristensen et al., 2009; Roche et al., 2009a,b; Zhu et al., 2011). Calculating yields from liquid-phase concentrations alone, without accounting for the presence of the solid phase, can result in overestimation of conversion yield by as much as 30% (Kristensen et al., 2009). Instead, a mass-based fractional conversion yield equation is recommended, given by
ξ =
∑
iri(fi−fi,0) fis,0∑
jxj,0 , (4.1)
whereξis the mass fraction of insoluble polysaccharides that have been hydrolyzed to soluble sugars,fiis the mass fraction of soluble sugari(per unit mass of slurry),fis is the mass fraction of insoluble solids,xjis the mass fraction of polysaccharidejin the solid phase (per unit mass of insoluble solids), andriis the molecular weight ratio of a polysaccharide unit to its corresponding hydrated (soluble) uniti. The additional
“0” in a subscript denotes the value at a reference point, typically the start of a batch reaction. Soluble sugars that are usually measured and accounted for are glucose, cellobiose, and xylose, and polysaccharide components that are usually included are glucan and xylan. Additional sugars, such as arabinose and xylooligomers, and polysaccharides, such as arabinan, may be included if they are present in significant amounts. A conversion yield calculation accounting only for the conversion of glucan to glucose would be given as
ξG= rg(fg−fg,0)
fis,0xG,0 . (4.2)
The mass fraction of a sugar in the slurry (g/g) may be calculated from its liquid-phase concentrationci(g/L) using the relationship
fi−fi,0= ci
ρliqfliq− ci,0
ρliq,0fliq,0, (4.3)
wherefliq=1−fis is the mass fraction of the liquid phase. The liquid densityρliq depends on the concentration of dissolved sugars, where a simple linear relationship has been shown to agree well with measured values (Zhu et al., 2011). Finally, the mass fraction of liquid in the slurry is related to sugar concentrations by the equation
fliq= fliq,0(1−∑
irici,0∕ρliq,0) 1−∑
irici∕ρliq . (4.4)
To determine the extent of conversion of an enzymatic hydrolysis reaction using this approach, measured liquid concentration values are first used with Equations 4.3 and 4.4 to obtain values for the mass fractions of liquid and soluble sugars in the slurry. These values are then used with Equation 4.1 to calculate the conversion yield.
4.3.2 Product Inhibition of Enzymes
It is well known that fungal hemicellulases and cellulases are inhibited by their reaction products. Cross inhibition, for example, inhibition of cellobiohydrolase by xylooligomers, has also been demonstrated to contribute significantly to reduced reaction rates and conversion yields (Qing et al., 2010). Reducing the water con- tent of hydrolyzing biomass slurries results in an inversely proportional increase in the concentrations of soluble oligosaccharides and monomeric sugar products.
Although product inhibition is inherent to fungal enzymes and cannot be avoided, a few approaches can be used to alleviate it. Through targeted genetic engineering, for example, it may be possible to modify the enzymes themselves to exhibit less inhi- bition (Bu et al., 2011). Commercial enzyme cocktails now include many different cellulases and hemicellulases that act synergistically and prevent a buildup of any intermediate sugar or oligosaccharide family. Enzyme mixtures can also be tailored to the feedstock and pretreatment conditions (Banerjee et al., 2010). Nevertheless, product inhibition ofβ-glucosidase andβ-1,4-xylosidase by the monomeric sugars glucose and xylose will still be problematic unless these enzymes are overloaded, which is generally not economically feasible to do.
In order to truly mitigate product inhibition, the sugar products must be removed from the reaction as the reaction is taking place. One means of doing so is to use a process configuration based on simultaneous saccharification and (co-)fermentation (SSF or SSCF), a practice that has a long history in the laboratory (Ghosh et al., 1982; Wang et al., 2013) and has been scaled to pilot-plant operations (Dale and Moelhman, 2009). In SSF, the fermentative microorganism continuously metabolizes the sugars to product (e.g., ethanol), hence preventing the accumulation of high sugar concentrations that cause inhibition of the enzymes. However, the SSF must be operated at temperatures that are favorable for fermentation (typically 30–37◦C) rather than the optimum for enzymatic saccharification (typically 50◦C). This slows the rate of enzymatic conversion such that the system is typically in a sugar-depleted state, starving the fermentation. SSF may still be limited by fermentation product inhibition, both to the enzymes and the microorganism.
Substrate buffer cellulase
Reaction slurry
MF Membrane
Membrane reactor Soluble enzymes
Buffer
Permeate Stirred
reactor
FIGURE 4.1 Block diagram of combined enzymatic hydrolysis and two-stage membrane separation (microfiltration (MF) and ultrafiltration) to remove sugars while retaining undigested biomass and enzymes. Reprinted from Andric et al. (2010b) with permission from Elsevier.
Membrane filtration is another potential way to remove sugars during enzymatic hydrolysis while retaining undigested solid biomass and enzymes (Knutsen and Davis, 2004; Gan et al., 2005). Andric et al. (2010b) provide a review of several combined reaction and separation processes for enzymatic hydrolysis that were stud- ied in the laboratory, and a diagram of one such process is shown in Figure 4.1. In addition to reducing enzyme inhibition, these so-called “membrane reactors” have the potential for continuous operation with the retention or recycle of enzymes. How- ever, they have not yet been sufficiently developed for scale-up and routine use in pilot-scale operations. Few studies have used lignocellulosic substrates, where the buildup of lignin is likely to be problematic.
There have been numerous empirical and semi-empirical models proposed for the enzymatic hydrolysis of cellulose that include inhibition terms (Andric et al., 2010a,b). The use of these models can be helpful with process design and economic calculations where it is necessary to account for the limitations imposed by inhibition.
More rigorous mechanistic models for enzymatic hydrolysis have also included terms for product inhibition, as described in section 4.4.
4.3.3 Slurry Transport and Mixing
Pretreated biomass slurries at high solids concentrations have highly non-Newtonian rheology (Knutsen and Liberatore, 2009; Stickel et al., 2009; Ehrhardt et al., 2010).
Dilute-acid pretreated corn stover slurries exhibit a yield stress at concentrations above 5% insoluble solids. The slurries are shear-thinning after yielding, although measuring shear-flow profiles of sufficient quality for fitting to constitutive models has
FIGURE 4.2 Yield stress evolution through the course of enzymatic hydrolysis of pre- treated corn stover, starting at 20% insoluble solids content and varying enzyme loading (mass enzyme/mass cellulose), as a function of biomass conversion. The predictive model is described by Roche et al. (2009a).
proved challenging. The yield stress increases by orders of magnitude as the insoluble solids concentration increases. Above 20% insoluble solids, air voids appear in the slurry as most of the water resides inside the pores of the biomass. These three-phase (air–liquid–solid) materials obey the principles of wet granular materials rather than hydrodynamics. During enzymatic hydrolysis, the insoluble cellulose is hydrolyzed to soluble species, effectively transferring solid-phase material to the liquid phase.
Consequently, as enzymatic hydrolysis proceeds, the slurry thins out considerably, and the yield stress decreases by orders of magnitude as illustrated in Figure 4.2 (Roche et al., 2009a). Transporting high solids slurries by pump (or other means) and mixing such slurries can be highly energy intensive. Mixing of pretreated biomass that has a high yield stress (initially) is ineffective using traditional impellers in vertical stirred tanks. Mixing in horizontal reaction vessels by axial rotation has been proposed by a few investigators and has been shown to scale well (Jứrgensen et al., 2007; Roche et al., 2009a,b). Some types of water-soluble polymers and surfactants have been shown to act as rheology modifiers for biomass slurries, effectively reducing their yield stress (Knutsen and Liberatore, 2010; Samaniuk et al., 2012).
4.3.4 Heat and Mass Transport
As recently reviewed by Viamajala et al. (2010), the inherently multi-scale structure of lignocellulosic biomass can limit heat and mass transport in reacting biomass slurries. Although heat transfer is of more concern for pretreatment, it should not be neglected when designing large-scale enzymatic hydrolysis reactors where heating or cooling the slurries could take considerably more time than for more typical dilute aqueous streams. Mass transport, however, is a significant issue for enzymatic hydrolysis, especially at high solids concentrations. A few experimental studies have demonstrated the need for effective bulk mixing of enzymes with the pretreated
biomass, especially at the start of the reaction (Mais et al., 2002; Roche et al., 2009b;
Lavenson et al., 2012). Slow continuous mixing or moderate intermittent mixing has been shown to be sufficient to achieve good conversion yields, provided mixing occurs throughout the reactor with few dead zones. Mixing with overly high shear rates has been shown to deactivate cellulases (Cao and Tan, 2004).
Diffusive transport of enzymes and sugars to the surface of and within the porous structure of biomass particles is also an important consideration. Grethlein (1985) demonstrated experimentally that the initial rate of enzymatic hydrolysis corre- lates strongly with the volume of pores that are larger than the size of cellulases.
Luterbacher et al. (2012) used confocal fluorescent microscopy to observe that digest- ing biomass particles do not shrink but rather fade, indicating that the pore structure enlarges during enzymatic digestion. Roberts et al. (2011) used NMR to probe water mobility and diffusivity of solutes in cellulose suspensions. They found that the amount of water associated with the cellulose, supposed to be water in the pores, decreased with increasing solids concentration. They also found that the diffusivity of sugar and enzyme surrogate (bovine serum albumin) through the cellulose suspen- sion decreased with increasing solids concentration. It has been hypothesized that product inhibition is amplified when there is poor mass transfer in the reacting slurry (Hodge et al., 2008; Viamajala et al., 2010). As enzymes hydrolyze the polysac- charides to sugars, locally high concentrations of the sugars may accumulate near the enzymes unless they are transported away by diffusive and convective transport.
Because mixing becomes more difficult at high solids loading, product inhibition and ineffective transport are antagonistic to the overall reaction rates and yields.