Climate changes and shortage of fossil fuels have sparked a growing demand for liquid biofuels which in turn has increased the amount of research into the production of lignocellulose-derived bioethanol [1
]. However, being an insoluble and highly heterogeneous substrate, lignocellulosic materials pose several challenges in conversion to fermentable sugars. In addition to understanding complex enzyme system kinetics, these biomass-related challenges include recalcitrance to hydrolysis [3
] and mixing difficulties [4
]. Water content in the hydrolysis slurry is directly correlated to rheology, that is, viscosity and shear rate during mixing [5
], important for the interaction between lignocellulose and cell wall-degrading enzymes. Thus, water is not only critical in hydrolysis being a substrate and a prerequisite for enzyme function, but is also crucial for enzyme transport mechanisms throughout hydrolysis as well as mass transfer of intermediates and end-products [6
]. Maintaining high substrate concentrations throughout the conversion process from biomass to ethanol is important for the energy balance and economic viability of bioethanol production.
High-solids enzymatic hydrolysis can be defined as taking place at solids levels where initially there are no significant amounts of free liquid water present [7
]. By increasing the solids loading, the resulting sugar concentration and consequently ethanol concentration increase, having significant effects on processing costs, in particular distillation [8
]. Furthermore, lower water content allows for a larger system capacity, less energy for heating and cooling of the slurry and less waste water [4
]. Model-based estimations have shown significant reductions of operating costs of simultaneous saccharification and fermentation (SSF) of pretreated softwood when the initial solids concentration was increased [8
]. Unfortunately, there are also disadvantages to increasing the substrate concentration. Concentrations of end products and inhibitors will increase, causing enzymes and fermenting organisms to not function optimally. Also, high-solids loadings can cause insufficient mixing, or mixing can be too energy-consuming in conventional stirred-tank reactors as the viscosity of slurries increases abruptly at increasing solids loadings, in particular over 20% solids [11
native cellulase systems have been reported to function at solids levels as high as 76% (all concentrations are given as total solids on a w/w
], indicating that enzymatic hydrolysis may be limited by the laboratory or industrial process set-up. Twelve to fifteen per cent total solids is often considered the upper limit at which pretreated biomass can be mixed and hydrolysed in conventional stirred-tank reactors [7
]. However, at the laboratory scale, enzymatic hydrolysis at up to 32% total solids has been reported [12
]. A number of studies have utilised fed-batch operations in order to increase the final solids loading [7
]. We have previously described a gravimetric mixing reactor design that allows batch enzymatic liquefaction and hydrolysis of pretreated wheat straw at up to 40% solids concentration [4
]. This is a significant increase from what has previously been possible, and thus significantly increases the techno-economic potential of the whole process. The gravimetric mixing principle has been up-scaled and used in a pilot plant for several years [19
During the work with high solids concentrations we found that the enzymatic conversion (percent of theoretical) linearly decreased with increasing solids concentration (constant enzyme to substrate ratio) [4
]. This decrease partly offsets the advantages of running at high solids concentrations. As seen in Figure , the effect has been observed in both enzymatic hydrolysis and SSF by several groups working with various kinds of biomass [12
]. Although several of these studies were conducted at less than 10% initial solids content, the phenomenon appears to be an intrinsic or generic effect of enzymatic hydrolysis at increasing solids levels. In this paper, the decrease in yield at high solids concentrations is referred to as the solids effect.
Figure 1 Decreasing conversion of biomass. Results collected from several publications indicate that decreasing conversion at increasing solids content is a general effect. Results are for different kinds of biomass and for both enzymatic hydrolysis and simultaneous (more ...)
Some groups have suggested that the mechanism behind the decreasing conversion is product inhibition [12
] or inhibition by other compounds such as sugar-derived inhibitors (furfural and hydroxymethylfurfural (HMF)) [26
] and lignin [27
]. Others have suggested it may be explained by mass transfer limitations or other effects related to the increased content of insoluble solids, such as non-productive adsorption of enzymes [14
]. However, the specific mechanism(s) responsible for the decreasing hydrolytic efficiency are still uncertain [4
It should be noted that inhibition primarily affects the hydrolysis rate and not the maximum conversion or yield, given sufficient time. With limited reaction times and not fully converted, the conversion will correspond to the inhibition, that is, the conversion being a measure of the 'accumulated' inhibition. Working with initial reaction velocities in high-solids hydrolysis involves great difficulties due to the non-liquid properties of the substrate. For that reason, degree of conversion has been used to estimate the increased inhibition that appears to take place at elevated solids contents.
In this paper the possible mechanisms behind the solids effect have been divided into the following four categories: Compositional and substrate effects; product inhibition; water concentration; and cellulase adsorption. These four topics will be introduced below.
Compositional and substrate effects
The heterogeneity and structure of lignocellulosic biomass means that high viscosity prevents efficient mixing at high solids concentrations when performed in conventional stirred-tank reactors [14
]. The viscosity of lignocellulosic slurries increases sharply over a certain threshold (typically around 20% solids) but, despite the extreme difference in viscosity between, for example, 5% and 40% solids loading, the conversion of lignocellulosics as a function of solids appears to be linear (Figure ). Although mixing of substrate and enzymes is crucial for an efficient liquefaction, our previous findings showed that it does not appear that lack of mixing is the cause of the decreasing conversion, at least not at the solids levels documented [4
]. This is in accordance with the recent findings of Hodge and co-workers who concluded that possible mass transfer limitations caused by insoluble solids were not apparent at below 20% insoluble solids content [25
]. At very high solids levels (above 20 to 30% dry matter), a mass transfer limitation may be involved in the lower yield, but the linearity of the solids effect over a large range of conditions with a number of substrates (wheat and barley straw [4
], corn stover [17
], softwood [22
], hardwood [16
] and an industrial ethanol fermentation residue (vinasse) [28
]) indicates that a single factor may be responsible for the effect (all the way from, for example, 5% to 40% dry matter).
In order to be able to establish that the solids effect is not caused by lignin adsorption or lignin-derived inhibitors (phenolics), experiments for this paper were carried out with filter paper. Filter paper has the advantage of containing no lignin yet still retains the secondary cell wall structure, as opposed to Sigmacell and Avicel, for example.
End-product inhibition plays an important role in enzymatic hydrolysis as glucose, cellobiose and ethanol have demonstrated their ability to significantly inhibit endoglucanases, cellobiohydrolases and β
]. However, working with an insoluble substrate and kinetics that do not follow the Michaelis-Menten model, the exact type of inhibition is difficult to determine [33
]. The decrease in hydrolysis rate over time has been attributed to inhibition by the accumulated end products [34
]. Others conclude that when hydrolysing natural, lignocellulosic substrates, cellulases are more resistant to product inhibition than with amorphous reference materials and that the early stage decrease in hydrolysis rate is not caused by product inhibition [35
]. In high-solids enzymatic hydrolysis of pretreated corn stover, Hodge and co-workers recently found that increased sugar concentrations were the primary cause of performance inhibition [25
]. Based on the above, we have investigated the inhibitory effect of increased sugar concentration in connection with high-solids enzymatic hydrolysis.
Working with a system with low water content may directly affect enzyme performance. Not only is water a substrate for the hydrolysis but it is also the solvent that allows the function of enzymes, contact between enzymes and substrate and transport of products [37
]. We have previously investigated the role of water in enzymatic hydrolysis [6
]. In this study, we wanted to investigate if the solids effect was related to a lower concentration of water in relation to solids. As mentioned, hydrolysis is possible at very high solids concentrations but the rate of reaction may be impaired under such conditions [13
We have investigated the role of water concentration by replacing various amounts of the water in enzymatic hydrolysis with oleyl alcohol, an inert oil that does not directly affect the function of the enzymes [38
Cellulose accessibility and degree of adsorption of cellulases are well known as controlling factors for conversion rates and yields [40
]. It has long been known that certain hydrolysis products are able to inhibit cellulase adsorption [42
]. It has, however, recently been shown that glucose and especially cellobiose strongly inhibit cellulase adsorption in a linear fashion [43
]. This adsorption inhibition can be seen as a sub-class of product inhibition where the catalytic site may not necessarily be involved. In order to investigate whether adsorption (or lack thereof) could possibly be involved in the observed solids effect, the adsorption of enzyme was measured in hydrolysis of filter paper at different solids contents.