Investigating the role of mechanics in lignocellulosic biomass degradation during hydrolysis: Part II

Abstract Lignocellulose breakdown in biorefineries is facilitated by enzymes and physical forces. Enzymes degrade and solubilize accessible lignocellulosic polymers, primarily on fiber surfaces, and make fibers physically weaker. Meanwhile physical forces acting during mechanical agitation induce tearing and cause rupture and attrition of the fibers, leading to liquefaction, that is, a less viscous hydrolysate that can be further processed in industrial settings. This study aims at understanding how mechanical agitation during enzymatic saccharification can be used to promote fiber attrition. The effects of reaction conditions, such as substrate and enzyme concentration on fiber attrition rate and hydrolysis yield were investigated. To gain insight into the fiber attrition mechanism, enzymatic hydrolysis was compared to hydrolysis by use of hydrochloric acid. Results show that fiber attrition depends on several factors concerning reactor design and operation including drum diameter, rotational speed, mixing schedule, and concentrations of fibers and enzymes. Surprisingly, different fiber attrition patterns during enzymatic and acid hydrolysis were found for similar mixing schedules. Specifically, for tumbling mixing, slow continuous mixing appears to function better than faster, intermittent mixing even for the same total number of drum revolutions. The findings indicate that reactor design and operation as well as hydrolysis conditions are key to process optimization and that detailed insights are needed to obtain fast liquefaction without sacrificing saccharification yields.

concentration and, depending on the type of biomass, free water might be absent at concentrations above 15-40% insoluble solids. 4,5 Water is essential for biomass hydrolysis not only because it is involved in the hydrolysis reaction, but also as it acts as a dispersing agent for biomass particles and provides a diffusion medium for enzymes and soluble hydrolysis products. 6 A limited amount or absence of free water during hydrolysis is therefore associated with limited hydrolysis efficiency and challenging processing. 4,7 This includes ineffective heat and mass transfer, 6 end-product inhibition, 8 biomass agglomeration, 9 decreased mixing efficacy and, at the same time, increased energy requirements for mixing. 10,11 Many different strategies have been used to increase biomass degradation rate, including the use of more efficient enzyme formulations, 12 higher enzyme dosages, 13 addition of supplemental agents (e.g., surfactants), 14,15 as well as improving reactor design and operation in order to maximize physical breakdown of biomass particles. 2,16 The importance of mechanical agitation during hydrolysis of lignocellulose is increasingly recognized. 9,17,18 This is primarily due to the findings that enzymes and mechanical loading act synergistically in biomass degradation and that mixing can significantly affect biomass degradation rate. 2,17 In particular, it has been found that free fall agitation, that is, tumbling, where mixing relies on gravity, is much more effective than stirring for hydrolysis at high biomass concentration. 2,16 It is suggested that more efficient agitation promotes heat and mass transfer, including enzyme redistribution and dispersal of hydrolysis products away from the active sites of enzymes, hereby alleviating end product inhibition. 19,20 Notably, physical forces acting on fibers during mixing also directly contribute to fiber attrition. 9,17,20 During tumbling mixing, the fibers are continuously subjected to the impact force which is generated once the falling fibers hit the bottom side of a reactor. Equation (1) defines the impact force of a falling object and indicates which parameters influence the impact force and thus physical fiber attrition, where P (N) specify impact force, m (kg) is object mass, g (m/s 2 ) gravitational acceleration, h (m) fall height and s (m) slow down distance.
Fiber attrition, that is, the reduction in size of fiber fragments during agitation, does not necessary lead to higher hydrolysis yields, although it increases the surface area and potentially enables higher surface coverage with enzymes. 9,20 However, shorter fibers are less prone to entangle. 21 As a result, shortening of fibers during hydrolysis typically leads to liquefaction, that is, a less viscous fiber suspension, which can be pumped easier between different parts of the process equipment. 21,22 Consequently, the risk of pipe blockage is reduced.
On the other hand, enzymes are sensitive biological molecules that can be negatively affected by process related factors, including mixing. Mixing induces shear forces and leads to aeration of enzymes, which accelerates enzyme inactivation. [23][24][25] Due to the complex synergistic and antagonistic effects of agitation on biomass degradation, it is necessary to determine the optimal reactor design and operation conditions for achieving fast liquefaction and optimal hydrolysis yields. 9 In the first part of this study we showed that neither mechanical agitation alone nor enzymatic treatment without mechanical agitation has any noteworthy effect on flax fiber attrition. It was found that successive treatment where enzymatic hydrolysis was preceded by mechanical agitation did not induce any substantial segmentation (i.e., attrition or shortening) of flax fibers. However, we showed that fibers subjected to prolonged hydrolysis where more susceptible to fracture during proceeding mechanical treatment than untreated fibers. Attrition of fibers was gradual, indicating a fatigue type of fail-

| Effect of reactor design and operation
Three different laboratory-scale agitation systems were used: a roller bottle reactor and two horizontally rotating tumbling reactors ( Table 1).
The latter two only differed in the drum diameter (30 and 15 cm) and in the length of the integrated paddle (5 and 2.5 cm, respectively) that, during rotation, lifted and dropped the bottles placed within the drums (37 cm in length). 26 In the roller bottle reactor, bottles were not subjected to tumbling, but instead were horizontally oriented and Note: Symbols * and indicate that hydrolysis was carried out using enzymes or HCl acid, respectively. Twenty milliliters hydrolysis vessels were subjected to tumbling inside 30 or 15 cm diameter reactors. The length of the tumbling reactors was the same-37 cm. The size (diameter) of the bottles schematically shown inside the tumbling reactors is kept proportional to the size (diameter) of the reactors and the length of integrated paddles. In the roller bottle reactor, bottles were not subjected to tumbling, but instead were horizontally oriented and rotated around their axis.
flax fibers were pre-incubated with hydrochloric acid solution for 12 hr before initiating the intermitted mechanical treatment.

| Effect of enzyme loading and substrate concentration
To investigate the effect of enzyme and substrate concentration on flax fiber attrition and saccharification, three doses of Cellic CTec2 enzyme preparation (4, 8, and 16 mg protein/g flax) and two doses of substrate loading (25 and 10% DM) were tested (Table 1).

| Analytical methods
Flax fiber dimensions were analyzed with a PulpEye (Eurocon Analyser AB, Sweden) particle analyzer in manual mode. 26

| Effect of reactor design
The flax fiber attrition data obtained from three different reactor setups (Table 1)   was not only exposed to the impact force, but also to the rotational  9 This leads to a substantial reduction of the substrate volume within the reaction vessels, and in such a way permits easier substrate movement within the vessel. Due to the aforementioned effects it is challenging to precisely estimate the magnitude of impact force to which fibers were exposed during tumbling.
The roller bottle results from this study are in contrast with those of Roche et al who found that rotational mixing within 125 and 250 ml reaction vessels lead to efficient liquefaction of dilute sulfuric acid pretreated corn stover, even at 30% substrate loading. 16 The present observation of limited fiber attrition (Figure 1a) as well as absence of fiber agglomeration (visual observation) indicate that forces acting during rotational mixing were insufficient to induce fiber breakage or even a movement of relatively long flax fibers within the 20 ml reaction vessels, that is, they were insufficient to overcome the strength of the fiber network and its association with the vessel surface. Noteworthy, the large void volume required to achieve efficient biomass mixing within tumbling type reactors is a considerable drawback of this reactor system. 2 The contrasting outcome of the two studies can potentially be attributed to the different void volumes within the reaction vessels, but also to the specific biomass properties, for example, fiber morphology, initial fiber length distribution, chemical composition, as well as the reaction conditions, that is, substrate concentration, reactor and enzyme specifics. 16 In this study, it was also attempted to vary the mass of the hydrolysate within the reaction vessels, that is, 5 or 10 g, in order to  (1)).
In summary, the results suggest that fall height is the most decisive parameter of the reactor design with regards to fiber attrition.

| Effect of agitation intensity
The effect of mixing intensity on the attrition of flax fibers was examined by reducing agitation rate 10 times, from 30 to 3 rpm. During the acid hydrolysis, mixing was also carried out at 15 rpm.
From Figure 2a, it can be noted that tenfold reduction in agitation intensity during enzymatic hydrolysis considerably slowed down attrition of flax fibers. Agitation intensity during the acid hydrolysis impacted fiber breakage in a similar manner where both twofold (from T A B L E 2 Impact force dependence on fall height illustrated for two different substrate loadings (10 and 25%) when the same amount of a substrate is used (1 g) and assuming that all water is associated with the substrate and a slow down distance is the same, that is, 2 cm  Figure 2b). During the time period studied, acid hydrolysis without agitation did not result in substantial fiber attrition (only a share of long, i.e., 2.5-5 mm fibers is shown in Figure 2b). This finding is in agreement with previously published data showing that enzymatic hydrolysis without agitation, although it leads to significant hydrolysis yields, do not induce considerable attrition of flax fibers. 9,28 It is worth noting that extensive fiber attrition during acid hydrolysis only occurred after a delay phase of app. 12 hr, unlike for simultaneous enzymatic and mechanical treatment. The long stagnant phase seen before the fast fiber attrition is likely a result of physical and catalytic differences between the catalysts. H 3 O + ions are significantly smaller, $0.100 nm, 29 than enzymes (typical cellulase has a diameter of 4-6.5 nm and is 18-21.5 nm long), 30 and can therefore diffuse and penetrate plant cell walls more evenly than enzymes. We speculate that this implies that it takes longer time for a sufficient degree of hydrolysis to take place in locations prone to breakage.
Enzymes are on the other hand limited to diffusing into larger pores or other defects in the cell walls. 31 The results above indicate that there is a simple relationship between agitation speed and the rate at which attrition happens during enzymatic hydrolysis. The design challenge is thus to find an agitation speed that gives sufficiently fast liquefaction without inducing unacceptable deactivation rates of the enzyme preparation used.

| Effect of intermittent tumbling
Intermittent tumbling was tested as an attempt to introduce a milder, but still efficient, agitation scheme. Intermittent tumbling during enzymatic or acid hydrolysis was carried out following three different schedules: (i) for 6 min each hr, (ii) for 12 min every 2 hr, or (iii) for 24 min every 4 hr in the tumbling reactor, Ø 30 cm, rotating at a speed of 30 rpm. This implies that independently of the agitation intervals used, samples were exposed to the same magnitude of mechanical treatment over a 12 hr period (i.e., 2,160 reactor revolutions in total), which also corresponds to the agitation intensity obtained during continuous mixing at 3 rpm. In these experiments, higher enzyme loading, 16 mg/g of substrate, was used.
It was found that even though the total number of reactor revolutions was the same in all cases, the fiber attrition profiles during enzymatic hydrolysis were different (see Figure 3a). The attrition of flax fibers turned out to be progressively slower with increasing time periods between agitation cycles. The trend is best represented by attrition of long, that is, 2.5-5 mm flax fibers. Noteworthy, the continuous mixing at 3 rpm accelerated flax fiber attrition the most. The observed difference in fiber attrition during enzymatic hydrolysis cannot be attributed to the mechanical forces since the magnitude of agitation, that is, the fatigue damage due to cyclic loading during the intermittent tumbling in all instances was the same. Contrary to the enzymatic-intermittent tumbling, the trends of fiber attrition profiles were somewhat similar during acidintermittent tumbling (Figure 3b). Therefore, other factors, most likely related to the enzymes, lead to the aforementioned differences. During the intermitted tumbling, enzymes are given periods of time to hydrolyze the substrate undisturbed by mechanical forces. 9 Processive enzymes, such as typical cellobiohydrolases, remain active unless they are physically obstructed, for example by lignin, and continue to hydrolyze cellulose even in the absence of mixing. 32 A static hydrolysis has its benefits since it has been shown that mechanical agitation may reduce enzyme activity. 9 Lack of agitation, on the other hand, also leads to accumulation of reaction products in the vicinity of enzymes (end product inhibition) and may lead to a lack of redistribution of non-processive enzymes, the diffusion of which toward accessible substrate areas potentially becomes F I G U R E 2 Effect of mixing intensity on flax fiber attrition during (a) enzymatic and (b) acid hydrolysis. Hydrolysis experiments were carried out at 25% substrate loading in the tumbling reactor having a diameter of 30 cm. The enzyme loading during hydrolysis was 16 mg/g DM flax. In subplot (b), single black line with stars represent attrition of the fibers ranging between 2.5 and 5 mm when acid hydrolysis was carried out without agitation. Four different colors of symbols and lines are used to represent different fiber length ranges limited. Endoglucanases, for instance, are often non-processive enzymes, but they play a key role in fiber attrition. 22 Endoglucanases are known to preferentially attack less crystalline cellulose regions (often called amorphous regions) and break β-(1,4)glycosidic linkages internally. 33 The regions in plant fibers where cellulose microfibrils are less ordered, that is, dislocations, are often crack initiation sites during mechanical loading. 34 Consequently, endoglucanases additionally weaken these fiber regions and makes fibers more vulnerable to mechanical failure. 17,35 In a recent study, Ciesielski et al employed direct nano-manipulation of cellulose nanofibrils and showed that fibrils can be kinked by applied mechanical force to such an extent that breakages in the glucan chains are introduced, which then provides initiation sites for processive, exo-active cellobiohydrolases (i.e., Cel7A). 36 The study further supports the finding that enzymes and mechanics act synergistically in fiber attrition. The progressively slower fiber attrition seen during the intermittent tumbling suggests that such agitation is less efficient than continuous, but slower, agitation in ensuring redistribution of enzymes, in particular, endoglucanases.
As described previously, prior to intermittent tumbling the fibers where subjected to 12 hr static acid hydrolysis. This step was introduced because no significant fiber attrition occurred during the initial 12 hr of simultaneous acid and mechanical treatment ( Figure 2b). Figure 3 suggest that in order to achieve fast fiber attrition, continuous rather than intermitted agitation should be applied during enzymatic hydrolysis of lignocellulose.

| Effect of enzyme concentration
Data in Figure 4a,b indicate that fiber attrition as well as saccharification could be considerably promoted by increasing enzyme loading.
When the enzyme concentration was increased from 4 mg/g DM to The phenomenon that increasing enzyme concentration enhances holocellulose conversion only to a certain extent has been noted previously. 13,23,38 The absence of accessible surface areas of the substrate, that is, lack of potential enzyme binding sites, has been proposed to be responsible for the observed retardation of hydrolysis rate at high enzyme loading. 23 Dissociation of flax fiber bundles and fragmentation of fibers, as seen in Figure 4a, during the treatment potentially increased accessible surface area of the sub- strate. Yet, the result that similar glucose yields were obtained when the enzyme concentration was either 8 mg/g DM or 16 mg/g DM flax, indicates that enzymes at these hydrolysis conditions were in excess.

| Effect of substrate concentration
The substrate concentration was another factor that considerably influenced fragmentation and saccharification of flax fibers, Figure 5a, b, respectively. A decrease in the substrate concentration from 25 to 10% w/v resulted in considerably faster fiber attrition as well as higher hydrolysis yield, that is, glucose concentration was 12% higher after 24 hr of hydrolysis. The effect that increasing lignocellulose concentration during hydrolysis negatively affects hydrolysis yield has been routinely observed in similar studies. 2,7,39 It has been suggested that reduced enzyme mobility through the medium containing limited amounts of free water, changing rheological properties, heat and hydrolysis product transfer, end-product inhibition, as well as other factors could be responsible for the observed decrease in saccharification rates. 7,13,40 However, more recent studies have pointed out that the relationship between sugar yields and solids contents may be less straight forward than hitherto assumed and for example depends on the enzyme preparation used. 4

| CONCLUSIONS
It was found that both reactor design, for example, the diameter of tumbling type reactors, as well as operating conditions, that is, rotational speed and intermittent mixing, affect the attrition rate of flax fibers during hydrolysis. Different fiber attrition patterns were observed despite the same mixing intensities applied during enzymatic and acid hydrolysis experiments, indicating that enzymes target and damage lignocellulose more selectively and less evenly than hydrochloric acid. Results confirmed that increased enzyme concentration (to a certain limit) as well as reduced substrate loading during hydrolysis leads to faster fiber attrition and higher saccharification yields. To summarize, the findings of this work indicate that reactor design and operation as well as hydrolysis conditions are all important factors, which needs to be optimized meticulously to obtain efficient lignocellulose depolymerization and low operating costs of a biorefinery facility. The results suggest that fiber attrition, and thus liquefaction, in biorefineries can be improved by increasing the fall distance of a fibrous substrate within a reactor, which means the need F I G U R E 5 Effect of substrate concentration on (a) flax fiber attrition and (b) saccharification rate during enzymatic hydrolysis at 10% and 25% (w/w) solid loadings. Please note that error bars in the subplot (b) represent SD of only two measurements. Four different colors of symbols and lines are used to represent different fiber length ranges