Laboratory-scale method for enzymatic saccharification of lignocellulosic biomass at high-solids loadings

<p>Abstract</p> <p>Background</p> <p>Screening new lignocellulosic biomass pretreatments and advanced enzyme systems at process relevant conditions is a key factor in the development of economically viable lignocellulosic ethanol. Shake flasks, the reaction vessel commonly used for screening enzymatic saccharifications of cellulosic biomass, do not provide adequate mixing at high-solids concentrations when shaking is not supplemented with hand mixing.</p> <p>Results</p> <p>We identified roller bottle reactors (RBRs) as laboratory-scale reaction vessels that can provide adequate mixing for enzymatic saccharifications at high-solids biomass loadings without any additional hand mixing. Using the RBRs, we developed a method for screening both pretreated biomass and enzyme systems at process-relevant conditions. RBRs were shown to be scalable between 125 mL and 2 L. Results from enzymatic saccharifications of five biomass pretreatments of different severities and two enzyme preparations suggest that this system will work well for a variety of biomass substrates and enzyme systems. A study of intermittent mixing regimes suggests that mass transfer limitations of enzymatic saccharifications at high-solids loadings are significant but can be mitigated with a relatively low amount of mixing input.</p> <p>Conclusion</p> <p>Effective initial mixing to promote good enzyme distribution and continued, but not necessarily continuous, mixing is necessary in order to facilitate high biomass conversion rates. The simplicity and robustness of the bench-scale RBR system, combined with its ability to accommodate numerous reaction vessels, will be useful in screening new biomass pretreatments and advanced enzyme systems at high-solids loadings.</p

Structure and dynamics of colloidal depletion gels: coincidence of transitions and heterogeneity

Transitions in structural heterogeneity of colloidal depletion gels formed through short-range attractive interactions are correlated with their dynamical arrest. The system is a density and refractive index matched suspension of 0.20 volume fraction poly(methyl methacyrlate) colloids with the non-adsorbing depletant polystyrene added at a size ratio of depletant to colloid of 0.043. As the strength of the short-range attractive interaction is increased, clusters become increasingly structurally heterogeneous, as characterized by number-density fluctuations, and dynamically immobilized, as characterized by the single-particle mean-squared displacement. The number of free colloids in the suspension also progressively declines. As an immobile cluster to gel transition is traversed, structural heterogeneity abruptly decreases. Simultaneously, the mean single-particle dynamics saturates at a localization length on the order of the short-range attractive potential range. Both immobile cluster and gel regimes show dynamical heterogeneity. Non-Gaussian distributions of single particle displacements reveal enhanced populations of dynamical trajectories localized on two different length scales. Similar dependencies of number density fluctuations, free particle number and dynamical length scales on the order of the range of short-range attraction suggests a collective structural origin of dynamic heterogeneity in colloidal gels.Comment: 14 pages, 10 figure

GSK3-mediated raptor phosphorylation supports amino acid-dependent Q2 mTORC1-directed signalling

The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser(859). We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1 (ribosomal S6 kinase 1) and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation

Origins of heterogeneous dynamics in colloidal gels.

Heterogeneous dynamics of collodial depletion gels arises directly from structural features in some specific cases identified in this thesis. Our experimental system consists of sterically stabilized, fluorescent poly(methyl methacrylate) spheres dispersed in a density and refractive index matched solvent of cyclohexyl bromide and decalin. Volume fraction is fixed (phi c = 0.20) at a midrange value far from low volume fractions modeled by fractal gels or high volume fractions near the attractive glass transition. The depletion interaction is induced with non-adsorbing polystyrene. For the first study, the range of inter-particle attraction was fixed (Rg/a = 0.043) while the strength of pair attraction was varied (c/c* = 0.15--1.54). With increasing attraction, a maximum in structural heterogeneity is found locally by examining contact number distributions and, on a longer range, using number density fluctuations. The maximum in structural heterogeneity correlates with a plateau of dynamic arrest dividing our attractive system into a cluster phase and a network phase. We further analyze the relationship between local structure and dynamics by selecting a test case from each of the two regimes identified: weak cluster and strong network gels. Contact number distinguishes a number of dynamic features in the cluster gel regime. Mean squared displacement decreases with increasing contact number. Particles with a lower contact number took longer to reach the time scale on which they achieve their maximum heterogeneity and had a larger magnitude of non-Gaussian character in the cluster sample. Contact number showed no effect in dividing the dynamics of network gels. All structural classifications of particles studied in either regime produce heterogeneous dynamics. The nature of that heterogeneity is different between cluster and network gels. We apply an electric field to a sample equivalent to one of the strongest gels formed in the cluster regime. We observe and quantify both formation and breakage of inter-particle bonds in a preliminary analysis to identify characteristics for automatic event recognition. Particle pairs that had a breaking bond elongated significantly in the one frame where the event was identified. Particles forming a bond compressed evenly but significantly over the four frames tracked. Images are acquired at 0.1 s per frame. More displacements of particles involved in breakage or healing events tend to be in the mid-range of displacements that is unfavorable to a quiescent sample and might be analogous to an activated complex in a transition state.Ph.D.Applied SciencesChemical engineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126410/2/3253260.pd