A pore‐hindered diffusion and reaction model can help explain the importance of pore size distribution in enzymatic hydrolysis of biomass

Until now, most efforts to improve monosaccharide production from biomass through pretreatment and enzymatic hydrolysis have used empirical optimization rather than employing a rational design process guided by a theory‐based modeling framework. For such an approach to be successful a modeling framework that captures the key mechanisms governing the relationship between pretreatment and enzymatic hydrolysis must be developed. In this study, we propose a pore‐hindered diffusion and kinetic model for enzymatic hydrolysis of biomass. When compared to data available in the literature, this model accurately predicts the well‐known dependence of initial cellulose hydrolysis rates on surface area available to a cellulase‐size molecule. Modeling results suggest that, for particles smaller than 5 × 10^?3 cm, a key rate‐limiting step is the exposure of previously unexposed cellulose occurring after cellulose on the surface has hydrolyzed, rather than binding or diffusion. However, for larger particles, according to the model, diffusion plays a more significant role. Therefore, the proposed model can be used to design experiments that produce results that are either affected or unaffected by diffusion. Finally, by using pore size distribution data to predict the biomass fraction that is accessible to degradation, this model can be used to predict cellulose hydrolysis with time using only pore size distribution and initial composition data. Biotechnol. Bioeng. 2013; 110: 127–136. © 2012 Wiley Periodicals, Inc.     

Observing and modeling BMCC degradation by commercial cellulase cocktails with fluorescently labeled Trichoderma reseii Cel7A through confocal microscopy

Understanding the depolymerization mechanisms of cellulosic substrates by cellulase cocktails is a critical step towards optimizing the production of monosaccharides from biomass. The Spezyme CP cellulase cocktail combined with the Novo 188 β‐glucosidase blend was used to depolymerize bacterial microcrystalline cellulose (BMCC), which was immobilized on a glass surface. The enzyme mixture was supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A, which served as a reporter to track cellulase binding onto the physical structure of the cellulosic substrate. Both micro‐scale imaging and bulk experiments were conducted. All reported experiments were conducted at 50°C, the optimal temperature for maximum hydrolytic activity of the enzyme cocktail. BMCC structure was observed throughout degradation by labeling it with a fluorescent dye. This method allowed us to measure the binding of cellulases in situ and follow the temporal morphological changes of cellulose during its depolymerization by a commercial cellulase mixture. Three kinetic models were developed and fitted to fluorescence intensity data obtained through confocal microscopy: irreversible and reversible binding models, and an instantaneous binding model. The models were successfully used to predict the soluble sugar concentrations that were liberated from BMCC in bulk experiments. Comparing binding and kinetic parameters from models with different assumptions to previously reported constants in the literature led us to conclude that exposing new binding sites is an important rate‐limiting step in the hydrolysis of crystalline cellulose. Biotechnol. Bioeng. 2013; 110: 108–117. © 2012 Wiley Periodicals, Inc.     

Biomass responses in a temperate European grassland through 17 years of elevated CO2

Future increase in atmospheric CO₂ concentrations will potentially enhance grassland biomass production and shift the functional group composition with consequences for ecosystem functioning. In the “GiFACE” experiment (Giessen Free Air Carbon dioxide Enrichment), fertilized grassland plots were fumigated with elevated CO₂ (eCO₂) year‐round during daylight hours since 1998, at a level of +20% relative to ambient concentrations (in 1998, aCO₂ was 364 ppm and eCO₂ 399 ppm; in 2014, aCO₂ was 397 ppm and eCO₂ 518 ppm). Harvests were conducted twice annually through 23 years including 17 years with eCO₂ (1998 to 2014). Biomass consisted of C3 grasses and forbs, with a small proportion of legumes. The total aboveground biomass (TAB) was significantly increased under eCO₂ (p = .045 and .025, at first and second harvest). The dominant plant functional group grasses responded positively at the start, but for forbs, the effect of eCO₂ started out as a negative response. The increase in TAB in response to eCO₂ was approximately 15% during the period from 2006 to 2014, suggesting that there was no attenuation of eCO₂ effects over time, tentatively a consequence of the fertilization management. Biomass and soil moisture responses were closely linked. The soil moisture surplus (c. 3%) in eCO₂ manifested in the latter years was associated with a positive biomass response of both functional groups. The direction of the biomass response of the functional group forbs changed over the experimental duration, intensified by extreme weather conditions, pointing to the need of long‐term field studies for obtaining reliable responses of perennial ecosystems to eCO₂ and as a basis for model development.     

The origin of Jurassic reefs: Current research developments and results

Summary In order to elucidate the control of local, regional and global factors on occurrence, distribution and character of Jurassic reefs, reefal settings of Mid and Late Jurassic age from southwestern Germany, Iberia and Romania were compared in terms of their sedimentological (including diagenetic), palaeoecological, architectural, stratigraphic and sequential aspects. Upper Jurassic reefs of southern Germany are dominated by siliceous sponge—microbial crust automicritic to allomicritic mounds. During the Oxfordian these form small to large buildups, whereas during the Kimmeridgian they more frequently are but marginal parts of large grain-dominated massive buildups. Diagenesis of sponge facies is largely governed by the original composition and fabric of sediments. The latest Kimmeridgian and Tithonian spongiolite development is locally accompanied by coral facies, forming large reefs on spongiolitic topographic elevations or, more frequently, small meadows and patch reefs within bioclastic to oolitic shoal and apron sediments. New biostratigraphic results indicate a narrower time gap between Swabian and Franconian coral development than previously thought. Palynostratigraphy and mineralostratigraphy partly allow good stratigraphic resolution also in spongiolitic buildups, and even in dolomitised massive limestones. Spongiolite development of the Bajocian and Oxfordian of eastern Spain shares many similarities. They are both dominated by extensive biostromal development which is related to hardground formation during flooding events. The Upper Jurassic siliceous sponge facies from Portugal is more localised, though more differentiated, comprising biostromal, mudmound and sponge-thrombolite as well as frequent mixed coral-sponge facies. The Iberian Upper Jurassic coral facies includes a great variety of coral reef and platform types, a pattern which together with the analysis of coral associations reflects the great variability of reefal environments. Microbial reefs ranging from coralrich to siliceous sponge-bearing to pure thrombolites frequently developed at different water depths. Reef corals even thrived within terrigeneous settings. In eastern Romania, small coral reefs of various types as well as larger siliceous sponge-microbial crust mounds grew contemporaneously during the Oxfordian, occupying different bathymetric positions on a homoclinal ramp. Application of sequence stratigraphic concepts demonstrates that onset or, in other cases, maximum development of reef growth is related to sea level rise (transgressions and early highstand) which caused a reduction in allochthonous sedimentation. The connection of reef development with low background sedimentation is corroborated by the richness of reefs in encrusting organisms, borers and microbial crusts. Microbial crusts and other automicrites can largely contribute to the formation of reef rock during allosedimentary hiatuses. However, many reefs could cope with variable, though reduced, rates of background sedimentation. This is reflected by differences in faunal diversities and the partial dominance of morphologically adapted forms. Besides corals, some sponges and associated brachiopods show distinct morphologies reflecting sedimentation rate and substrate consistency. Bathymetry is another important factor in the determination of reefal composition. Not only a generally deeper position of siliceous sponge facies relative to coral facies, but also further bathymetric differentiation within both facies groups is reflected by changes in the composition, diversity and, partly, morphology of sponges, corals, cementing bivalves and microencrusters. Criteria such as authigenic glauconite, dysaerobic epibentic bivalves,Chondrites burrows or framboidal pyrite in the surrounding sediments of many Upper Jurassic thrombolitic buildups suggest that oxygen depletion excluded higher reefal metazoans in many of these reefs. Their position within shallowing-upwards successions and associated fauna from aerated settings show that thrombolitic reefs occurred over a broad bathymetric area, from moderately shallow to deep water. Increases in the alkalinity of sea water possibly enhanced calcification. Reefs were much more common during the Late Jurassic than during the older parts of this period. Particularly the differences between the Mid and Late Jurassic frequencies of reefs can be largely explained by a wider availability of suitable reef habitats provided by the general sea level rise, rather than by an evolutionary radiation of reef biota. The scarcity of siliceous sponge reefs on the tectonically more active southern Tethyan margin as well as in the Lusitanian Basin of west-central Portugal reflects the scarcity of suitable mid to outer ramp niches. Coral reefs occurred in a larger variety of structural settings. Upper Jurassic coral reefs partly grew in high latitudinal areas suggesting an equilibrated climate. This appears to be an effect of the buffering capacity of high sea level. These feedback effects of high sea level also may have reduced oceanic circulation particularly during flooding events of third and higher order, which gave rise to the development of black shales and dysaerobic thrombolite reefs. Hence, the interplay of local, regional and global factors caused Jurassic reefs to be more differentiated than modern ones, including near-actualistic coral reefs as well as non-actualistic sponge and microbial reefs.     

Dependence of the red blood cell calcium pump on the membrane potential

(1) It is shown that the rate of calcium extrusion from intact human red cells is faster at a membrane potential of approximately +50 mV (inside) than at approximately -50 mV. (2) The positive potential applied was the chloride potential of KCl cells in a K-gluconate medium when the Ca2+ sensitive K+ channel was blocked by 0.3mM quinidine. The negative potential resulted from the high K+ permeability in Ca2+ loaded cells (the cells were loaded to a Ca2+ activity in the cell water of about 50 microM). (3) It is further demonstrated that the Ca2+ affinity of the pump ATPase is decreased both at the internal (high affinity) and external (low affinity) site by increasing the proton concentration. Acidification thus inhibits internally and stimulates externally. (4) An indirect effect of the membrane potential on the pump activity via the accompanying pH shifts on either side of the membrane could be ruled out by choosing Ca2+ concentrations which are fully activating at the internal Ca2+ binding site at pH 6.5 and not yet inhibitory at the external Ca2+ binding site at pH 8. (5) The result is compatible with the assumption that the human red cell Ca-pump is exchanging Ca2+ for protons, yet is electrogenic by virtue of a stoichiometry of 1H+:1Ca2+ for this exchange.     

The marine paleogene of the tremp region (NE Spain)-depositional sequences,facies history,biostratigraphy and controlling factors

Summary The marine Paleogene of the Tremp Basin in the Central Southern Pyrenees corresponds to four depositional sequences which are related to global eustatic third order cycles (Tejas A 2.3–2.6). Associated transgressive and downlap surfaces coincide with boundaries of biozones. Lowstand systems tracts consist of estuarine and braid delta systems. Transgressive and highstand systems tracts are composed of carbonate banks and reefs. Slow thrust-induced changes of the basin topography conditioned the basic type and the areal distribution of carbonate highstand and clastic lowstand systems. Rapid relative sea level changes controlled the activity and internal dynamic of the depositional systems. E-W directed blind thrust anticlines are covered during highstand periods by carbonate fringing banks withNummulites bars. N-S orientation of thrust anticlines leads to the evolution of reef-dominated barrier banks and shelf lagoonal homoclinal ramps. On-bank transport of carbonate sands dominates during transgressions, off-bank transport during highstand periods. Continuous thrusting during the Ilerdian caused angular unconformities only in combination with relative sea level fall. Sequence-internal onlap configurations result from contemporaneous tectonic tilting. Fourth order carbonate bank margin cycles contain well developed lowstand tracts due to increased subsidence rates. Fourth order flooding surfaces are marked by paleosoil horizons at their landward continuation.     

Two-temperature stage biphasic CO2-H2O pretreatment of lignocellulosic biomass at high solid loadings

Most biomass pretreatment processes for monosaccharide production are run at low-solid concentration (<10 wt%) and use significant amounts of chemical catalysts. Biphasic CO(2) -H(2) O mixtures could provide a more sustainable pretreatment medium while using high-solid contents. Using a stirred reactor for high solids (40 wt%, biomass water mixture) biphasic CO(2)-H(2) O pretreatment of lignocellulosic biomass allowed us to explore the effects of particle size and mixing on mixed hardwood and switchgrass pretreatment. Subsequently, a two-temperature stage pretreatment was introduced. After optimization, a short high-temperature stage at 210°C (16 min for hardwood and 1 min for switchgrass) was followed by a long low-temperature stage at 160°C for 60 min. Glucan to glucose conversion yields of 83% for hardwood and 80% for switchgrass were obtained. Total molar sugar yields of 65% and 55% were obtained for wood and switchgrass, respectively, which consisted of a 10% points improvement over those obtained during our previous study despite a 10-fold increase in particle size. These yields are similar to those obtained with other major pretreatment technologies for wood and within 10% of major technologies for switchgrass despite the absence of chemical catalysts, the use of large particles (0.95 cm) and high solid contents (40 wt%).