Although measurements of crystallinity index (CI) have a long history, it has been found that CI varies significantly depending on the choice of measurement method. In this study, four different techniques incorporating X-ray diffraction and solid-state 13C nuclear magnetic resonance (NMR) were compared using eight different cellulose preparations. We found that the simplest method, which is also the most widely used, and which involves measurement of just two heights in the X-ray diffractogram, produced significantly higher crystallinity values than did the other methods. Data in the literature for the cellulose preparation used (Avicel PH-101) support this observation. We believe that the alternative X-ray diffraction (XRD) and NMR methods presented here, which consider the contributions from amorphous and crystalline cellulose to the entire XRD and NMR spectra, provide a more accurate measure of the crystallinity of cellulose. Although celluloses having a high amorphous content are usually more easily digested by enzymes, it is unclear, based on studies published in the literature, whether CI actually provides a clear indication of the digestibility of a cellulose sample. Cellulose accessibility should be affected by crystallinity, but is also likely to be affected by several other parameters, such as lignin/hemicellulose contents and distribution, porosity, and particle size. Given the methodological dependency of cellulose CI values and the complex nature of cellulase interactions with amorphous and crystalline celluloses, we caution against trying to correlate relatively small changes in CI with changes in cellulose digestibility. In addition, the prediction of cellulase performance based on low levels of cellulose conversion may not include sufficient digestion of the crystalline component to be meaningful.

Greater understanding of the mechanisms contributing to chemical and enzymatic solubilization of plant cell walls is critical for enabling cost-effective industrial conversion of cellulosic biomass to biofuels. Here, we report the use of correlative imaging in real time to assess the impact of pretreatment, as well as the resulting nanometer-scale changes in cell wall structure, upon subsequent digestion by two commercially relevant cellulase systems. We demonstrate that the small, noncomplexed fungal cellulases deconstruct cell walls using mechanisms that differ considerably from those of the larger, multienzyme complexes (cellulosomes). Furthermore, high-resolution measurement of the microfibrillar architecture of cell walls suggests that digestion is primarily facilitated by enabling enzyme access to the hydrophobic cellulose face. The data support the conclusion that ideal pretreatments should maximize lignin removal and minimize polysaccharide modification, thereby retaining the essentially native microfibrillar structure.

Nature has evolved multiple enzymatic strategies for the degradation of plant cell wall polysaccharides, which are central to carbon flux in the biosphere and an integral part of renewable biofuels production.Many biomass-degrading organisms secrete synergistic cocktails of individual enzymes with one or several catalytic domains per enzyme, whereas a few bacteria synthesize large multi-enzyme complexes, termed cellulosomes, which contain multiple catalytic units per complex. Both enzyme systems employ similar catalytic chemistries; however, the physical mechanisms by which these enzyme systems degrade polysaccharides are still unclear. Here we examine a prominent example of each type, namely a freeenzyme cocktail expressed by the fungus Hypocrea jecorina and a cellulosome preparation secreted from the anaerobic bacterium Clostridium thermocellum. We observe striking differences in cellulose saccharification exhibited by these systems at the same protein loading. Free enzymes are more active on pretreated biomass and in contrast cellulosomes are much more active on purified cellulose. When combined, these systems display dramatic synergistic enzyme activity on cellulose. To gain further insights, we imaged free enzyme-and cellulosome-digested cellulose and biomass by transmission electron microscopy, which revealed evidence for different mechanisms of cellulose deconstruction by free enzymes and cellulosomes. Specifically, the free enzymes employ an ablative, fibril-sharpening mechanism, whereas cellulosomes physically separate individual cellulose microfibrils from larger particles resulting in enhanced access to cellulose surfaces. Interestingly, when the two enzyme systems are combined, we observe changes to the substrate that suggests mechanisms of synergistic deconstruction. Insight into the different mechanisms underlying these two polysaccharide deconstruction paradigms will eventually enable new strategies for enzyme engineering to overcome biomass recalcitrance. Broader contextIndustrial conversion of plant biomass into transportation fuels will likely be a vital component of the global renewable energy portfolio to reduce both greenhouse gas emissions and fossil fuel utilization for mankind's energy needs. However, plants have evolved considerable defense mechanisms against deconstruction of their cell wall polysaccharides into sugars, and as such, depolymerization to sugars for subsequent biological or catalytic conversion to fuels remains a signicant technical challenge with major cost implications in biomass conversion. In nature, many microorganisms evolved a strategy wherein plant cell wall-active enzymes are secreted as a cocktail of individual enzymes that work synergistically to depolymerize biomass, referred to as a "free enzyme" paradigm. Some ruminal microorganisms conversely evolved a strategy wherein their plant cell wall-active enzymes are tethered to large scaffolds, which are linked to the cells for sugar production in close proximity for uptake, termed the "cellulosome". Here, we mecha...

Biodegradation of plant biomass is a slow process in nature, and hydrolysis of cellulose is also widely considered to be a rate-limiting step in the proposed industrial process of converting lignocellulosic materials to biofuels. It is generally known that a team of enzymes including endo- and exocellulases as well as cellobiases are required to act synergistically to hydrolyze cellulose to glucose. The detailed molecular mechanisms of these enzymes have yet to be convincingly elucidated. In this report, atomic force microscopy (AFM) is used to image in real-time the structural changes in Valonia cellulose crystals acted upon by the exocellulase cellobiohydrolase I (CBH I) from Trichoderma reesei. Under AFM, single enzyme molecules could be observed binding only to one face of the cellulose crystal, apparently the hydrophobic face. The surface roughness of cellulose began increasing after adding CBH I, and the overall size of cellulose crystals decreased during an 11-h period. Interestingly, this size reduction apparently occurred only in the width of the crystal, whereas the height remained relatively constant. In addition, the measured cross-section shape of cellulose crystal changed from asymmetric to nearly symmetric. These observed changes brought about by CBH I action may constitute the first direct visualization supporting the idea that the exocellulase selectively hydrolyzes the hydrophobic faces of cellulose. The limited accessibility of the hydrophobic faces in native cellulose may contribute significantly to the rate-limiting slowness of cellulose hydrolysis.

This study tested hypothesized relationships between noise exposure and auditory deficits. Both retrospective assessment of potential associations between noise exposure history and performance on an audiologic test battery and prospective assessment of potential changes in performance after new recreational noise exposure were completed.Methods: 32 participants (13M, 19F) with normal hearing (25-dB HL or better, 0.25–8 kHz) were asked to participate in 3 pre- and post-exposure sessions including: otoscopy, tympanometry, distortion product otoacoustic emissions (DPOAEs) (f2 frequencies 1–8 kHz), pure-tone audiometry (0.25–8 kHz), Words-in-Noise (WIN) test, and electrocochleography (eCochG) measurements at 70, 80, and 90-dB nHL (click and 2–4 kHz tone-bursts). The first session was used to collect baseline data, the second session was collected the day after a loud recreational event, and the third session was collected 1-week later. Of the 32 participants, 26 completed all 3 sessions.Results: The retrospective analysis did not reveal statistically significant relationships between noise exposure history and any auditory deficits. The day after new exposure, there was a statistically significant correlation between noise “dose” and WIN performance overall, and within the 4-dB signal-to-babble ratio. In contrast, there were no statistically significant correlations between noise dose and changes in threshold, DPOAE amplitude, or AP amplitude the day after new noise exposure. Additional analyses revealed a statistically significant relationship between TTS and DPOAE amplitude at 6 kHz, with temporarily decreased DPOAE amplitude observed with increasing TTS.Conclusions: There was no evidence of auditory deficits as a function of previous noise exposure history, and no permanent changes in audiometric, electrophysiologic, or functional measures after new recreational noise exposure. There were very few participants with TTS the day after exposure - a test time selected to be consistent with previous animal studies. The largest observed TTS was approximately 20-dB. The observed pattern of small TTS suggests little risk of synaptopathy from common recreational noise exposure, and that we should not expect to observe changes in evoked potentials for this reason. No such changes were observed in this study. These data do not support suggestions that common, recreational noise exposure is likely to result in “hidden hearing loss”.