Properties important for solid-liquid separations change during the enzymatic hydrolysis of pretreated wheat straw.

OBJECTIVES: The biochemical conversion of lignocellulosic biomass into renewable fuels and chemicals provides new challenges for industrial scale processes. One such process, which has received little attention, but is of great importance for efficient product recovery, is solid-liquid separations, which may occur both after pretreatment and after the enzymatic hydrolysis steps. Due to the changing nature of the solid biomass during processing, the solid-liquid separation properties of the biomass can also change. The objective of this study was to show the effect of enzymatic hydrolysis of cellulose upon the water retention properties of pretreated biomass over the course of the hydrolysis reaction. RESULTS: Water retention value measurements, coupled with 1H NMR T2 relaxometry data, showed an increase in water retention and constraint of water by the biomass with increasing levels of cellulose hydrolysis. This correlated with an increase in the fines fraction and a decrease in particle size, suggesting that structural decomposition rather than changes in chemical composition was the most dominant characteristic. CONCLUSIONS: With increased water retained by the insoluble fraction as cellulose hydrolysis proceeds, it may prove more difficult to efficiently separate hydrolysis residues from the liquid fraction with improved hydrolysis.

Using electrophoretic exclusion to manipulate small molecules and particles on a microdevice.

Electrophoretic exclusion, a novel separations technique that differentiates species in bulk solution using the opposing forces of electrophoretic velocity and hydrodynamic flow, has been adapted to a microscale device. Proof-of-principle experiments indicate that the device was able to exclude small particles (1 µm polystyrene microspheres) and fluorescent dye molecules (rhodamine 123) from the entrance of a channel. Additionally, differentiation of the rhodamine 123 and polystyrene spheres was demonstrated. The current studies focus on the direct observation of the electrophoretic exclusion behavior on a microchip.

Examining serum amyloid P component microheterogeneity using capillary isoelectric focusing and MALDI-MS.

Serum amyloid P component (SAP) is a glycoprotein of interest due to its presence in amyloid plaque formations. As with most glycoproteins, SAP can possibly vary greatly in its isoforms, which can be an important factor toward understanding the role of SAP. Interestingly, previous characterizations suggest varying degrees of microheterogeneity, some of which are in conflict. In this work, we provide new information to clarify SAP's microheterogeneity profile using CIEF to carefully analyze pooled samples and by studying individual samples across populations with mass spectrometric immunoassay. With respect to CIEF, a single pI band was observed suggesting that human SAP does not have extensive heterogeneity concluded from gel IEF experiments in the past. Additionally, this is supported by a population study, which revealed an overwhelming degree of uniformity. Overall, this work corroborates the idea that SAP is relatively consistent across the population and with respect to microheterogeneity.

Dielectrophoretic mobility determination in DC insulator-based dielectrophoresis.

Insulator-based dielectrophoresis (iDEP) is a powerful tool for separating and characterizing particles, yet it is limited by a lack of quantitative characterizations. Here, this limitation is addressed by employing a method capable of quantifying the DEP mobility of particles. Using streak-based velocimetry the particle properties are deduced from their motion in a microfluidic channel with a constant electric field gradient. From this approach, the DEP mobility of 1 µm polystyrene particles was found to be -2±0.4 10(-8) cm4 /(V2 s). In the future, such quantitative treatment will allow for the elucidation of unique insights and rational design of devices.

Isoelectric focusing in a drop.

A novel approach to molecular separations is investigated using a technique termed droplet-based isoelectric focusing. Drops are manipulated discretely on a superhydrophobic surface, subjected to low voltages for isoelectric focusing, and split-resulting in a preparative separation. A universal indicator dye demonstrates the generation of stable, reversible pH gradients (3-10) in ampholyte buffers, and these gradients lead to protein focusing within the drop length. Focusing was visually characterized, spectroscopically verified, and assessed quantitatively by noninvasive light scattering measurements. It was found to correlate with a quantitative model based on 1D steady-state theory. This work illustrates that molecular separations can be deployed within a single open drop, and the differential fractions can be separated into new discrete liquid elements.

Arthroscopic proximal row carpectomy.

PURPOSE: Proximal row carpectomy is an effective procedure for treating a variety of wrist pathologies. To date, all outcome studies have reported on the results of an open procedure, with a dorsal capsulotomy. We present our technique and early results of arthroscopic proximal row carpectomy. METHODS: A total of 17 consecutive patients (10 men and 7 women) underwent arthroscopic proximal row carpectomy. After routine arthroscopy, the proximal carpal row was removed with an arthroscopic bur, with care being taken to protect the articular cartilage of the head of the capitate and the lunate fossa. The average time of the procedure was 70 minutes (range, 34-110 min). Range of motion exercise was initiated 2 days postoperatively. We assessed clinical follow-up objectively by evaluating range of motion and grip strength. We assessed subjective outcomes with the Disabilities of the Arm, Shoulder, and Hand questionnaire and with a patient-centered questionnaire assessing satisfaction, return to prior employment, and pain. RESULTS: A total of 16 patients were available, with an average follow-up of 24 months (range, 12-48 mo). There were no complications, no revisions were required, and no arthroscopic procedure was converted to open technique. At final follow-up, the average wrist flexion-extension arc was 80% of the contralateral side, and grip strength averaged 81% of the contralateral side. The mean Disabilities of the Arm, Shoulder, and Hand score was 21. All 16 patients rated themselves as satisfied or very satisfied with the procedure. Eleven patients had no work restrictions and 13 were able to return to previous employment. Ten patients rated themselves as having mild or no pain. CONCLUSIONS: Arthroscopic proximal row carpectomy appears to be a safe, effective, and reliable procedure for a variety of wrist conditions, and it allows for rapid mobilization of the wrist compared with the open procedure. Range of motion and grip strength compare favorably with existing values in the literature for the open technique.

Chemical and Structural Changes in Corn Stover After Ensiling: Influence on Bioconversion.

Production of biofuels, bioproducts, and bioenergy requires a well-characterized, stable, and reasonably uniform biomass supply and well-established supply chains for shipping biomass from farm fields to biorefineries, while achieving year-round production targets. Preserving and stabilizing biomass feedstock during storage is a necessity for cost-effective and sustainable biofuel production. Ensiling is a common storage method used to preserve and even improve forage quality; however, the impact of ensiling on biomass physical and chemical properties that influence bioconversion processes has been variable. Our objective in this work was to determine the effects of ensiling on lignocellulosic feedstock physicochemical properties and how that influences bioconversion requirements. We observed statistically significant decreases (p < 0.05) in the content of two major structural carbohydrates (glucan and xylan) of 5 and 8%, respectively, between the ensiled and non-ensiled materials. We were unable to detect differences in sugar yields from structural carbohydrates after pretreatment and enzymatic hydrolysis of the ensiled materials compared to non-ensiled controls. Based on this work, we conclude that ensiling the corn stover did not change the bioconversion requirements compared to the control samples and incurred losses of structural carbohydrates. At the light microscopy level, ensiled corn stover exhibited little structural change or relocation of cell wall components as detected by immunocytochemistry. However, more subtle structural changes were revealed by electron microscopy, as ensiled cell walls exhibit ultrastructural characteristics such as wall delimitation intermediate between non-ensiled and dilute-acid-pretreated cell walls. These findings suggest that alternative methods of conversion, such as deacetylation and mechanical refining, could take advantage of lamellar defects and may be more effective than dilute acid or hot water pretreatment for biomass conversion of ensiled materials.

Enzymatic hydrolysis is limited by biomass-water interactions at high-solids: improved performance through substrate modifications.

BACKGROUND: To improve process economics for production of fuels and chemicals from lignocellulosic biomass, high solids concentrations are applied in enzymatic hydrolysis, to increase product concentration and reduce energy input. However, increasing solids concentrations decrease cellulose conversion yields, the so called 'high-solids effect.' Previous work suggests that product inhibition and mixing contribute, but an understanding of how biomass properties influence the high-solids effect, is lacking. RESULTS: Cellulose hydrolysis yields with an industrial cellulase (Ctec2) were measured on pretreated wheat straw and spruce from 5 to 30% dry matter (DM), and compared to yields of an older industrial cellulase mixture (Celluclast 1.5L/Novozym188). For Ctec2, yield was independent of DM below 15-18% DM, while yields decreased with increasing DM above this range, but at different rates for each biomass. For Celluclast 1.5L/Novozym188, yields decreased already from the lowest DM, suggesting that the high-solids effect was more a function of product inhibition, while the yields of the newer Ctec2 mixture were driven more by biomass-water interactions. LF-NMR relaxometry showed that the onset of the high-solids effect for Ctec2 corresponded to the disappearance of free water from the system, and a decrease in water self-diffusion rates. While the spruce had higher yields at low-solids, the wheat straw had higher yields at high-solids conditions, exhibiting that relative yields at low and high-solids are not related. Higher yields corresponded to increased water constraint by the biomass at high-solids conditions. Modifications to the pretreated wheat straw resulted in improved yields, and changes to the inflection point and intensity of the high-solids effect, showing that this effect can be reduced. CONCLUSIONS: The high-solids effect is both enzyme and substrate dependent, and can be reduced by modifying the pretreated biomass, suggesting that pretreatment processes can be designed to achieve similar effects. Yields at low and high-solids concentrations do not correlate for a given biomass, and thus industrial evaluation of biomass recalcitrance should be carried out at high-solids conditions.

Percutaneous Release of Trigger Digits.

Stenosing flexor tenosynovitis, or trigger finger, has been treated with open, endoscopic, and percutaneous tendon sheath incisions. A comparison of techniques highlights increased cost, procedure time, and postoperative recovery associated with open surgical treatment. We conducted a study of the efficacy of percutaneous tendon sheath incision as a treatment for trigger digit. Using percutaneous tendon sheath incisions, a single surgeon treated 596 trigger digits (429 patients) over a 10-year period. Successful release was defined as symptom relief on follow-up, or patient request for no further treatment. There were 537 successful releases (90.1%). Of the 59 failures, 17 underwent another percutaneous procedure (15 successful) and 40 underwent open surgical release (100% successful); 2 patients requested no further treatment. There were no significant differences in digit success rates and no complications. The success rate of this method was comparable to rates reported for open surgical procedures, but there were no instances of infection, chronic pain, or tendon or neurovascular damage. Percutaneous release is a safe, effective, and economical treatment option for trigger digits.

Biomass-water interactions correlate to recalcitrance and are intensified by pretreatment: An investigation of water constraint and retention in pretreated spruce using low field NMR and water retention value techniques.

The underlying mechanisms of the recalcitrance of biomass to enzymatic deconstruction are still not fully understood, and this hampers the development of biomass based fuels and chemicals. With water being necessary for most biological processes, it is suggested that interactions between water and biomass may be key to understanding and controlling biomass recalcitrance. This study investigates the correlation between biomass recalcitrance and the constraint and retention of water by the biomass, using SO2 pretreated spruce, a common feedstock for lignocellulosic biofuel production, as a substrate to evaluate this relationship. The water retention value (WRV) of the pretreated materials was measured, and water constraint was assessed using time domain Low Field Nuclear Magnetic Resonance (LFNMR) relaxometry. WRV increased with pretreatment severity, correlating to reduced recalcitrance, as measured by hydrolysis of cellulose using commercial enzyme preparations. Water constraint increased with pretreatment severity, suggesting that a higher level of biomass-water interaction is indicative of reduced recalcitrance in pretreated materials. Both WRV and water constraint increased significantly with reductions in particle size when pretreated materials were further milled, suggesting that particle size plays an important role in biomass water interactions. It is suggested that WRV may be a simple and effective method for measuring and comparing biomass recalcitrance. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:146-153, 2017.

Enzymatic lignocellulose hydrolysis: Improved cellulase productivity by insoluble solids recycling.

BACKGROUND: It is necessary to develop efficient methods to produce renewable fuels from lignocellulosic biomass. One of the main challenges to the industrialization of lignocellulose conversion processes is the large amount of cellulase enzymes used for the hydrolysis of cellulose. One method for decreasing the amount of enzyme used is to recycle the enzymes. In this study, the recycle of enzymes associated with the insoluble solid fraction after the enzymatic hydrolysis of cellulose was investigated for pretreated corn stover under a variety of recycling conditions. RESULTS: It was found that a significant amount of cellulase activity could be recovered by recycling the insoluble biomass fraction, and the enzyme dosage could be decreased by 30% to achieve the same glucose yields under the most favorable conditions. Enzyme productivity (g glucose produced/g enzyme applied) increased between 30 and 50% by the recycling, depending on the reaction conditions. While increasing the amount of solids recycled increased process performance, the methods applicability was limited by its positive correlation with increasing total solids concentrations, reaction volumes, and lignin content of the insoluble residue. However, increasing amounts of lignin rich residue during the recycle did not negatively impact glucose yields. CONCLUSIONS: To take advantage of this effect, the amount of solids recycled should be maximized, based on a given processes ability to deal with higher solids concentrations and volumes. Recycling of enzymes by recycling the insoluble solids fraction was thus shown to be an effective method to decrease enzyme usage, and research should be continued for its industrial application.

Gaze cues influence memory but not for long.

Many factors influence the manner in which material is encoded into memory, with one of the most important determinants of subsequent memorability being the degree to which an item is attended at study. Attentional gaze manipulations - which occur when a task-irrelevant face at fixation looks towards or away from a target - have been shown to enhance attention such that stimuli that are gazed at elicit quicker responses. In the present study, four experiments were conducted to determine whether attentional gaze cues can also influence the recall of items appearing at gazed-at or gazed-away from locations. In Experiment 1, an irrelevant gaze cue at fixation preceded the presentation of to-be-remembered items, with each item remaining on screen for 1000ms. Gaze direction had no effect on memory for words. In Experiment 2, the presentation time for to-be-remembered items was reduced to 250ms or 500ms. Now gazed at items were more memorable. In Experiment 3, we manipulate the intentionality of the memory instruction and demonstrate that gaze cues influence memory even when participants are not explicitly attempting to memorize items. Finally, Experiment 4 demonstrates that these findings are specific to gaze cues as no memory effect is observed when arrow cues are presented. It is argued that gaze cues can modify memory for items, but that this effect is primarily attributable to shifts of attention away from target items when a gaze cue is invalid.

Cutting a drop of water pinned by wire loops using a superhydrophobic surface and knife.

A water drop on a superhydrophobic surface that is pinned by wire loops can be reproducibly cut without formation of satellite droplets. Drops placed on low-density polyethylene surfaces and Teflon-coated glass slides were cut with superhydrophobic knives of low-density polyethylene and treated copper or zinc sheets, respectively. Distortion of drop shape by the superhydrophobic knife enables a clean break. The driving force for droplet formation arises from the lower surface free energy for two separate drops, and it is modeled as a 2-D system. An estimate of the free energy change serves to guide when droplets will form based on the variation of drop volume, loop spacing and knife depth. Combining the cutting process with an electrofocusing driving force could enable a reproducible biomolecular separation without troubling satellite drop formation.

Spatio-temporal image analysis of particle streaks in micro-channels for low-cost electro-hydrodynamic flow characterization.

Flow characterization is a primary analytical method for performance evaluation of microfluidic devices. With the increasing prevalence of microfluidic devices in recent years, there is a growing need for simple methods of automated flow estimation. In this work, a novel flow diagnostic technique based on image analysis of particle streaks is introduced, to characterize local flow velocities. While 1D velocimetry using particle tracks has occasionally been discussed for macro-scale environments, the use of particle streaks for 2-D flow characterization in micro-channels has not been explored. The proposed technique is qualitatively validated against electrokinetic experiment and numerically validated with simulated flows.

Impact of corn stover composition on hemicellulose conversion during dilute acid pretreatment and enzymatic cellulose digestibility of the pretreated solids.

This study assessed the impact of corn stover compositional variability on xylose conversion yields during dilute acid pretreatment and on enzymatic cellulose digestibility of the resulting pretreated solids. Seven compositionally-different stovers obtained from various locations throughout the United States were pretreated at three different conditions in triplicate in a pilot-scale continuous reactor. At the same pretreatment severity, a 2-fold increase in monomeric xylose yield and a 1.5-fold increase in enzymatic cellulose digestibility from their lowest values were found. Similar results were observed at the other pretreatment conditions. It was found that xylose conversion yields decreased with increasing acid neutralization capacity or soil content of the corn stover. Xylose yields also increased with increasing xylan content. No other significant correlations between corn stover's component concentrations and conversion yields were found.

Capillary isoelectric focusing coupled offline to matrix assisted laser desorption/ionization mass spectrometry.

This work presents several critical details for making cIEF-MALDI-MS a robust technique which will allow for more routine application and aid in automation. This includes emphasis on the hardware necessary for syringe pump mobilization and proper protocol for preventing disruption from gas bubbles. Following these guidelines, excellent elution time reproducibility is demonstrated for six pI markers (RSD <5%). Additionally, the pI markers are used to calibrate the pH gradient and determine experimental pIs of proteins detected offline by mass spectrometry. This was demonstrated using a standard protein mixture of myoglobin and two forms of beta-lactoglobulin. Experimental determination of protein pIs and molecular weights were found to be in agreement with literature values. The technical details discussed provide a sound foundation for applying the offline coupling of MALDI-MS with cIEF.

A simplified method for the measurement of insoluble solids in pretreated biomass slurries.

The biochemical conversion of cellulosic biomass to liquid transportation fuels includes the breakdown of biomass into its soluble, fermentable components. Pretreatment, the initial step in the conversion process, results in heterogeneous slurry comprised of both soluble and insoluble biomass components. For the purpose of tracking the progress of the conversion process, it is important to be able to accurately measure the fraction of insoluble biomass solids in the slurry. The current standard method involves separating the solids from the free liquor and then repeatedly washing the solids to remove the soluble fraction, a laborious and tedious process susceptible to operator variations. In this paper, we propose an alternative method for calculating the fraction of insoluble solids which does not require a washing step. The proposed method involves measuring the dry matter content of the whole slurry as well as the dry matter content in the isolated liquor fraction. We compared the two methods using three different pretreated biomass slurry samples and two oven-drying techniques for determining dry matter content, an important measurement for both methods. We also evaluated a large set of fraction insoluble solids data collected from previously analyzed pretreated samples. The proposed new method provided statistically equivalent results to the standard washing method when an infrared balance was used for determining dry matter content in the controlled measurement experiment. Similarly, in the large historical data set, there was no statistical difference shown between the wash and no-wash methods. The new method is offered as an alternative method for determining the fraction of insoluble solids.

High xylose yields from dilute acid pretreatment of corn stover under process-relevant conditions.

Pretreatment experiments were carried out to demonstrate high xylose yields at high solids loadings in two different batch pretreatment reactors under process-relevant conditions. Corn stover was pretreated with dilute sulfuric acid using a 4-l Steam Digester and a 4-l stirred ZipperClave reactor. Solids were loaded at 45% dry matter (wt/wt) after sulfuric acid catalyst impregnation using nominal particle sizes of either 6 or 18 mm. Pretreatment was carried out at temperatures between 180 and 200 degrees C at residence times of either 90 or 105 s. Results demonstrate an ability to achieve high xylose yields (>80%) over a range of pretreatment conditions, with performance showing little dependence on particle size or pretreatment reactor type. The high xylose yields are attributed to effective catalyst impregnation and rapid rates of heat transfer during pretreatment.