Rose Bengal Labeled Bovine Serum Albumin for Protein Transport Imaging in Subcutaneous Tissues Using Computed Tomography and Fluorescence Microscopy.

Subcutaneous (SC) injection of protein-based therapeutics is a convenient and clinically established drug delivery method. However, progress is needed to increase the bioavailability. Transport of low molecular weight (Mw) biotherapeutics such as insulin and small molecule contrast agents such as lipiodol has been studied using X-ray computed tomography (CT). This analysis, however, does not translate to the investigation of higher Mw therapeutics, such as monoclonal antibodies (mAbs), due to differences in molecular and formulation properties. In this study, an iodinated fluorescein analog rose bengal (RB) was used as a radiopaque and fluorescent label to track the distribution of bovine serum albumin (BSA) compared against unconjugated RB and sodium iodide (NaI) via CT and confocal microscopy following injection into ex vivo porcine SC tissue. Importantly, the high concentration BSA-RB exhibited viscosities more like that of viscous biologics than the small molecule contrast agents, suggesting that the labeled protein may serve as a more suitable formulation for the investigation of injection plumes. Three-dimensional (3D) renderings of the injection plumes showed that the BSA-RB distribution was markedly different from unconjugated RB and NaI, indicating the need for direct visualization of large protein therapeutics using conjugated tags rather than using small molecule tracers. Whereas this proof-of-concept study shows the novel use of RB as a label for tracking BSA distribution, our experimental approach may be applied to high Mw biologics, including mAbs. These studies could provide crucial information about diffusion in SC tissue and the influence of injection parameters on distribution, transport, and downstream bioavailability.

Concentration-dependent diffusion of unlabeled protein within an in vitro hyaluronic acid matrix.

Diffusion and movement of subcutaneously injected biologics and high-concentration immunoglobulin G (IgG) therapeutics away from the injection site and through the subcutaneous (SC) tissue may be concentration dependent. This possibility was confirmed by in situ measurement of diffusion coefficients of unlabeled bovine IgG in phosphate-buffered saline within an in vitro hyaluronic acid matrix that represents the SC electrostatic environment. Diffusion decreased from 2.67 to 0.05 × 10-7 cm2 /s when IgG concentration increased from 25 to 73 mg/mL. The results demonstrated that in situ detection of unlabeled proteins within an in vitro SC environment provides another useful tool for the preclinical characterization of injectable biologics.

In vitro measurement of concentration of unlabeled protein within a hyaluronic acid matrix.

There are currently more than 560 therapeutic monoclonal antibodies (mAbs) at various stages of research and clinical testing, including candidates for administration by subcutaneous (SC) injection. Preclinical studies based on in vitro measurements of high molecular weight proteins within simulated SC matrices are assisting laboratory studies of interactions of injectable biotherapeutic proteins within the SC environment in relation to bioavailability. We report a new method for directly measuring diffusion of unlabeled, high molecular weight proteins injected into an in vitro matrix that simulates the negatively charged environment of the SC. The matrix consists of 10 mg/ml HA in a repurposed cell culture chamber. The measurement consists of pipetting triplicate 20 µl protein samples into the matrix, placing the chamber in a laboratory scanner, activating tryptophan residues in the protein at 280 nm, and imaging the resulting protein fluorescence at 384 nm over a 0.5-4 h time period thus tracking protein movement. This facile approach enables mapping of protein concentration as a function of time and distance within the matrix, and determination of diffusion coefficients, D, within ±10%. Bovine IgG and BSA gave D = 2.3 ± 0.2*10-7 and 4.6 ± 0.2*10-7 cm2 /s at 24°C, respectively, for initial protein concentrations of 21 mg/mL.

Hydrolysis-determining substrate characteristics in liquid hot water pretreated hardwood.

Fundamental characterization of pretreated hardwood and its interactions with cellulolytic enzymes has confirmed that a pathway exists for dramatically reducing the loading of cellulase required for hydrolysis of pretreated biomass. We demonstrate that addition of protein effecting a seven-fold decrease in the specific activity of cellulases enables a ten-fold reduction in enzyme loading while maintaining a high level of cellulose hydrolysis in pretreated hardwood. While use of protein and other additives that adsorb on lignin have been reported previously, the current work demonstrates the effect in a dramatic manner and brings the rationale for this change into clear focus. The key to this result is recognizing and mitigating the pretreatment conundrum where increasingly severe pretreatment conditions enhance accessibility of the enzymes not only to cellulose, but also to lignin. The lignin adsorbs enzyme protein causing loss of cellulase activity. More enzyme, added to compensate for this lost activity, results in a higher cellulase loading. The addition of a different protein, such as BSA, prevents cellulase adsorption on lignin and enables the enzyme itself to better target its glucan substrate. This effect dramatically reduces the amount of cellulase for a given level of conversion with enzyme loadings of 15 FPU and 1.3 FPU/g solids both achieving 80% conversion. The understanding of this phenomenon reinvigorates motivation for the search for other approaches that prevent cellulase adsorption on lignin in order to achieve high glucose yields at low enzyme loadings for pretreated lignocellulose.

Adsorption of enzyme onto lignins of liquid hot water pretreated hardwoods.

The adsorption of cellulase enzymes onto lignin is shown to be non-productive and therefore reduces enzymatic hydrolysis of liquid hot water pretreated cellulose. Among the enzyme components of Trichoderma reesei cellulase cocktail, ß-glucosidase showed the strongest adsorption onto lignin. Only 2-18% of the initial ß-glucosidase activity remained in the supernatant while 50-60% of cellobiohydrolase and endoglucanase activities were recovered after incubation with lignin. By increasing the pH to 5.5 and adding NaCl to a 200 mM, the free enzymes in the supernatant were increased but hydrolysis was not enhanced since optimal pH for enzymatic hydrolysis is at 4.8. Electrostatic interactions contributed to enzyme adsorption and their effect was most pronounced for T. reesei ß-glucosidase which had high molecular weights (78-94 kDa) and high isoelectric points (pI 5.7-6.4). Since the enzyme components which are required to synergistically hydrolyze cellulose have different profiles (molecular weight, hydrophobicity and pI), they exhibit different adsorption behaviors with lignin, and thereby change the ratio of enzyme activities needed for synergism during cellulose hydrolysis. ß-glucosidase from Aspergillus niger exhibits less adsorption than ß-glucosidase from T. reesei. Supplemental addition of A. niger ß-glucosidase to the enzyme mixture increases hydrolysis of pretreated hardwood by a factor of two. The analysis presented in this paper shows that lignins with higher guaiacyl content adsorb more cellulase enzymes, particularly ß-glucosidase, and that adsorption of ß-glucosidase onto lignin indirectly suppresses enzymatic hydrolysis of cellulose in pretreated hardwoods due to decreased hydrolysis of cellobiose which in turn accumulates and inhibits CBH.

Effect of liquid hot water pretreatment severity on properties of hardwood lignin and enzymatic hydrolysis of cellulose.

Lignin, one of the major components of lignocellulosic biomass, plays an inhibitory role on the enzymatic hydrolysis of cellulose. This work examines the role of lignin in pretreated hardwood, where extents of cellulose hydrolysis decrease, rather than increase with increasing severity of liquid hot water pretreatment. Hardwood pretreated with liquid hot water at severities ranging from log Ro = 8.25 to 12.51 resulted in 80-90% recovery of the initial lignin in the residual solids. The ratio of acid insoluble lignin (AIL) to acid soluble lignin (ASL) increased and the formation of spherical lignin droplets on the cell wall surface was observed as previously reported in the literature. When lignins were isolated from hardwoods pretreated at increasing severities and characterized based on glass transition temperature (Tg ), the Tg of isolated lignins was found to increase from 171 to 180°C as the severity increased from log Ro = 10.44 to 12.51. The increase in Tg suggested that the condensation reactions of lignin molecules occurred during pretreatment and altered the lignin structure. The contribution of the changes in lignin properties to enzymatic hydrolysis were examined by carrying out Avicel hydrolysis in the presence of isolated lignins. Lignins derived from more severely pretreated hardwoods had higher Tg values and showed more pronounced inhibition of enzymatic hydrolysis.

Severity factor coefficients for subcritical liquid hot water pretreatment of hardwood chips.

Single stage and multi-stage liquid hot water pretreatments of mixed hardwood pinchips were investigated at various severities (log R0 = 3.65-4.81) to assess the efficiencies of the pretreatments with respect to achieving high pentose sugar yields and improved enzymatic digestibility of pretreated cellulose. We investigate the effect of pretreatment parameters that is, temperature, and time, as expressed in the severity factor, on the recovery of sugars and hydrolyzability of pretreated cellulose. We find the severity factor, in its widely used form, is an incomplete measure for evaluating the pretreatment efficiencies and predicting overall sugar yields when pretreatment temperatures above 200°C are used. Corrections to the severity factor and its correlation to the measured pretreatment responses (% xylan solubilization, xylan recovery as fermentable sugars, cellulose enzymatic digestibility) indicate a greater influence of temperature on the pretreatment efficiencies than predicted by the commonly used severity factor. A low temperature, long residence time is preferred for hemicellulose dissolution during the pretreatment since the condition favors oligosaccharide and monomeric sugar formation over sugar degradation. On the contrary, high cellulose hydrolyzability is achieved with a high temperature (>200°C), high severity pretreatment when pretreatment is followed by enzyme hydrolysis. In multi-stage pretreatment, the first low-severity pretreatment is optimized for solubilizing fast-hydrolyzing hemicellulose while minimizing formation of furans. The subsequent pretreatment is carried out at over 200°C to recover the difficult-to-hydrolyze hemicellulose fraction as well as to increase susceptibility of pretreated cellulose to enzymes. High recovery (>92%) of hemicellulose-derived pentose sugars and enhanced enzymatic hydrolysis of pretreated cellulose (where >80% glucose yield results with 20 FPU = 32 mg protein/g glucan or 10-13 mg/g initial hardwood) are achieved by applying a multi-stage pretreatment. This work shows how the severity equation may be used to obtain a single characteristic curve that correlate xylan solubilization and enzymatic cellulose hydrolysis as a function of severity at pretreatment temperatures up to 230°C.

Effect of flux and shear rate on E. coli recovery in tangential flow filtration through a single hollow fiber.

Pathogenic bacteria which enter a viable but non-culturable (VBNC) state impede efforts to reach detectable concentrations required for PCR methods. This motivated a strategy for tangential flow filtration to concentrate bacteria in aqueous samples while maintaining the bacteria in a viable state, maximizing their recovery and achieving high fluxes through a single hollow fiber membrane. Filtrations were carried out for green fluorescent protein (GFP) E. coli at high shear rates (up to 27,000 sec-1) through 0.2 µm cut-off polyethersulfone (PES) microfilter membranes or 50 kDa polysulfone (PS) ultrafilter membranes. High shear minimized bacterial attachment on membrane surfaces, which would otherwise occur due to forced convection of the particles to the membrane surface at high flux conditions. Single fiber filter modules were constructed to facilitate concentration of Escherichia coli at fluxes ranging from 55 to 4500 L m-2 h-1. The effect of high shear rates on bacterial viability was found to be minimal with bacterial losses during filtration caused principally by their accumulation on the membrane surface. Recoveries of 90% were achievable at high shear rates when the average flux was ≤300 L m-2 h-1. This corresponded to a 3-h filtration time for a 225 mL sample through a single hollow fiber. Detectable bacteria concentrations of 1800 colony-forming unit (CFU)/mL were achieved for starting concentrations of 140 CFU/mL.

Rapid sample processing for detection of food-borne pathogens via cross-flow microfiltration.

This paper reports an approach to enable rapid concentration and recovery of bacterial cells from aqueous chicken homogenates as a preanalytical step of detection. This approach includes biochemical pretreatment and prefiltration of food samples and development of an automated cell concentration instrument based on cross-flow microfiltration. A polysulfone hollow-fiber membrane module having a nominal pore size of 0.2 µm constitutes the core of the cell concentration instrument. The aqueous chicken homogenate samples were circulated within the cross-flow system achieving 500- to 1,000-fold concentration of inoculated Salmonella enterica serovar Enteritidis and naturally occurring microbiota with 70% recovery of viable cells as determined by plate counting and quantitative PCR (qPCR) within 35 to 45 min. These steps enabled 10 CFU/ml microorganisms in chicken homogenates or 10(2) CFU/g chicken to be quantified. Cleaning and sterilizing the instrument and membrane module by stepwise hydraulic and chemical cleaning (sodium hydroxide and ethanol) enabled reuse of the membrane 15 times before replacement. This approach begins to address the critical need for the food industry for detecting food pathogens within 6 h or less.

Inhibition of enzyme hydrolysis of cellulose by phenols from hydrothermally pretreated sugarcane straw.

Relatively few studies have addressed the characterization of sugarcane straw (SCS) for production of fermentable sugars through enzyme hydrolysis. Straw is a major co-product of the sugarcane harvest in Brazil that has potential to sustainably increase cellulosic feedstocks in Brazil by 50%. Pretreatment of 10% w/v straw with liquid hot water (LHW) at 180 °C for 50 min (severity, So, of 4.05), solubilizes hemicellulose, preserves glucan, and generates 4.49 g/L soluble phenolic compounds in the resulting liquid. Extracts from washing pretreated solids with excess hot water followed by acetone resulted in 1.10 and 0.83 g/L phenolics, respectively. Acetone-derived extracts were more inhibitory and decreased glucose yield for enzyme hydrolysis of Solka Floc (a lignin-free cellulose) by 42%. In comparison, pretreated straw washed with hot water or acetone was readily hydrolyzed to 92% and 97% by cellulase enzyme. Hydrothermally treated SCS has the potential to provide a valuable and added source of fermentable sugars suitable for bioprocessing into biofuels and bioproducts when cellulase enzyme inhibitors are removed after pretreatment.

Effect of pelleting on the enzymatic digestibility of corn stover.

Pelleting of lignocellulosic biomass to improve its transportation, storage and handling impacts subsequent processing and conversion. This work reports the role of high moisture pelleting in the enzymatic digestibility of corn stover prior to pretreatment, together with associated substrate characteristics. Pelleting increases the digestibility of unpretreated corn stover, from 8.2 to 15.5% glucan conversion, at 5% solid loading using 1 FPU Cellic® CTec2 per g solids. Compositional analysis indicates that loose and pelleted corn stover have similar non-dissolvable compositions, although their extractives are different. Enzymatic hydrolysis of corn stover after size reduction to normalize particle sizes and removal of extractives confirms that pelleting improves corn stover digestibility. Such differences may be explained by the decreased particle size, improved substrate accessibility, and hydrolysis of cross-linking structures induced by pelleting. These findings are useful for the development of processing schemes for sustainable and efficient use of lignocellulose.

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: enzymatic hydrolysis (part 1).

Lignin content, composition, distribution as well as cell wall thickness, structures, and type of tissue have a measurable effect on enzymatic hydrolysis of cellulose in lignocellulosic feedstocks. The first part of our work combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. The extent of enzyme hydrolysis follows the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Physical fractionation of corn stalks or other C(4) grasses into soft and hard tissue types could reduce cost of cellulose conversion by enabling reduced enzyme loadings to hydrolyze soft tissue, and directing the hard tissue to other uses such as thermal processing, combustion, or recycle to the land from which the corn was harvested.

Tissue-specific biomass recalcitrance in corn stover pretreated with liquid hot-water: SEM imaging (part 2).

In the first part of our work, we combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. Hydrolysis experiments coupled with chemical analysis of the different fractions of corn stover showed significant differences in cell wall structure before and after liquid hot water pretreatment. The extent of enzyme hydrolysis followed the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Since similar lignin contents remained after liquid hot water pretreatment of leaves, rind, and pith, our results indicated that the amount of lignin alone is not sufficient to explain the different enzymatic hydrolysis characteristics of the fractions. While the role of structural characteristics on enzyme hydrolysis of cell walls is measured as described in part I, the SEM images presented in this part II of our work show that sugar yields from enzymatic hydrolysis of corn fractions correlate with changes in plant cell wall structure both before and after liquid hot water pretreatment.

Screening method for Enzyme-based liquefaction of corn stover pellets at high solids.

Liquefaction of high solid loadings of unpretreated corn stover pellets has been demonstrated with rheology of the resulting slurries enabling mixing and movement within biorefinery bioreactors. However, some forms of pelleted stover do not readily liquefy, so it is important to screen out lots of unsuitable pellets before processing is initiated. This work reports a laboratory assay that rapidly assesses whether pellets have the potential for enzyme-based liquefaction at high solids loadings. Twenty-eight pelleted corn stover (harvested at the same time and location) were analyzed using 20 mL enzyme solutions (3 FPU cellulase/ g biomass) at 30 % w/v solids loading. Imaging together with measurement of reducing sugars were performed over 24-hours. Some samples formed concentrated slurries of 300 mg/mL (dry basis) in the small-scale assay, which was later confirmed in an agitated bioreactor. Also, the laboratory assay showed potential for optimizing enzyme formulations that could be employed for slurry formation.

Doppler imaging detects bacterial infection of living tissue.

Living 3D in vitro tissue cultures, grown from immortalized cell lines, act as living sentinels as pathogenic bacteria invade the tissue. The infection is reported through changes in the intracellular dynamics of the sentinel cells caused by the disruption of normal cellular function by the infecting bacteria. Here, the Doppler imaging of infected sentinels shows the dynamic characteristics of infections. Invasive Salmonella enterica serovar Enteritidis and Listeria monocytogenes penetrate through multicellular tumor spheroids, while non-invasive strains of Escherichia coli and Listeria innocua remain isolated outside the cells, generating different Doppler signatures. Phase distributions caused by intracellular transport display Lévy statistics, introducing a Lévy-alpha spectroscopy of bacterial invasion. Antibiotic treatment of infected spheroids, monitored through time-dependent Doppler shifts, can distinguish drug-resistant relative to non-resistant strains. This use of intracellular Doppler spectroscopy of living tissue sentinels opens a new class of microbial assay with potential importance for studying the emergence of antibiotic resistance.

Rheology of enzyme liquefied corn stover slurries: The effect of solids concentration on yielding and flow behavior.

The measurement of yield stress and shear thinning flow behavior of slurries formed from unpretreated corn stover at solids loadings of 100-300 g/L provides a key metric for the ability to move, pump, and mix this lignocellulosic slurry, particularly since corn stover slurries represent a major potential feedstock for biorefineries. This study compared static yield stress values and flow hysteresis of corn stover slurries of 100, 150, 200, 250, and 300 g/L, after these slurries were formed by adding pellets to a cellulase enzyme solution (Celluclast 1.5 L) in a fed-batch manner. A rotational rheometer was used to quantitate relative yield stress and its dependence on processing history at insoluble solids concentrations of 4%-21% (wt/vol). Key findings confirmed previous observations that yield stress increases with solids loadings and reaches ~3000 Pa at 25% (wt/vol) solids concentration compared to ~200 Pa after enzyme liquefaction. While optimization of slurry forming (i.e., liquefaction) conditions remains to be done, metrics for quantifying liquefaction extent are needed. The method for obtaining comparative metrics is demonstrated here and shows that the yield stress, shear thinning and shear thickening flow behaviors of enzyme liquefied corn stover slurries can be analyzed using a wide-gap rheometry setup with relative measuring geometries to mimic the conditions that may exist in a mixing vessel of a bioreactor while applying controlled and precise levels of strain.

Cellulolytic enzymes production guided by morphology engineering.

Endoglucanase and xylanase are critical enzymes for liquefaction and enzyme hydrolysis of high solids lignocellulosic biomass to facilitate its transport and production of desired derived products. Here is reported how combinations of different spore concentrations and pH influence microbial morphology, and how this may be used to direct expression and secretion of enzymes by Aspergillus niger. While xylanase production is not affected by A. niger morphology changes, endoglucanase production is enhanced under conditions of lower stress and by morphology that results in pellets. ß-glucosidase production is enhanced under dispersed morphology, which results in up to fourfold increase of this enzyme production under the tested experimental conditions. A morphologic scale (Y) is proposed based on a form factor that considers the size and frequency of each morphology class, and that points to conditions that result in high selectivity for either endoglucanase or ß-glucosidase production. An equation proposed to relate enzyme activity to morphology provides a useful tool for tuning enzyme production of A. niger, where morphology is a first indication of relative enzyme activities in a fermentation broth.

Severity factor kinetic model as a strategic parameter of hydrothermal processing (steam explosion and liquid hot water) for biomass fractionation under biorefinery concept.

Hydrothermal processes are an attractive clean technology and cost-effective engineering platform for biorefineries based in the conversion of biomass to biofuels and high-value bioproducts under the basis of sustainability and circular bioeconomy. The deep and detailed knowledge of the structural changes by the severity of biomasses hydrothermal fractionation is scientifically and technological needed in order to improve processes effectiveness, reactors designs, and industrial application of the multi-scale target compounds obtained by steam explosion and liquid hot water systems. The concept of the severity factor [log10 (Ro)] established>30 years ago, continues to be a useful index that can provide a simple descriptor of the relationship between the operational conditions for biomass fractionation in second generation of biorefineries. This review develops a deep explanation of the hydrothermal severity factor based in lignocellulosic biomass fractionation with emphasis in research advances, pretreatment operations and the applications of severity factor kinetic model.

Protective effects of non-catalytic proteins on endoglucanase activity at air and lignin interfaces.

The manner in which added non-catalytic proteins during enzymatic hydrolysis of lignocellulosic substrates enhances hydrolysis mechanisms is not completely understood. Prior research has indicated that a reduction in the non-specific adsorption of enzymes on lignin, and deactivation of enzymes exposed to air-liquid interface provide rationale. This work investigated root causes including effects of the air-liquid interface on non-catalytic proteins, and effects of lignin on endoglucanase. Three different experimental designs and three variables (air-liquid interfacial area, the types of lignin (acid or enzymatic lignin), and the presence of non-enzymatic protein (bovine serum albumin [BSA] or soy proteins ) were used. The results showed that acid isolated lignin adsorbed almost all endoglucanase activity initially present in supernatant, independent of air interface conditions (25 or 250 ml flasks) with the presence of BSA preventing this effect. Endoglucanase lost 30%-50% of its activity due to an air-liquid interface in the presence of lignin while addition of non-enzymatic protein helped to preserve this enzyme's activity. Langmuir and Freundlich models applied to experimental data indicated that the adsorption increases with increasing temperature for both endoglucanase and BSA. Adsorption of the enzyme and protein were endothermic with an increase in entropy. These results, combined, show that hydrophobicity plays a strong role in the adsorption of both endoglucanase and BSA on lignin.

New strategy for liquefying corn stover pellets.

The movement of solid material into and between unit operations within a biorefinery is a bottleneck in reaching design capacity, with formation of biomass slurries needed to introduce feedstock. Corn stover slurries have been achieved from dilute acid, pretreated materials resulting in slurry concentrations of up to about 150 g/L, above which flowability is compromised. We report a new strategy to liquefy corn stover at higher solids concentration (300 g/L) by initially cooking it with the enzyme mimetic maleic acid at 40 mM and 150 °C. This is followed by 6 h of enzymatic modification at 1 FPU (2.2 mg protein)/g solids, resulting in a yield stress of 171 Pa after 6 h and 58 Pa in 48 h compared to 6806 Pa for untreated stover. Mimetic treatment of corn stover pellets minimizes the inhibitory effect of xylo-oligomers on hydrolytic enzymes. This strategy allows for the delivery of solid lignocellulosic slurry into a pretreatment reactor by pumping, improving operability of a biorefinery.