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.

Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant.

Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

Adaptive laboratory evolution of nanocellulose-producing bacterium.

Adaptive laboratory evolution through 12 rounds of culturing experiments of the nanocellulose-producing bacterium Komagataeibacter hansenii ATCC 23769 in a liquid fraction from hydrothermal pretreatment of corn stover resulted in a strain that resists inhibition by phenolics. The original strain generated nanocellulose from glucose in standard Hestrin and Schramm (HS) medium, but not from the glucose in pretreatment liquid. K. hansenii cultured in pretreatment liquid treated with activated charcoal to remove inhibitors also converted glucose to bacterial nanocellulose and used xylose as carbon source for growth. The properties of this cellulose were the same as nanocellulose generated from media specifically formulated for bacterial cellulose formation. However, attempts to directly utilize glucose proved unsuccessful due to the toxic character of the lignin-derived phenolics, and in particular, vanillan and ferulic acid. Adaptive laboratory evolution at increasing concentrations of pretreatment liquid from corn stover in HS medium resulted in a strain of K. hansenii that generated bacterial nanocellulose directly from pretreatment liquids of corn stover. The development of this adapted strain positions pretreatment liquid as a valuable resource since K. hansenii is able to convert and thereby concentrate a dilute form of glucose into an insoluble, readily recovered and value-added product-bacterial nanocellulose.

Impact of protein blocking on enzymatic saccharification of bagasse from sugarcane clones.

Lignin plays an important functional and structural role in plants, but also contributes to the recalcitrance of lignocellulosic biomass to hydrolysis. This study addresses the influence of lignin in hydrolysis of sugarcane bagasse from conventional bred lines (UFV260 and UFV204) that were selected from 432 field-grown clones. In addition to higher sugar production, bagasse clone UFV204 had a small, but statistically significant, lower insoluble lignin content compared with clone UFV260 (15.5% vs, 16.6%) and also exhibited a significantly higher cellulose conversion to glucose (81.3% vs. 63.3%) at a cellulase loading of 5 (filter paper unit) FPU/g of glucan or 3 FPU/g total solids for liquid hot water pretreated bagasse (200°C, 10 min). The enzyme loading was further decreased by 50% to 2.5 FPU/g glucan and resulted in a similar glucan conversion (88.5%) for clone UFV204 when the bagasse was preincubated with bovine serum albumin at pH 4.8 and nonproductive binding of cellulase components was blocked. Comparison of Langmuir adsorption isotherms and differential adsorption of the three major cellulolytic enzyme components endoglucanase, cellobiohydrolase, and ß-glucosidase help to explain differences due to lignin content.

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure.

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content.

Human pathogens in plant biofilms: Formation, physiology, and detection.

Fresh produce, viewed as an essential part of a healthy life style is usually consumed in the form of raw or minimally processed fruits and vegetables, and is a potentially important source of food-borne human pathogenic bacteria and viruses. These are passed on to the consumer since the bacteria can form biofilms or otherwise populate plant tissues, thereby using plants as vectors to infect animal hosts. The life cycle of the bacteria in plants differs from those in animals or humans and results in altered physiochemical and biological properties (e.g., physiology, immunity, native microflora, physical barriers, mobility, and temperature). Mechanisms by which healthy plants may become contaminated by microorganisms, develop biofilms, and then pass on their pathogenic burden to people are explored in the context of hollow fiber microfiltration by which plant-derived microorganisms may be recovered and rapidly concentrated to facilitate study of their properties. Enzymes, when added to macerated plant tissues, hydrolyze or alter macromolecules that would otherwise foul hollow-fiber microfiltration membranes. Hence, microfiltration may be used to quickly increase the concentration of microorganisms to detectable levels. This review discusses microbial colonization of vegetables, formation and properties of biofilms, and how hollow fiber microfiltration may be used to concentrate microbial targets to detectable levels. The use of added enzymes helps to disintegrate biofilms and minimize hollow fiber membrane fouling, thereby providing a new tool for more time effectively elucidating mechanisms by which biofilms develop and plant tissue becomes contaminated with human pathogens. Biotechnol. Bioeng. 2017;114: 1403-1418. © 2017 Wiley Periodicals, Inc.

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.

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 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.

Intramodule pressure profiles and protein accumulation during tangential flow filtration.

Tangential flow filtration (TFF) through a 30 kDa nominal molecular weight cut-off (MWCO) ultrafiltration membrane is widely employed to concentrate purified monoclonal antibodies (mAbs) to levels required for their formulation into injectable biologics. While TFF has been used to remove casein from milk for cheese production for over 35 years, and in pharmaceutical manufacture of biotherapeutic proteins for 20 years, the rapid decline in filtration rate (i.e., flux) at high protein concentrations is a limitation that still needs to be addressed. This is particularly important for mAbs, many of which are 140-160 kDa immunoglobulin G (IgG) type proteins recovered at concentrations of 200 mg/mL or higher. This work reports the direct measurement of local transmembrane pressure drops and off-line confocal imaging of protein accumulation in stagnant regions on the surface of a 30 kDa regenerated cellulose membrane in a flat-sheet configuration widely used in manufacture of biotherapeutic proteins. These first-of-a-kind measurements using 150 kDa bovine IgG show that while axial pressure decreases by 58 psi across a process membrane cassette, the decrease in transmembrane pressure drop is constant at about 1.2 psi/cm along the 20.7 cm length of the membrane. Confocal laser scanning microscopy of the membrane surface at the completion of runs where retentate protein concentration exceeds 200 mg/mL, shows a 50 µm thick protein layer is uniformly deposited. The localized measurements made possible by the modified membrane system confirm the role of protein deposition on limiting ultrafiltration rate and indicate possible targets for improving membrane performance.

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.

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.

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.

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.