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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Effect of using a nitrogen atmosphere on enzyme hydrolysis at high corn stover loadings in an agitated reactor.

A comprehensive review of the literature shows that enzyme hydrolysis efficiency decreases with increased solids loadings at constant enzyme:cellulose ratios for pretreated lignocellulosic substrates. In seeking a mechanistic explanation for this phenomenon, we found that a nitrogen atmosphere enhances enzyme hydrolysis and minimizes the decrease in glucose yields as solids loadings are increased in an agitated bioreactor. For liquid hot water pretreated corn stover, at solids loadings of both 100 and 200 g/L and hydrolyzed for 72 hr in a 1 L bioreactor at pH 5.0 with 3.6 mg protein per g biomass, glucose yields were 55% in a nitrogen atmosphere versus 45% in air with agitation and about 34% without agitation. While mixing promotes biomass/enzyme contact and disperses sugars released during hydrolysis that would otherwise cause product inhibition, nitrogen gas displaces air, avoiding deactivation of cellulases by oxygen. The nitrogen effect points to a facile approach of enhancing hydrolysis at high solids loadings.

Construction and operation of a multiplexed microfiltration device to facilitate rapid pathogen detection.

Millions of Americans contract food poisoning or are affected by microbial pathogens each year. Rapid, sensitive detection of dilute levels of pathogens in foods, produce, water, and biomanufacturing process samples is key to consumer protection; however, current enrichment methods require as much as a full day to enrich viable bacterial pathogens to detectable levels. Our lab previously demonstrated the ability to concentrate and detect dilute levels of pathogens, within 8 hr, from various food matrices using microfiltration in our continuous cell concentration device (i.e., C3D) with one or two filter modules. This short communication describes the design, materials and construction, layout, and operational characteristics of a four filter module multiplexed system based on a four channel device. Benefits are a 2× greater sample capacity than an equivalent duplex system (achieving the same time to result of less than 8 hr from sample preparation to detection), simpler operation, and a footprint enabling operation inside a biosafety cabinet instead of requiring a BSL-2 room. Flow rate variability through four channels fit within an operational envelope of ±3%; flow rates are reproducible from one run to the next thus ensuring relatively simple, concurrent processing of samples.

Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach.

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.

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.

Lignin-Enzyme Interactions in the Hydrolysis of Lignocellulosic Biomass.

Lignin is central to overcoming recalcitrance in the enzyme hydrolysis of lignocellulose. While the term implies a physical barrier in the cell wall structure, there are also important biochemical components that direct interactions between lignin and the hydrolytic enzymes that attack cellulose in plant cell walls. Progress toward a deeper understanding of the lignin synthesis pathway - and the consistency between a range of observations over the past 40 years in the very extensive literature on cellulose hydrolysis - is resulting in advances in reducing a major impediment to cellulose conversion: the cost of enzymes. This review addresses lignin and its role in the hydrolysis of hardwood and other lignocellulosic residues.

Accelerated Sample Preparation for Fast Salmonella Detection in Poultry Products.

Salmonella is the most burdensome foodborne pathogen in the USA and a major causal agent of foodborne outbreaks. Detection of a pathogen such as Salmonella can be achieved within a few hours using commercially available rapid methods, but the sample preparation is time consuming and may require multiple days. We have developed and successfully tested an accelerated sample preparation method based on microfiltration, in some cases preceded by a short enrichment step, for the rapid detection of selected pathogens. The time-frame of the overall process, from sample preparation (i.e., food rinse or homogenate preparation, microbial enrichment, and filtration steps) to detection is 8 h or less. While microfiltration has been practiced for 70 years, the complex interactions between food substances and filter membrane surfaces have shown that food pretreatment methods need to be developed on a case by case basis for the recovery of bacteria from food homogenates and/or rinses. We have also demonstrated that addition of protease to treat homogenates of different poultry products prior to microfiltration avoids the rapid decrease in flux that otherwise occurs during microfiltration. This protease treatment minimizes filter clogging, so that the microbial concentration, recovery and detection of 1 to 10 CFU/g of Salmonella in poultry products is possible in less than 8 h.

Temperature dependent cellulase adsorption on lignin from sugarcane bagasse.

Extents of adsorption of cellulolytic enzymes on lignin, derived from sugarcane bagasse, were an inverse function of incubation temperature and varied with type of lignin extraction. At 45 °C, lignin derived from acid hydrolyzed liquid hot water pretreated bagasse completely adsorbed cellulolytic enzymes from Trichoderma reesei within 90 min. Lignin derived from enzyme hydrolyzed liquid hot water pretreated bagasse adsorbed only 60% of T. reesei endoglucanase, exoglucanase and ß-glucosidase activities. ß-Glucosidase from Aspergillus niger was not adsorbed. At 30 °C, adsorption of all of the enzymes was minimal and enzyme hydrolysis at 30 °C approached that at 45 °C after 168 h. Hence, temperature provided an approach to decrease loss of enzyme activity by reducing enzyme adsorption on lignin. This helps to explain why simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP), both carried out at 30-32 °C, could offer viable options for mitigating lignin-derived inhibition effects.