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.

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.

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.

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.

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.

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.

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.

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.

Deactivation and activation of lignocellulose degrading enzymes in the presence of laccase.

Cellulase and hemicellulase activities in a 1:1 ratio of enzymes extracted from Chrysoporthe cubensis and Penicillium pinophilum were evaluated in the presence of known monocomponent phenolic inhibitors and also with phenol mixtures derived from alkali pretreated sugarcane bagasse. The cellulolytic activities from C. cubensis:P. pinophilum displayed a much higher tolerance to phenolic inhibitors than equivalent enzyme activities obtained from Trichoderma reesei and Aspergillus niger. Enzymes from T. reesei and A. niger were deactivated at 0.3 and 1.5mg phenols/mg protein, respectively, as reported previously, while enzymes from C. cubensis:P. pinophilum resisted deactivation at 35mg phenols/mg protein. However, tolerance of xylanase with respect to phenols required the presence of laccase. Removal of laccase (enzyme) activity using sodium azide resulted in a 2x higher xylanase deactivation (from 40% to 80%). This paper identifies enzymes that are phenol tolerant, and whose adoption for lignocellulose hydrolysis could contribute to reductions in enzyme loading needed to hydrolyze alkali pretreated lignocellulosic substrates in the presence of lignin derived phenols.

Cellulose conversion of corn pericarp without pretreatment.

We report enzyme hydrolysis of cellulose in unpretreated pericarp at a cellulase loading of 0.25FPU/g pericarp solids using a phenol tolerant Aspergillus niger pectinase preparation. The overall protein added was 5mg/g and gave 98% cellulose conversion in 72h. However, for double the amount of enzyme from Trichoderma reesei, which is significantly less tolerant to phenols, conversion was only 16%. The key to achieving high conversion without pretreatment is combining phenol inhibition-resistant enzymes (such as from A. niger) with unground pericarp from which release of phenols is minimal. Size reduction of the pericarp, which is typically carried out in a corn-to-ethanol process, where corn is first ground to a fine powder, causes release of highly inhibitory phenols that interfere with cellulase enzyme activity. This work demonstrates hydrolysis without pretreatment of large particulate pericarp is a viable pathway for directly producing cellulose ethanol in corn ethanol plants.