Kinetic characterization for pretreatment of timber varieties and switchgrass using diluted acid hydrolysis

In recent years, growing attention has been devoted to the use of lignocellulosic biomass as a feedstock to produce renewable carbohydrates as a source of energy products, including liquid alternatives to fossil fuels. The benefits of developing woody biomass to ethanol technology are to increase the long-term national energy security, reduce fossil energy consumption, lower greenhouse gas emissions, use renewable rather than depletable resources, and create local jobs. Currently, research is driven by the need to reduce the cost of biomass-ethanol production. One of the preferred methods is to thermochemically pretreat the biomass material and subsequently, enzymatically hydrolyze the pretreated material to fermentable sugars that can then be converted to ethanol using specialized microorganisms. The goals of pretreatment are to remove the hemicellulose fraction from other biomass components, reduce bioconversion time, enhance enzymatic conversion of the cellulose fraction, and, hopefully, obtain a higher ethanol yield. The primary goal of this research is to obtain kinetic detailed data for dilute acid hydrolysis for several timber species from the Upper Peninsula of Michigan and switchgrass. These results will be used to identify optimum reaction conditions to maximize production of fermentable sugars and minimize production of non-fermentable byproducts. The structural carbohydrate analysis of the biomass species used in this project was performed using the procedure proposed by National Renewable Energy Laboratory (NREL). Subsequently, dilute acid-catalyzed hydrolysis of biomass, including aspen, basswood, balsam, red maple, and switchgrass, was studied at various temperatures, acid concentrations, and particle sizes in a 1-L well-mixed batch reactor (Parr Instruments, ii Model 4571). 25 g of biomass and 500 mL of diluted acid solution were added into a 1-L glass liner, and then put into the reactor. During the experiment, 5 mL samples were taken starting at 100°C at 3 min intervals until reaching the targeted temperature (160, 175, or 190°C), followed by 4 samples after achieving the desired temperature. The collected samples were then cooled in an ice bath immediately to stop the reaction. The cooled samples were filtered using 0.2 μm MILLIPORE membrane filter to remove suspended solids. The filtered samples were then analyzed using High Performance Liquid Chromatography (HPLC) with a Bio-Rad Aminex HPX-87P column, and refractive index detection to measure monomeric and polymeric sugars plus degradation byproducts. A first order reaction model was assumed and the kinetic parameters such as activation energy and pre-exponential factor from Arrhenius equation were obtained from a match between the model and experimental data. The reaction temperature increases linearly after 40 minutes during experiments. Xylose and other sugars were formed from hemicellulose hydrolysis over this heat up period until a maximum concentration was reached at the time near when the targeted temperature was reached. However, negligible amount of xylose byproducts and small concentrations of other soluble sugars, such as mannose, arabinose, and galactose were detected during this initial heat up period. Very little cellulose hydrolysis yielding glucose was observed during the initial heat up period. On the other hand, later in the reaction during the constant temperature period xylose was degraded to furfural. Glucose production from cellulose was increased during this constant temperature period at later time points in the reaction. The kinetic coefficient governing the generation of xylose from hemicellulose and the generation of furfural from xylose presented a coherent dependence on both temperature and acid concentration. However, no effect was observed in the particle size. There were three types of biomass used in this project; hardwood (aspen, basswood, and red maple), softwood (balsam), and a herbaceous crop (switchgrass). The activation energies and the pre-exponential factors of the timber species and switchgrass were in a range of 49 - 180 kJ/mol and from 7.5x104 - 2.6x1020 min-1, respectively, for the xylose formation model. In addition, for xylose degradation, the activation energies and the preexponential factors ranged from 130 - 170 kJ/mol and from 6.8x1013 - 3.7x1017 min-1, respectively. The results compare favorably with the literature values given by Ranganathan et al, 1985. Overall, up to 92 % of the xylose was able to generate from the dilute acid hydrolysis in this project.

Ethanol Production from Hardboard Manufacturing Process Wastewater: Methods for Improving Product Yields from Hemicellulose Hydrolysates

Waste effluents from the forest products industry are sources of lignocellulosic biomass that can be converted to ethanol by yeast after pretreatment. However, the challenge of improving ethanol yields from a mixed pentose and hexose fermentation of a potentially inhibitory hydrolysate still remains. Hardboard manufacturing process wastewater (HPW) was evaluated at a potential feedstream for lignocellulosic ethanol production by native xylose-fermenting yeast. After screening of xylose-fermenting yeasts, Scheffersomyces stipitis CBS 6054 was selected as the ideal organism for conversion of the HPW hydrolysate material. The individual and synergistic effects of inhibitory compounds present in the hydrolysate were evaluated using response surface methodology. It was concluded that organic acids have an additive negative effect on fermentations. Fermentation conditions were also optimized in terms of aeration and pH. Methods for improving productivity and achieving higher ethanol yields were investigated. Adaptation to the conditions present in the hydrolysate through repeated cell sub-culturing was used. The objectives of this present study were to adapt S. stipitis CBS6054 to a dilute-acid pretreated lignocellulosic containing waste stream; compare the physiological, metabolic, and proteomic profiles of the adapted strain to its parent; quantify changes in protein expression/regulation, metabolite abundance, and enzyme activity; and determine the biochemical and molecular mechanism of adaptation. The adapted culture showed improvement in both substrate utilization and ethanol yields compared to the unadapted parent strain. The adapted strain also represented a growth phenotype compared to its unadapted parent based on its physiological and proteomic profiles. Several potential targets that could be responsible for strain improvement were identified. These targets could have implications for metabolic engineering of strains for improved ethanol production from lignocellulosic feedstocks. Although this work focuses specifically on the conversion of HPW to ethanol, the methods developed can be used for any feedstock/product systems that employ a microbial conversion step. The benefit of this research is that the organisms will the optimized for a company's specific system.

Specificity, duplex degradation and subcellular localization of antagomirs

MicroRNAs (miRNAs) are an abundant class of 20-23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed 'antagomirs'. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings.

Appraising and selecting conservation measures to mitigate desertification and land degradation based on stakeholder participation and global best practices

Most desertification research focuses on degradation assessments without putting sufficient emphasis on prevention and mitigation strategies, although the concept of Sustainable Land Management (SLM) is increasingly being acknowledged. A variety of already applied conservation measures exist at the local level, but they are not adequately recognised, evaluated and shared, either by land users, technicians, researchers, or policy makers. Likewise, collaboration between research and implementation is often insufficient. The aim of this paper is to present a new methodology for a participatory process of appraising and selecting desertification mitigation strategies, and to present first experiences from its application in the EU-funded DESIRE project. The methodology combines a collective learning and decision approach with the use of evaluated global best practices. In three parts, it moves through a concise process, starting with identifying land degradation and locally applied solutions in a stakeholder workshop, leading to assessing local solutions with a standardised evaluation tool, and ending with jointly selecting promising strategies for implementation with the help of a decision support tool. The methodology is currently being applied in 16 study sites. Preliminary analysis from the application of the first part of the methodology shows that the initial stakeholder workshop results in a good basis for stakeholder cooperation, and in promising land conservation practices for further assessment. Study site research teams appreciated the valuable results, as burning issues and promising options emerged from joint reflection. The methodology is suitable to initiate mutual learning among different stakeholder groups and to integrate local and scientific knowledge.

Protein changes and proteolytic degradation in red and white clover plants subjected to waterlogging

Red (Trifolium pratense L., cv. “Start”) and white clover varieties (Trifolium repens L., cv. “Debut” and cv. “Haifa”) were waterlogged for 14 days and subsequently recovered for the period of 21 days. Physiological and biochemical responses of the clover varieties were distinctive, which suggested different sensitivity toward flooding. The comparative study of morphological and biochemical parameters such as stem length, leaflet area, dry weight, protein content, protein pattern and proteolytic degradation revealed prominent changes under waterlogging conditions. Protease activity in the stressed plants increased significantly, especially in red clover cv. “Start”, which exhibited eightfold higher azocaseinolytic activity compared to the control. Changes in the protein profiles were detected by SDS-PAGE electrophoresis. The specific response of some proteins (Rubisco, Rubisco-binding protein, Rubisco activase, ClpA and ClpP protease subunits) toward the applied stress was assessed by immunoblotting. The results characterized the red clover cultivar “Start” as the most sensitive toward waterlogging, expressing reduced levels of Rubisco large and small subunits, high content of ClpP protease subunits and increased activity of protease isoforms.

The Effect of Heat Treatment of Forages on Degradation Kinetics and Escape Protein Concentration

An in situ study was conducted to evaluate the effects of heat treatments on the degradation kinetics and escape protein concentrations of forages (alfalfa and berseem clover). Alfalfa collected at 4 and 7 weeks post-harvest and berseem clover collected at 5 and 7 weeks postharvest were freeze-dried and then heated to 100, 125, and 150o C for 2 hours. Heat treatment effects were determined by placing two bags of sample (for each treatment, maturity, and forage species for a given incubation times) into the rumen of one fistulated steer fed alfalfa hay. Bags were incubated for periods of 0 to 48 hours. Increasing levels of heat treatments of forages increased concentrations of neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent insoluble nitrogen (ADIN) and non-degradable protein (NDP), potentially degradable protein proportion (PDP), and protein escaping rumen degradation (PEP) while decreasing water soluble protein (WSP) and the rates of crude protein (CP), except immature berseem clover and cell wall (CW) degradation. PEP was greater and rate of CP degradation was lower at 100 and 150o C compared to 125o C in immature berseem clover.

The Effect of Maturity and Frost Killing of Forages on Degradation Kinetics and Escape Protein Concentration

Two consecutive in situ studies were conducted to determine the effects of maturity and frost killing of forages (alfalfa and berseem clover) on degradation kinetics and escape protein concentrations. Four maturities (3, 5, 7, and 9 weeks after second harvest) of forages collected from three locations were used to determine the effects of maturity. Four weeks after a killing frost (-2o C), berseem clover was harvested from the same locations previously sampled. To evaluate maturity, 336 DacronÒ bags containing all maturities of either alfalfa or berseem clover were placed into the rumen of two fistulated steers fed alfalfa-grass hay. Frost killing effects of berseem clover were compared with maturecut berseem clover by placing DacronÒ bags into the rumen of one fistulated steer fed alfalfa hay. Bags were incubated for periods of 0 to 48 hours. With increasing maturity, the proportion of non-degradable protein (NDP) and the rate of crude protein (CP) degradation increased in both forages. While the rate of neutral detergent fiber (NDF) degradation and potentially degradable protein proportion (PDP) increased with increasing maturity in alfalfa, the rate of NDF degradation and PDP proportion decreased and proportion of water soluble protein (WSP) increased in berseem clover. The proportion of protein escaping rumen degradation (PEP) was greater in berseem clover than alfalfa, but was not affected by maturity. Frost killing of mature berseem clover decreased WSP proportion and increased PDP proportion compared to mature berseem clover harvested live. Even though ADIN concentration was higher for frost-killed berseem clover, PEP and total escape protein concentration (CEP) was also higher for frostkilled berseem clover than mature berseem clover harvested live, due to decreases in the rate of ruminal N degradation with frost-killing.

Tools for better SLM knowledge management and informed decision-making in addressing land degradation at different scales: the WOCAT–LADA–DESIRE methodology

Desertification research conventionally focuses on the problem – that is, degradation – while neglecting the appraisal of successful conservation practices. Based on the premise that Sustainable Land Management (SLM) experiences are not sufficiently or comprehensively documented, evaluated, and shared, the World Overview of Conservation Approaches and Technologies (WOCAT) initiative (www.wocat.net), in collaboration with FAO’s Land Degradation Assessment in Drylands (LADA) project (www.fao.org/nr/lada/) and the EU’s DESIRE project (http://www.desire-project.eu/), has developed standardised tools and methods for compiling and evaluating the biophysical and socio-economic knowledge available about SLM. The tools allow SLM specialists to share their knowledge and assess the impact of SLM at the local, national, and global levels. As a whole, the WOCAT–LADA–DESIRE methodology comprises tools for documenting, self-evaluating, and assessing the impact of SLM practices, as well as for knowledge sharing and decision support in the field, at the planning level, and in scaling up identified good practices. SLM depends on flexibility and responsiveness to changing complex ecological and socioeconomic causes of land degradation. The WOCAT tools are designed to reflect and capture this capacity of SLM. In order to take account of new challenges and meet emerging needs of WOCAT users, the tools are constantly further developed and adapted. Recent enhancements include tools for improved data analysis (impact and cost/benefit), cross-scale mapping, climate change adaptation and disaster risk management, and easier reporting on SLM best practices to UNCCD and other national and international partners. Moreover, WOCAT has begun to give land users a voice by backing conventional documentation with video clips straight from the field. To promote the scaling up of SLM, WOCAT works with key institutions and partners at the local and national level, for example advisory services and implementation projects. Keywords: Sustainable Land Management (SLM), knowledge management, decision-making, WOCAT–LADA–DESIRE methodology.

THE UBIQUITIN LIGASE UBE4B IS REQUIRED FOR EFFICIENT EPIDERMAL GROWTH FACTOR RECEPTOR DEGRADATION

The length of time that integral membrane proteins reside on the plasma membrane is regulated by endocytosis, a process that can inactivate these proteins by removing them from the membrane and may ultimately result in their degradation. Proteins are internalized and pass through multiple distinct intracellular compartments where targeting decisions determine their fate. Membrane proteins initially enter early endosomes, and subsequently late endosomes/multivesicular bodies (MVBs), before being degraded in the lysosome. The MVB is a subset of late endosomes characterized by the appearance of small vesicles in its luminal compartment. These vesicles contain cargo proteins sorted from the limiting membrane of the MVB. Proteins not sorted into luminal vesicles remain on the MVB membrane, from where they may be recycled back to the plasma membrane. In the case of receptor tyrosine kinases (RTKs), such as epidermal growth factor (EGF) receptor, this important sorting step determines whether a protein returns to the surface to participate in signaling, or whether its signaling properties are inactivated through its degradation in the lysosome. Hrs is a protein that resides on endosomes and is known to recruit sorting complexes that are vital to this sorting step. These sorting complexes are believed to recognize ubiquitin as sorting signals. However, the link between MVB sorting machinery and the ubiquitination machinery is not known. Recently, Hrs was shown to recruit and bind an E3 ubiquitin ligase, UBE4B, to endosomes. In an assay that is able to measure cargo movement, the disruption of the Hrs-UBE4B interaction showed impaired sorting of EGF receptor into MVBs. My hypothesis is that UBE4B may be the connection between MVB sorting and ubiquitination. This study addresses the role of UBE4B in the trafficking and ubiquitination of EGF receptor. I created stable cell lines that either overexpresses UBE4B or expresses a UBE4B with no ligase activity. Levels of EGF receptor were analyzed after certain periods of ligand-induced receptor internalization. I observed that higher expression levels of UBE4B correspond to increased degradation of EGF receptor. In an in vitro ubiquitination assay, I also determined that UBE4B mediates the ubiquitination of EGF receptor. These data suggest that UBE4B is required for EGFR degradation specifically because it ubiquitinates the receptor allowing it to be sorted into the internal vesicles of MVBs and subsequently degraded in lysosomes.

Dynamic Remodeling of the Stressed Heart: Role of Protein Degradation Pathways

The heart is a remarkable organ. In order to maintain its function, it remodels in response to a variety of environmental stresses, including pressure overload, volume overload, mechanical or pharmacological unloading and hormonal or metabolic disturbances. All these responses are linked to the inherent capacity of the heart to rebuild itself. Particularly, cardiac pressure overload activates signaling pathways of both protein synthesis and degradation. While much is known about regulators of protein synthesis, little is known about regulators of protein degradation in hypertrophy. The ubiquitin-proteasome system (UPS) selectively degrades unused and abnormal intracellular proteins. I speculated that the UPS may play an important role in both qualitative and quantitative changes in the composition of heart muscle during hypertrophic remodeling. My study hypothesized that cardiac remodeling in response to hypertrophic stimuli is a dynamic process that requires activation of highly regulated mechanisms of protein degradation as much as it requires protein synthesis. My first aim was to adopt a model of left ventricular hypertrophy and determine its gene expression and structural changes. Male Sprague-Dawley rats were submitted to ascending aortic banding and sacrificed at 7 and 14 days after surgery. Sham operated animals served as controls. Effective aortic banding was confirmed by hemodynamic assessment by Doppler flow measurements in vivo. Banded rats showed a four-fold increase in peak stenotic jet velocities. Histomorphometric analysis revealed a significant increase in myocyte size as well as fibrosis in the banded animals. Transcript analysis showed that banded animals had reverted to the fetal gene program. My second aim was to assess if the UPS is increased and transcriptionally regulated in hypertrophic left ventricular remodeling. Protein extracts from the left ventricles of the banded and control animals were used to perform an in vitro peptidase assay to assess the overall catalytic activity of the UPS. The results showed no difference between hypertrophied and control animals. Transcript analysis revealed decreases in transcript levels of candidate UPS genes in the hypertrophied hearts at 7 days post-banding but not at 14 days. However, protein expression analysis showed no difference at either time point compared to controls. These findings indicate that elements of the UPS are downregulated in the early phase of hypertrophic remodeling and normalizes in a later phase. The results provide evidence in support of a dynamic transcriptional regulation of a major pathway of intracellular protein degradation in the heart. The discrepancy between transcript levels on the one hand and protein levels on the other hand supports post-transcriptional regulation of the UPS pathway in the hypertrophied heart. The exact mechanisms and the functional consequences remain to be elucidated.

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.