2.1.2 Pretreatment Facility for Research and Science

Pretreatment is the first step in lignocellulosic biomass processing and is essential for enzyme effectiveness. This facility provides the GLBRC with a means to pretreat the various cellulosic feedstocks that will be investigated.

Pretreatment opens up the cell-wall structure of lignocellulosic biomass so that enzymes can act upon the structural carbohydrates more readily and release sugars for biofuel production. Pretreatment of lignocellulosic biomass is a critical and integral part of the GLBRC; without pretreatment, the biomass materials from Thrust 1 will not give adequate sugar yields to make the bioenergy and fuel production in Thrust 3 possible. Without pretreatment, commercial lignocellulases are only able to convert 30% of the cellulose in corn stover to glucose (xylose conversions are less than 20%). Following pretreatment (Ammonia Fiber Expansion - AFEX, in this case), glucose yields approach 100% and xylose yields are near 70%. Also, supplementing lignocellulase with small amounts of xylanase pushes xylose yields over 90% while reducing lignocellulase loadings required for complete hydrolysis. 

There are many methods of lignocellulosic biomass pretreatment. AFEX was chosen as the standard pretreatment method of the GLBRC because it provides high sugar conversions and minimal sugar degradation. It also does not require chemical neutralization or wash streams. In order to better understand the fundamental principles of pretreatment, we are also examining another alkaline pretreatment (alkaline peroxide), as well as two other popular pretreatments, dilute acid and steam explosion.

Team Members and Roles

Bruce Dale MSU PI, project lead
Stephanie Crews, Graduate Student
Mary Ann Vigil, MSU technician
Charles Donald, MSU technician

2.2.1 Understanding the Effects of Alkaline Pretreatments on the Ultra-Structural and Chemical Properties of Biomass

The conversion of lignocellulosic biomass to biofuels has many advantages compared to conventional fuel production from crude oil. We are able to engineer feedstocks of suitable composition and ultrastructure to be more amenable to industrial processing. Petroleum crude oil refiners cannot do this. Native plant cell walls are recalcitrant to hydrolysis due to the inherent nature of the substrate and hence require pretreatment to enhance enzymatic digestibility and the subsequent release of fermentable sugars.

In order to selectively breed and/or genetically engineer plant cell walls that can be easily broken down following a mild pretreatment process, it is necessary to better understand the fundamental physicochemical factors that limit biomass deconstruction. However, this understanding will only be possible when the engineered/selectively bred plant materials are chemically processed as they would be in the biorefinery. The research carried out within this project will inform plant biologists about the appropriate structures within the plant cell wall that can be re-engineered to facilitate future low severity, low-cost pretreatment and enzymatic hydrolysis.

The proposed work will be initiated with ammonia-based pretreatments and, in subsequent years, extended to improve our understanding of other alkaline pretreatments (e.g., alkaline peroxide). This project will allow us to gain a fundamental understanding and inform other researchers within/outside the GLBRC of the cell wall characteristics that limit its digestibility and how that recalcitrance can be overcome by alkaline-based pretreatments. Secondly, the knowledge gained will help inform plant biologists and breeders (in Thrust 1 and elsewhere) to select or develop biomass that is more amenable to industrial processing.

The goals of this project are: 

1. To develop a fundamental understanding of the mechanism of alkali-based pretreatments which would allow significant improvements to the existing process.
    
2. To analyze engineered and/or selectively bred plant materials that have been developed with divergent characteristics to determine their impact on reducing pretreatment severity and improving overall process yields.

Team Members and Roles

Bruce Dale MSU Co-PI, project lead
John Ralph UW Co-PI, project lead
Venkatesh Balan, MSU investigator
Fachuang Lu, UW investigator
Hoon Kim, UW investigator
Shishir Chundawat, MSU post-doc
Ali Azarpira, UW post-doc
Rebecca Garlock, MSU graduate student
Lily Wei, UW graduate student
Leonardo Sousa, MSU graduate student
Ramin Vismeh, MSU graduate student
Jorge Rencoret Pazo, UW post-doc
Nirmal Uppugundia, MSU technician

2.2.4 Optimization of enzymes for biomass conversion

Enzymes that release fermentable sugars from biomass feedstocks are one of the major costs in converting lignocellulose to ethanol. Currently available commercial enzymes preparations are complex mixtures of hundreds of proteins, and the proportions of the constituent enzymes do not take into account what is or might actually be needed for efficient degradation of industrially relevant lignocellulosic substrates. In addition, these mixtures have been tested for use on lignocellulose pretreated with acid, which removes most of the hemicelluloses. Since the GLBRC has committed itself to alkaline pretreatments, which largely do not remove hemicelluloses, existing enzyme mixtures are not adequate for the need.

The major goal of this project is to build superior enzyme mixtures for alkaline-pretreated biomass.  This is done by first defining a “minimal enzyme set” composed of those enzymes that are almost certainly essential for lignocellulose degradation. The second step is to develop an enzyme mixture in which additional enzymes, called “accessory” enzymes, are added to the minimal set to improve its performance.

The goals of this project are: 

1. To determine which enzymes are important, and unimportant, for degradation of corn stover and other lignocellulosic materials subjected to alkaline pretreatments.
    
2. To construct a minimal core set that provides a platform from which to systematically evaluate accessory enzymes and alternative core enzymes. Alternative enzymes are derived from fungi, bacteria, or plants and are supplied by this and other projects within the GLBRC.
    
3. Enzymes that are demonstrated to be important from goals 1 and 2 become the subject of bioprospecting screens for improved versions through genome sequencing, database mining, and metagenomics in collaboration with other GLBRC scientists. They also become the target for improvement by protein engineering and for expression in consolidated bioprocessing (CBP) organisms, in collaboration with researchers in Thrust 3.

Team Members and Roles

Jonathan Walton, MSU Co-PI, project lead
Bruce Dale MSU Co-PI, project lead
Venkatesh Balan, MSU investigator
John Scott-Craig, MSU investigator
Shishir Chundawat, MSU post-doc
Goutami Banerjee, MSU post-doc
Melissa Borrusch, MSU technician
Suzana Car, MSU research scholar
Nirmal Uppugundla, MSU technician
Dahai Gao, MSU graduate student
Rebecca Garlock, MSU graduate student
James Humpula, MSU technician
Suzana Car, MSU graduate student
Sau Jesjarm, MSU post-doc

2.2.5 Alkaline Peroxide Pretreatment

In contrast to the acidic pretreatment processes, where much of the research for pretreatments of lignocellulosic biomass for biofuels has been targeted, alkaline and oxidative processes specifically address biomass recalcitrance by focusing on delignification. A wide range of alkaline and oxidative treatments of lignocellulose are known from the chemical pulping and bleaching industry, and from more recent cellulosic biofuels applications. These treatments include sulfur as well as alkaline/oxidative biomass treatment chemistries. While diverse, these chemical treatments differ from acid processes in that these all target overcoming biomass recalcitrance by solubilizing lignin. Significant research effort has been put into understanding these reaction chemistries in terms of their applications in pulping and bleaching. However, their application for pretreatment technologies requires further research before these have the potential to develop into mature technologies. For many alkaline pretreatments, the liquid phase after pretreatment contains both solubilized hemicellulose and lignin together with any inorganics used in pretreatment, which presents a challenge to hemicellulose recovery or biological utilization for a biofuels process.

The primary goal of this project is to study alkaline/oxidative pretreatment chemistries and develop these into feasible technologies for cellulosic biofuels processes, primarily through understanding their process chemistry and its impact on the unique process integration constraints for cellulosic biofuels technologies.  

The project is focused in two areas:

1. Study the delignification by alkaline-oxidative pretreatments that include alkaline hydrogen peroxide (AHP), hydrogen peroxide in aqueous ammonia, alkaline wet oxidation, and variations of the soda process. Complementary to this research focus is developing an improved understanding of how these processes can be applied with the specific application as pretreatment technologies. 
    
2. Investigate the suitability of utilizing these reaction chemistries as pretreatment technologies and develop feasible processes where all the unit operations are well integrated.

Team Members and Roles

David Hodge, MSU PI, project lead
Tongjun Liu, MSU post-doc
Dan Williams, MSU graduate student
Muyang Li, MSU graduate student

2.2.6 Improving Alkaline Pretreatment

Team Members and Roles

Eric Hegg, MSU project lead
Vaidyanathan Mathrubootham, MSU post-doc

2.3.1 Discovery, Identification, and Systems Analysis of Natural Cellulytic Microbes and Microbial Communities

Industrial bioenergy production is ultimately hindered by the inefficiencies of microbial conversion of plant biomass into intermediates that can be used to make ethanol.  Our long term efforts are centered on providing fundamental understanding of natural deconstruction of plant biomass within lignocellulose-rich communities and discovering microbes and enzymes with superior metabolic capabilities relevant to cost-effective and energy-efficient lignocellulosic ethanol production.

We believe that lignocellulose-rich niches should be a rich source of useful microbes, as microbes in these habitats have likely undergone extensive evolution toward more efficient plant biomass deconstruction.  We are investigating the microbial communities associated with herbivorous insects (fungus-growing ants, fungus-growing termites, fungus-growing beetles, bark beetles, and wood wasps), herbivore dung, and natural composts.  Based on our findings in the leaf-cutter ant system, we believe that microbes and their corresponding lignocellulases do exist in nature that are superior to the current suite of enzymes employed for plant biomass deconstruction in industry, and we believe that understanding how recalcitrant plant biomass is broken down in natural environments will lead to the discovery of novel enzymes relevant to cellulosic ethanol production.

Furthermore, through our focused efforts in leaf-cutter-ant-degrading habitats from temperate and tropical ecosystems, we are obtaining microbes that have high value for the GLBRC Enzyme Focus Group and that complement the bioprospecting efforts of the center.  However, we have only begun to screen the vast diversity of microbes, and our efforts in these niches are resulting in the discovery of novel cellulytic microbes from phyla that are under-represented in bioethanol research.  Considering the high costs associated with expressing and testing individual enzymes, it is critical to focus these efforts on very promising microbes from nature, and we intend to employ new assays we have developed to further identify very promising microbes.

Team Members and Roles

Cameron Currie, UW PI, project lead
David Mead, Lucigen investigator
Phil Brumm, C56 Technologies investigator
Paul Weimer, UW investigator
Garret Suen, UW post-doc
Michael Poulsen, UW post-doc
Sandye Adams, UW graduate student
Joe Moeller, UW technician
Frank Aylward, UW graduate student
Jarrod Scott, UW graduate student
Adam Book, UW graduate student
Heidi Horn, UW technician
Gina Lewin, UW graduate student
Evelyn Wendt-Pienkowski, UW technician
Shanti Bramhacharya, UW technician

2.3.3. Identification of Novel Microbial Enzymes

A key requirement for the production of inexpensive biofuels from lignocellulosic materials is the availability of low cost, high efficiency enzymes that produce simple sugars at a cost of approximately $0.10/gallon ethanol. The sugars produced by these enzymes can then be fermented by microbes to produce bioethanol or other biofuels. Currently available commercial lignocellulases are too expensive for use in a commercial process to produce biofuels.

Our approach is to harvest the immense genetic diversity of bacteria to discover a new set of high-specific-activity enzymes.  This screening work will result in the discovery of new and presumably better enzymes that will improve our understanding about the diversity and function of biomass-degrading enzymes and eventually to reduce the cost of the enzymes needed for biofuel production. 

We support other researchers within GLBRC by supplying purified enzymes for biomass deconstruction experiments, as well as genetic resources for enzyme secretory pathways and genes for carbohydrases and fatty acid synthesis.

Team Members and Roles

David Mead, Lucigen Co-PI, project lead
Phil Brumm, C56 Technologies Co-PI, project lead
Julie Boyum, Lucigen investigator
Colleen Drinkwater, Lucigen investigator
Krishne Gowda, Lucigen investigator
Larry Allen, Lucigen investigator
Jan Deneke, Lucigen investigator

2.3.4. Platform for combinatorial discovery of enzymes and proteins

Our project provides an integrative approach to better understand the multitude of natural strategies used by microbial organisms for biomass transformation, identify best performing individual enzymes and combinations of enzymes regardless of their origin, and provide information on the performance of enzyme combinations with diagnostic substrate analogs or purified cellulose compounds. One of the greatest strengths of our approach is to use natural biomass substrates as an integral part of our discovery efforts.

Our project defines a minimal enzyme set as a composition of enzymes that will release glucose, xylose, and other soluble sugars from a biomass substrate.  Ultimately, an ideal minimal set suitable for commercialization would give almost complete hydrolysis of biomass in the shortest time possible, at the lowest total protein loading, and with the minimal amount of pre-treatment. 

Our research is designed to provide early stage discovery of new enzymes. Within the Enzyme Focus Group, individual projects have demonstrated skills in the isolation of cellulolytic and pectinolytic organisms, genome sequencing, and genome- and metagenome-wide functional screening. These projects are now orienting toward discovery and categorization of new enzymatic architectures for cellulose deconstruction, and exploration of alternative taxonomic and ecological niches for sources of better enzymes. These efforts provide a rich and continually expanding source of genes of potential interest for improvement of biomass deconstruction. 

The goals of our project are:

1. To provide leadership for GLBRC enzyme research.
    
2. To evaluate alternative cellulases in minimal sets.
    
3. To evaluate eight categories of accessory enzymes (in collaboration with other groups in Thrust 2 of GLBRC).
    
4. To define rate-determining steps in release of soluble sugars from lignocellulosic substrates.
    
5. To obtain mRNA samples of at least one priority organism grown on lignocellulosic substrates and analyze microarray data from one priority organism.

Team Members and Roles

Brian Fox, UW PI, project lead
Abolfazi Arabshahi, UW investigator
Taichi Takasuka, UW post-doc
Allison Riederer, UW technician
Johnnie Walker, UW graduate student
Lai Bergeman, UW technician
Christopher Bianchetti, UW post-doc
Erin Bulleit, UW graduate student
Jonathan Wagner, UW technician

2.5.1 Fuel Production from Alkaline Pretreated Biomass

Feedstock pretreatment, enzymatic hydrolysis and microbial fermentation are three central processes to convert structural carbohydrates to fuel molecules. Existing technology has achieved sugar hydrolysis yields greater than 90% but at a low sugar concentration, which is not industrially‐relevant. To achieve a commercially‐viable production, the ethanol titer must be at least 40 g/L with an overall ethanol yield greater than 80 gal/ton biomass. This will most likely be achieved in an integrated biological process unit operating at high solids loading. Integrated configurations such as Simultaneous Saccharification and Co‐fermentation (SSCF) and Consolidated Bioprocessing (CBP) are more advantageous than separated processes due to lower capital and operating costs, potentially higher product yield and titer, and reduced enzyme cost. Hence, a key to improving lignocellulosic biofuel technology is to understand the fundamental engineering principles and science governing the interactions between pretreatment chemistries, enzymatic hydrolysis and fermentation.

Our project is in a unique position to help unravel and understand these complex interactions using an integrated perspective. Improved understanding will help us identify and reduce rate limiting processes based on a system‐wide approach. The results will be applicable regardless of microbial platforms used, pretreatment chemistry (alkaline or acidic) or other fermentation products produced from lignocellulosic biomass. 

Team Members and Roles
Bruce Dale, MSU PI, project lead
Venkatesh Balan, MSU investigator
Christa Gunawan, MSU technician
Mingjie Jin, MSU graduate student
Ming Woei Lau, MSU graduate student
Leonardo Sousa, MSU graduate student