# Presentation: 855509

## Breaking the Biological Barriers to Cellulosic Ethanol: A Joint research Agenda

- John Houghton  Sharlene Weatherwax   John Ferrell
-   Office of Science                   Office of Science                    EERE

- Betty Mansfield   Mike Knotek

## US Ethanol Plants

- AS OF: March 2006

- In operation

- Under construction

- Proposed

## Chemical structure of starch

- 1 ton produces about 90 gal ethanol

- http://www.scientificpsychic.com/fitness/carbohydrates1.html

- http://www.ucmp.berkeley.edu/monocots/corngrainls.jpg

- STARCH

## NCGA analysis for 2015/16 crop year:

|  | Harvested Acres     (mil acres) | Yield (bu/acre) | Total Production   (mil bu) | Ethanol Use       (mil bu) | Ethanol Conversion (gal/bu) | Ethanol Prod.   (mmg) |
| --- | --- | --- | --- | --- | --- | --- |
| HI CASE | 78.0 | 193.0 | 15,054 | 5,954 | 3.0 | 17,862 |
| MED CASE | 78.0 | 187.0 | 14,586 | 5,486 | 2.9 | 15,909.4 |
| LO CASE | 76.0 | 178.0 | 13,528 | 4,428 | 2.9 | 12,841.2 |

## The DOE Biofuels Vision

- Grain

- Cellulose

- 0

- 10

- 20

- 30

- 40

- 50

- 60

- 70

- 2000

- 2005

- 2010

- 2015

- 2020

- 2025

**Year**

**Ethanol (Billions of gal/yr)**

- EXISTING

- EMERGING

- Sugar

- Platform

- -New Enzymes

- -Pretreatment

- -Fermentation

- ADVANCED

- Fundamental

- Advances in

- Lignocellulose

- Processing

- and fermentation

- Modified from Richard Bain, NREL

**Notes:**
Cindy:

The basis for these projections:
The renewable fuel standard demand is based on the US Senate version of the Energy Policy Act of 2002 (S. 517) Section 820, generally referred to as the Renewable Fuel Standard.  The House version of the energy bill contains no such provision.  Section 820 calls for a ramp up of ethanol use from 2004 to 20012, starting at 2.4 bgy in 04 and reaching 5 bgy in 2012.  This corresponds to an increase of 300 to 400 million gal per yr each year.
 In this analysis, I assume that the bulk of that ethanol will come from corn grain.
The analysis further assumes that, by 2005, new technology for utilizing corn fiber or some non starch components in grain will be implemented.  These advanced technologies are assumed to add 10% to the yield of the grain ethanol operation.
From 2005 to 2012, I assume a linear increase in the adoption of this technology, with one facility starting up in 05, and all facilities adopting the advanced technology by 2012.
At the same time, the first stover ethanol plant is assumed to come on line at a capacity of 70 MM gal per year.  The number of stover plants doubles each year for the first four years, and then takes on a linear growth rate.
In 2015 the first advanced ethanol technology kicks in, with a similar doubling in the number of plants for the first few years, followed by a linear growth of switchgrass and corn stover facilities..
In 2025, utilization of switchgrass and stover supplies reaches about half of Marie Walsh’s estimates for total biomass supply.

## US Biomass inventory = 1.3 billion tons 

- Forest

- 12.8%

- Urban waste

- 2.9%

- Manure

- 4.1%

- Grains

- 5.2%

- Crop residues

- 7.6%

- Soy

- 6.2%

- Wheat straw

- 6.1%

- Corn stover

- 19.9%

- Perennial crops

- 35.2%

- From: Billion ton Vision, DOE & USDA 2005

- ~ 3 tons/acre

## Define how work at the frontiers of science can enable the lignocellulose biorefinery industry.
Define overarching technical challenges in the context of national & global energy policies 
Utilize consensus approach to define common sets of science and technology opportunities and barriers.
Develop “Barrier Technology Research Packages” (BTRP) from technical breakout sessions.
Define overall program goals and requirements from the BTRPs.
Workshop product: Roadmaps for new, coordinated research across DOE EERE and Office of Science.

- Define overarching technical challenges in the context of national & global energy policies
- Utilize consensus approach to define common sets of science and technology opportunities and barriers.
- Develop “Barrier Technology Research Packages” (BTRP) from technical breakout sessions.
- Define overall program goals and requirements from the BTRPs.
- Workshop product: Roadmaps for new, coordinated research across DOE EERE and Office of Science.

- Workshop Goals

## Agenda

- Goal setting: Ari Patrinos, Richard Moorer, John Ferrell, John Houghton
- The role of biofuels in transforming our energy future (Justin Adams, BP)
- Industry “Technology Pull”
- Development of poly-lactic acid polymers  (Doug Cameron, Cargill)
- Enzymatic Biomass Conversion (Bill Dean, Genencor)
- New Science from Systems Biology
- Realizing the Billion Ton Vision (Chris Somerville, Stanford/Carnegie)
- GTL Crosscutting Science Applied to Biomass (Jim Fredrickson, PNL)
- Genome-Scale Cellular Models (Christophe Schilling, Genomatica)
- Engineering Industrial Biocatalysts by Metabolic Engineering (Greg Stephanopoulos, MIT)
- Workshop Process: Mike Himmel (NREL), Mike Knotek
- Technical working groups meet
- Group 1:  Feedstock Genomics & Engineering
- Group 2:  Plant Cell Wall Deconstruction
- Group 3:  Biological Conversion of Biomass
- Group 4: Crosscutting technologies
- Reports and Recommendations

## Workshop Sessions*

- Session 1: Feedstock Genomics & Engineering
- Chairs: C. Somerville (Stanford), J. Tuskan (ORNL)
- Session 2: Plant Cell Wall Deconstruction
- Chairs: M. Himmel (NREL) and M. Ladisch (Purdue)
- Session 3: Biological Conversion of Sugars to Fuels
- Chairs: B. Davison (ORNL) and L. Ingram (Univ. of Florida)
- Session 4: Crosscutting Science & Technology (audited other sessions)
- Chairs: H. Chum (NREL) and J. Fredrickson (PNNL)

- *all sessions audited by USDA representatives

## Steps in cellulosic ethanol production

- From: Breaking the Biological Barriers to Cellulosic Ethanol

## Desirable improvements

- From: Breaking the Biological Barriers to Cellulosic Ethanol

## Properties of Optimal Feedstocks

- High Yield (>15 tons/acre/year)
- High water use efficiency
- Low Input (fertilizer, tillage, pesticides)
- High conversion efficiency
- Sustainable
- Stable quality and _**quantity**_ year to year

- Crop residues may play a part  but high
- capital costs favor dedicated energy crops

## Annual precipitation

## Progress in engineering crops plants for drought tolerance

- From Monsanto presentation to investors, May 8 2003

- The report assumes that existing ag research
- infrastructure will address generic aspects of
- plant growth and development

## Land use is fungible

- High plant productivity is equally important for food and energy production
- Plant productivity is a function of many aspects of  growth and development so a broad approach to knowledge creation is essential

## Effect of 50% stover removal on corn grain yields in eastern NE. (120kg N/ha)

**0**

**2000**

**4000**

**6000**

**8000**

**10000**

**12000**

**2000**

**2001**

**2002**

**2003**

**2004**

**0 stover removed**

**50% removed**

- K. Vogel et al., unpublished

- Kg/Ha

- Long-term research needed on effects of
- various cropping regimes on soil quality
- and microbial ecology
- Use metagenomics to understand soil microbial
- ccology in a range of cropping systems

## Geographic distribution of biomass crops

- Wright et al DOE-ORNL-EERE

## Perennials have more photosynthesis

- Courtesy of Steve Long, University of Illinois

**Notes:**
Note. Annualized net photosynthesis is proportional to the area under the curve. Thus, it can be seen that the perennial crops have much higher Annualized net photosynthesis than the annual crop (Zea mays)

Note from steve Long “You are very welcome to make use of the mineral recycling diagram and also the Miscanthus images.  We do also have images of Miscanthus and switchgrass growing side-by-side, if that is of help - let me know.  There was no attachment, but we do have actual data of radiation interception by Miscanthus and by corn crops on the South Farms that demonstrate your point on radiation use (our Miscanthus typically forms a closed canopy a month or two ahead of corn and remains green and photosynthetically active until the first heavy frost - usually early November; our corn usually looses photosynthetic activity and its chlorophyll in the last week of August).  I am not sure that I can obtain the data though before Tuesday, but if you need it sooner let me know; I will be in the office tomorrow.  In the meantime, I have attached a figure for corn and Miscanthus radiation interception from my earlier work in the UK; corn there is very late, grown for silage and usually green until the first frost.”

## Switchgrass (Panicum virgatum)

- USDA-NRCS PLANTS Database / **Hitchcock, A.S. (rev. A. Chase). 1950. **
***Manual of the grasses of the United States*****. USDA Misc. Publ. No. 200. ****Washington, DC.**

## Perennial C4 grasses have highest biomass production rates

- 26.5 dry tons/acre in Illinois

- Courtesy of Steve Long et al

- There has been relatively little research on
- the most promising energy crops. Thus, large
- gains  in productivity are possible
- DOE should develop tools and information (eg DNA
- sequence) to facilitate improvement of energy crops

## Plants are mostly composed of sugars

- Section of a pine board

- 3 nm

- Polymerized glucose

- Improved imaging methods are needed to facilitate
- understanding of how walls are made and decomposed

## Possible packing structures of cellulose

- 6(2x2)

- 6x6

- R. Atalla, unpublished

## Cellulose is recalcitrant to hydrolysis

- NREL

- Research objectives include: altering cellulose structure,
- Identifying new sources of cellulases, understanding
- cellulosome structure and function, structural studies,
- directed evolution studies, enzyme mixture studies

## Some cellulytic enzymes are components of a “molecular machine”

- From: Breaking the Biological Barriers to Cellulosic Ethanol

- Most studies to date have used single enzymes
- and single substrates.  New multifactorial
- approaches are needed in order to make
- progress with understanding real-world conditions

## Cellulose is occluded by other polymers 

- Cellulose

- Hemicellulose

- Cellulose is occluded by other polymers

**Notes:**
Layer 3.tif

## Xylanases exhibit different activities

- -

- 

- (1

- 

- 2)-

- Xyl

- Ara

- -

- 

- (1

- 

- 4)-

- Xyl

- Xyl

- -

- 

- (1

- 

- 4)-

- Xyl

- -

- 

- (1

- 

- 4)-

- Xyl

- -

- 

- (1

- 

- 4)-

- -

- 

- (1

- 

- 2)-

- Xyl

- -

- 

- (1

- 

- 4)-

- Xyl

- -

- 

- (1

- 

- 4)-

- Xyl

- -

- 

- (1

- 

- 4)-

- Xyl

- -

- 

- (1

- 

- 4)-

- Xyl

- -

- 

- (1

- 

- 4)-

- Xyl

- -

- 

- (1

- 

- 4)-

- -

- 

- (1

- 

- 4)-

- -

- 

- (1

- 

- 2)-

- -

- 

- (1

- 

- 4)-

- (1-2)

- (1-2)

- (1-2)

- 3613

- 1818

- Xyl

- Xyl

- ...

- ...

- Many enzymes are required to hydrolyze plant
- polysaccharides. Discovery of new enzymes has
- been enabled by genomics but many opportunities
- for improvements exist and some needed enzymes
- are not yet known

## A genetic defect in making 4-O-methyl glucuronyl xylan leads to loss of xylan

- Bauer et al., PNAS in press

- Most of the enzymes that make cell walls are not
- known and the processes that control wall
- polysaccharide composition are unknown.
- Genomics, proteomics and informatics tools
- can be used to facilitate rapid progress.

## Effect of lignin content on enzymatic recovery of sugars from Miscanthus

- D Vrije et al (2002) Int J Hydrogen Energy 27,1381

- Switchgrass
- composition

- cellulose

- Hemi
-   cellulose

## Lignin occludes polysaccharides 

- Lignin

- Hemicellulose

- Cellulose

## Sheep prefer low lignin

- Clint Chapple, Purdue

## Lignin biosynthesis

- Humphreys and Chapple, Curr Opin Plant Biol 5,224

## Cleavable Lignin Concept

- O

- O

- O

- O

- OH

- OH

- O

- O

- O

- OH

- OH

- Simplified degradation

- Modified synthesis

- Normal precursor

- Novel precursor

- Several game-changing concepts have emerged.
- Realization of these ideas would have qualitative
- rather than incremental effects on the overall process

## Linocellulose to ethanol process

- Conc. H

- 2

- SO

- 4

- Water

- Gypsum

- Water

- Purified

- Sugar Solution

- Lignin

- Utilization

- Ethanol
- recovery

- Fermentor

- Neutralization

- Tank

- Acid

- Reconcentration

- Acid/Sugar

- Separation

- Decrystallization

- Hydrolysis

- Hydrolysis

- Biomass

- Ethanol
- dehydration

## The challenge is efficient conversion

- Burning switchgrass (10 t/ha) yields 14.6-fold more energy than input to produce*
- But, converting switchgrass to ethanol calculated to consume 45% more energy than produced

- Energy consumption

- *Pimentel & Patzek, Nat Res Res 14,65 (2005)

## Linocellulose to Hydrophobe Process

- Conc. H

- 2

- SO

- 4

- Water

- Gypsum

- Water

- Purified

- Sugar Solution

- Lignin

- Utilization

- Hydrophobe

- Fermentor

- Neutralization

- Tank

- Acid

- Reconcentration

- Acid/Sugar

- Separation

- Decrystallization

- Hydrolysis

- Hydrolysis

- Biomass

- 69 kt Ca, K, Mg, P
- 66 kt lipids, waxes
- 171 kt protein

- Many theoretical opportunities exist to create
- fundamental changes in the overall process
- Fuels other than ethanol
- Altered biomass = altered preprocessing

## Fermentation of all sugars is essential

- Jeffries & Shi Adv Bioch Eng 65,118

- Systems biology and synthetic biology will be used
- to develop new organisms that can utilize all
- sugars and are resistant to inhibitors and products

## Propanediol: An example of synthetic biology(DuPont/Tate &Lyle/Genencor made 100 changes to E. coli)

- From: Breaking the Biological Barriers to Cellulosic Ethanol

## Example of a Biological Regulatory Circuit

- Davidson et al., Science 295,1669

- Elucidation of the regulatory circuits for microbial
- metabolism will enable large improvements
- in biofuel and bioproduct production

## Limited potential of biodiesel

- Current biodiesel

- Capacity

- US Diesel

- US Fuel

- 0

- 20

- 40

- 60

- 80

- 100

- 120

- 140

- 160

- Billion gallons

- 65 biodiesel companies in operation, 50 in construction 2006

**Notes:**
Total us animal and plant fat production 31.7 billion pounds
7.75 pounds lipid per gallon
Numbers from congressional research service 2006 report on bioenergy

## Biofeedstocks

_**Barriers**_
- _Controlling cell wall composition_ for energy production
- Developing appropriate _model systems_ to study energy crops (biological)
- _Improving quantity and quality of perennial_ herbaceous and woody biomass crops
- Developing practices to make corn stover, crop residues and biomass energy crops a _sustainable_ bioethanol feedstock
_**Transformational Challenges**_
- Development of a high productivity biodiesel crop
- Cell wall imaging tools

**Notes:**
Besides grass populus, miscanthus – invasive european grass
Model – brachypodium distachyon

## Cell Wall Decomposition

_**Barriers**_
- Fundamental Physical and Chemical Factors Limiting the Enzymatic Hydrolysis of Biomass
- Limiting the Denaturation of Native Celluloses and Related Cell Wall Polysaccharides Emphasis on the cellulose microcrystallite level
- Bioprocess Engineering and Systems Analysis
- Improvement and Integration of Enzymes Deconstructing the Non Cellulose Cell Wall Matrix
- Improvement of Enzymes Deconstructing Cell Wall Microfibrils: Protein Machines
- Metagenomics: Harvesting the Biochemical Potential of Microorganisms for Improved Biomass Conversion
_**Transformational Challenges**_
- Harvesting Operations Can Initiate Cell Wall Disassembly
- Cell Wall Deconstruction Enzymes Expressed in Plants*
- Develop Predictive Computational Models for Biology
- Understand the Dynamics of Microbial Colonization of Decaying Biomass
- Advanced Imaging for Cell Wall Deconstruction Research

## Bioconversion

_**Barriers**_
**Pushing the limits of biology for optimal ethanol producing strains **
**Utilization of all sugars**
**Inhibition of ethanol**
**Designer microbe for fermentative production of 40% ethanol from biomass sugars**
**Inhibition of hydrolyzate byproducts**
**Kinetics and volumetric productivity**
**Understanding all physiology and regulation**
**Model-driven design of cellular biocatalysts**
**Advanced microorganisms for process simplification**
**Proteomic and Genomic studies of industrial ethanol strains and process **
**Simultaneous saccharification and co-fermentation (SSCF)**
**Eliminate step for detoxification of pretreatment hydrolyzates  **
**Combine cellulase production, cellulose hydrolysis, and fermentation**
**Enabling technologies**
**gene transfer methods and expression of genes in non-conventional host organisms, **
**rapid tools for the analysis and modeling of cellular composition and physiological state, and **
**high throughput screening methods **
**Enabling Microbiological tools**
**Ethanol from biomass process modeling**
_**Transformational Challenges**_
**Direct bioproduction of energy rich fuels**
**Potential uses of CO2 (fermentation byproduct) or syngas from gasification**
**Engineered microbial communities for robust energy production**

## Full complement of “omics’ technologies
Rapid manipulation tools for novel organisms (microbes and plants)
Computational science and biomolecular structure
Deeper understanding of (relevant) biochemistry
Physical chemistry at the nanometer scale
Modeling cellular processes
Role of advanced imaging
Role of HTP methods
Biomass compositional analysis
Challenges of data handling and access

- Rapid manipulation tools for novel organisms (microbes and plants)
- Computational science and biomolecular structure
- Deeper understanding of (relevant) biochemistry
- Physical chemistry at the nanometer scale
- Modeling cellular processes
- Role of advanced imaging
- Role of HTP methods
- Biomass compositional analysis
- Challenges of data handling and access

- Cross Cutting Science and Technology

**Notes:**
Also ELSI
Microbial physiology no longer taught

## Vision of the future

## The future

- ?

- From: Breaking the Biological Barriers to Cellulosic Ethanol

## The 1.3 Billion Ton Biomass Scenario

- Based on ORNL & USDA Resource Assessment Study by Perlach et.al. (April 2005) http://www.eere.energy.gov/biomass/pdfs/final_billionton_vision_report2.pdf

- The 1.3 Billion Ton Biomass Scenario

- Billion Barrel of Oil Equivalents

## HISTORY OF US FEDERAL GOVERNMENT R & D

- JFK Apollo
- Program

- Carter Energy
- Program

- Reagan “Star Wars” Program

- M. Hoeffert

## Why is photosynthetic efficiency so low?

- Visible portion of spectrum
- Active areas of plants
- Photo-inhibition
- Antenna length
- Electron transfer losses

- Wes Hermann, Stanford

## Broad-spectrum disease resistance

**Notes:**
Data for G861 shown. G861 is from the MADS domain family of TFs.