Sandra T. Merino • Joel Cherry (И)

Novozymes Inc., 1445 Drew Ave., Davis, CA 95618, USA JRoC@novozymes. com

1 Introduction……………………………………………………………………………………………….. 96

2 Lignocellulosic Biomass to Ethanol Process Overview……………………………………. 97

2.1 Minimizing Yield Loss and Cost…………………………………………………………………… 99

3 Impact of Process Steps on Enzyme Dosage and Cost…………………………………. 100

3.1 Impact of Substrate Selection on Enzyme Cost……………………………………………. 101

3.2 Impact of Pretreatment Selection……………………………………………………………….. 102

3.3 The Impact of Process Integration on Enzyme Requirements………………………… 104

4 Enzyme Discovery: Catalytic Efficiency and Productivity…………………………….. 106

4.1 T. reesei Cellulases: The Current Industry Standard………………………………………. 106

4.2 Searching for Synergy………………………………………………………………………………… 107

4.2.1 P-Glucosidase………………………………………………………………………………………….. 108

4.2.2 Glycosyl Hydrolase Family 61 109

4.2.3 Synergistic Hemicellulases………………………………………………………………………… 111

5 Producing Enzymes Economically………………………………………………………………. 115

5.1 Reduced Enzyme Recovery………………………………………………………………………… 117

6 Conclusions………………………………………………………………………………………………. 118

References ……………………………………………………………………………………………………. 118

Abstract Enzymes play a critical role in the conversion of lignocellulosic waste into fu­els and chemicals, but the high cost of these enzymes presents a significant barrier to commercialization. In the simplest terms, the cost is a function of the large amount of enzyme protein required to break down polymeric sugars in cellulose and hemicellulose to fermentable monomers. In the past 6 years, significant effort has been expended to re­duce the cost by focusing on improving the efficiency of known enzymes, identification of new, more active enzymes, creating enzyme mixes optimized for selected pretreated substrates, and minimization of enzyme production costs. Here we describe advances in enzyme technology for use in the production of biofuels and the challenges that remain.

Keywords Biomass • Enzymes • Hydrolysis

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Introduction

The utilization of lignocellulose for the production of fuels and chemicals has the potential to change the world economically, socially, and environ­mentally. Today roughly 87% of the energy used in the world is derived from non-renewable sources such as oil, natural gas, and coal, with total en­ergy consumption increasing at approximately 4% per annum. About 28% of that energy consumption is in the form of liquid transportation fuels, de­rived almost entirely from petroleum [1]. The long-term cost of continued use of these finite fuel sources can already be seen in increased conflict over their control and distribution, climate change linked to increased greenhouse gas emissions, and increasing prices, all of which negatively impact people around the world every day. Lignocellulosic biomass, in the form of plant materials such as grasses, woods, and crop residues, offers a renewable, geo­graphically distributed, greenhouse-gas neutral source of sugars that can be converted to ethanol or other liquid fuels via microbial fermentation.

In the past 30 years, ethanol produced from corn starch and sugarcane has been established as an economically viable supplement to gasoline. In the USA over the past 5 years, production has increased from 175 million gallons per year to nearly 4.5 billion gallons last year, and is growing at more than 25% per year. In the near future, the use of sugar and starch as feedstocks for fuel production is expected to face increasing competition with their direct use as food and animal feed, impacting both availability and price. Current estimates suggest that in the USA, starch-based ethanol output will reach a maximum of between 12 and 15 billion gallons per year [2]. To significantly impact the use of petroleum in the USA, which uses approximately 140 bil­lion gallons of gasoline per year, additional sources of fermentable sugar for ethanol production will be required.

Lignocellulosic biomass has the potential to become a major source of these fermentable sugars in the future. It is estimated that in the USA alone, more than one billion tons per year of biomass could be sustainably harvested in the form of crop and forestry residues, replacing as much as 30% of the total US gasoline consumption [3].

To turn the prospect of replacing a significant proportion of the current liquid fuels into reality, the conversion of lignocellulose to ethanol must be­come less expensive in both operating cost and capital investment. Current estimates suggest that the cost of producing cellulosic ethanol is $1.80/gal — lon or higher, or almost twice as high as the cost of producing ethanol from starch [4]. Part of this high cost results from a significantly higher esti­mated capital investment for the construction of cellulosic plants compared to starch-based production facilities. Cellulose-to-ethanol plants in current design scenarios require more unit operations, must be larger to accom­modate more dilute sugar streams, and in some cases require acid-resistant construction materials, which in sum are projected to increase the invest­ment more than fourfold relative to current dry milling starch-based ethanol plants (from $1.10/gallon installed capacity to $4.70/gal) [4]. On the operat­ing cost side, equipment replacement may be more frequent due to processing materials that are more abrasive than seed, enzyme cost will be significantly higher due to the increased complexity of the substrate and higher enzyme dosage required to release the sugars, and higher water consumption may be required to remove compounds that interfere with the hydrolysis and fermen­tation processes.

Starch is present in plants as an energy source for growing seeds, while lignocellulose is present as a structural cell wall component to give the plant rigidity; therefore it should be no surprise that the latter is much more resis­tant to enzymatic attack. On a protein weight basis, it takes anywhere from 40-100 times more enzyme to break down cellulose than starch, yet the cost of enzyme production is not substantially different (Novozymes, unpublished data).

In 2001, Novozymes was awarded a research subcontract by the US Depart­ment of Energy with the goal of reducing the cost of cellulases for ethanol production from biomass. This effort, called the Cellulase Cost Reduction Project, was administered by the National Renewable Energy Laboratory (NREL), with Novozymes providing expertise for enzyme improvement and production, and NREL contributing expertise in biomass pretreatment and enzyme evaluation. The stated goal of the project was to achieve a tenfold reduction in the cost of enzymes for the conversion of acid pretreated corn stover to ethanol in laboratory-scale testing. At the beginning of this work, the cost of providing a commercial cellulase preparation for the conversion of 80% of the cellulose in acid pretreated corn stover to fermentable glucose was estimated to be $5.40/gallon ethanol produced. During the course of the contract, significant advances were made in improving the efficiency of the cellulases, increasing the yield in production, and reducing the cost of pro­duction. In addition, work focusing on other enzyme activities required for effective enzymatic hydrolysis of lignocellulosic substrates other than acid pretreated corn stover was successfully conducted. In this manuscript, we highlight some of those efforts that have contributed to making enzymes for lignocellulose hydrolysis more affordable.

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