The Important Role of Enzymes in Cellulosic Ethanol

By Emmanuel Petiot | October 06, 2008
The future of biofuels is getting closer as the industry works tirelessly to turn biomass into economical fuel ethanol, but many questions still need to be answered. One important piece of the puzzle is the enzyme hydrolysis step, which is key to making the feedstock available to allow an efficient fermentation of the lignocellulosic materials cellulose and/or hemicellulose.

The production of ethanol from biomass can be broken down into the steps shown below. Although the process consists of many of the same steps as current starch-based ethanol conversion, it is not as easy to perform and, as yet, no commercial process is available. To develop a successful commercial process, all of these steps need to be integrated because there is a complex interplay between them. One reason why this process is much more difficult than starch-based conversion is because biomass material is much more complicated than its starch-based counterparts.

The steps needed to convert lignocellulosic feedstock into ethanol are more complicated than corn-to-ethanol production.


Biomass contains lignocellulosic fibers, which are composed of three major fractions: cellulose, hemicellulose and lignin. While enzymes are able to attack the polymeric sugar chain in the cellulose and hemicellulose fractions, releasing monomeric sugars for fermentation, they cannot break down the lignin. The presence of this lignin, a complex natural polymer, makes processing biomass more difficult.

Solutions for a Complex Problem
It has been noted much in the news that one of the stumbling blocks for transforming biomass into biofuels is the lack of cost-efficient enzymes. Enzyme cost has already been dramatically reduced due to dedicated research, but it needs to be further reduced. This is due to the complexity of the biomass material discussed earlier. To solve this issue, new types of cellulases and hemicellulase activities are being developed. Unfortunately, a complete "one-stop solution" is not possible as the type of feedstock and its composition, as well as the steps prior to enzymatic hydrolysis, have a lot to do with which enzymes are needed to optimally perform.

Pretreatment is needed to open up the fibers and make the lignocellulosic substrate (cellulose and/or hemicellulose) accessible to enzyme action. A variety of pretreatment methods are available that normally depend on a mechanical and/or chemical disruption of the feedstock. These methods make the cellulose and/or hemicellulose accessible for the enzymatic action, but due to their harshness, loss of material and generation of inhibitory compounds often take place as well. So far there is no clear winner in pretreatment, and the relative severity of these technologies is a difficult concept to master. It is imperative that a maximum amount of sugars be made available without destroying any valuable material.

A major complexity to note is that the enzymes must match the selected technologiesthe feedstock used as well as the process. For example, if a dilute acid pretreatment is used, most of the hemicellulose is degraded and hemicellulases will not be needed. However, if an alkaline or neutral pretreatment is used, the hemicellulose still needs to be hydrolyzed and hemicellulases will be needed. The enzymes needed for cellulosic conversion must be tailor-made to fit the rest of the process.

Another difficulty is in the cellulose component of the biomass. In order to efficiently break it down, a mixture of several proteins with different activities is required. This cocktail includes three basic types of enzymes.

Endoglucanases break bonds between adjacent sugar molecules in a cellulose chain, fragmenting the chain into shorter lengths. Endoglucanases act somewhat randomly along the length of the cellulose chain, although they prefer amorphous regions where the chains are less crystalline.

Cellobiohydrolases attack cellulose chains from the ends of the chain. This exo- or processive action releases mainly cellobiose (composed of two glucose sugar units). Because endoglucanases create new ends for cellobiohydrolases to act upon, the two classes interact synergistically.

Beta-glucosidases break down short glucose chains, such as the glucose dimer cellobiose, to release glucose, a fermentable sugar. Beta-glucosidases are important as they act on cellobiose, which inhibits the action of the other cellulases as it builds up the hydrolysis reactor.

After successful enzymatic hydrolysis has taken place a further challenge is faced at the fermentation stage. The five-carbon sugars produced by the hydrolysis of hemicellulose cannot be fermented by any yeast or microorganism currently in useat least not in commercially relevant conditions. Research is proceeding to develop organisms that can effectively use these sugars in order to increase ethanol yields and make the whole process cost-efficient.

Sugarcane Bagasse, Corn Stover
Sugarcane bagasse is made up of approximately two-thirds carbohydrates. The remaining one-third is lignin and other materials. The second-generation process revolves around accessing the large amounts of cellulosic material blocked within the lignin-based shell and creating ethanol from it.

Theoretically, one ton of sugarcane bagasse produces up to 300 liters (79 gallons) of ethanol. In reality the yield depends on a number of parameters such as quality of feedstock and process efficiency.

Currently, 6,000 to 7,000 liters (1,600 to 1,800 gallons) of ethanol is produced from one hectare (2.47 acres) of sugarcane, not including the bagasse. When bagasse is included, the amount will as much as double: 12,000 to 15,000 liters per hectare (1,280 to 1,600 gallons per acre).

Sugarcane bagasse has the most positive net energy balance of the common feedstocks. Eight times more energy is produced from sugarcane than what is used in its creation, according to the Brazilian Sugarcane Industry Association. When bagasse is included in the equation, it is estimated that the number may increase to as much as 16 times.

Corn stover, which in a strict definition only includes leaves and stalks but also cobs when considered more broadly, is another feedstock of choice by some of the early industry movers. It is a relatively widespread feedstock used all over the world, and an abundant source of biomass.

One metric ton of corn stover can theoretically be converted into 375 liters (99 gallons) of ethanol, but the same processing constraints apply as those mentioned earlier for sugarcane bagasse. In practice, the best lab- and pilot-scale plants can only produce approximately 290 liters (77 gallons) worth of ethanol with an aim to be as close as possible to the maximum yield.

Corn stover consists of approximately 66 percent carbohydrates, with a good balance between five- and six-carbon sugars, which makes the efficient conversion of the latter as important as the former.

One metric ton of harvested corn crop equals one metric ton of available corn stover. At this point, all projects pertaining to this feedstock take into account stringent sustainability criteria. When sourcing the agricultural residue for a given plant, players actually ensure that enough stover is left in the fields to maintain nutrition parameters at appropriate levels and to prevent soil erosion. A lot of work is being put into collection and storage, which remains a challenge as corn stover has a high bulk density. This, by the way, limits the value one can make out of burning corn stover.

Although no one has a definitive answer to the question of when cellulosic ethanol will become economically viable, a reasonable answer is about two to three years from now. All of the elements are in place to almost certainly ensure eventual success for the industry. Within this timeframe it is probable that several plants will begin to produce cellulosic ethanol from feedstocks such as agricultural residues, wood residues and industrial waste. Ultimately, they will likely produce this on an industrial basis with 25 MMgy to 50 MMgy plants.

Novozymes is putting a lot of effort into making cost-effective lignocellulosic ethanol a reality. Currently, a wide range of cellulase and hemicellulase enzymes are in the experimental stage. Bringing the cost of these enzymes down is the major challenge. An estimate of two to three years to support economically viable processes is realistic.

Emmanuel Petiot is global biomass business development manager for Novozymes. Reach him at or (919) 494-3022.