January 2010

ALTERNATIVE ENERGY:

Searching for Rugged Enzymes Adapted to Switchgrass Processing

Most of the worlds's carbon supply comes from nonrenewable sources. Given the fact that, by definition, nonrenewable materials aren't in everlasting supply, there is great interest in obtaining carbon from renewable sources, such as crops and algae.

Burning such carbon for energy may serve as a realistic transition to nonpolluting sources of energy, such as hydrogen and solar power. In the long term, carbon will still be needed, even if not for energy; the carbon base of paints, pharmaceuticals, and plastics will still need to come from somewhere.

Switchgrass: Opportunities and challenges.

Switchgrass, a rugged plant native to North America, is an attractive renewable carbon source. Unfortunately, it's very challenging to break apart the sugar polymers in this and other grasses into synthetically-useful molecules.

Relevant to this issue, Yuan Kou (Peking University, China), Johannes Lercher (Technische Universität München, Germany), and coworkers have converted phenol and phenolic polymers (common components of biomass) into a stable fuel. Mark Mascal and Edward Nikitin at the University of California (Davis) have converted cellulose into a safe, high-energy fuel.

To convert biomass into synthetically-useful molecules, however, the final product doesn't have to be a fuel. If switchgrass is to be used as a carbon source, the sugar polymers must simply be broken up into simpler small molecules.

A possible solution to the switchgrass processing challenge.

Enzymes may be of use for this purpose. Enzymes are proteins that generally have a specific chemical function, and do it very efficiently, far more efficiently than the most advanced chemicals synthesized by chemists.

A limitation of enzymes in this context is that they are generally not functional under harsh conditions, such as dilute acid. Such conditions are typical of industrial-scale switchgrass carbon extraction protocols.

Rugged enzymes are needed if switchgrass is to be broken down into useful molecules. Philip Hugenholtz (Joint Bioenergy Institute and Joint Genome Institute, California) and coworkers have addressed this challenge, by discovering and characterizing an enzyme from bacteria that is adapted to decomposing switchgrass under rugged environmental conditions.

Designing a composting laboratory.

If one desires to find an enzyme ideally suited for breaking down switchgrass, a good place to begin the search is in compost. Bacteria present in compost are likely to be rugged, due to the range of heat, oxidation, and water conditions present in compost.

Consequently, the enzymes these bacteria use to obtain nutrients from compost are also likely to be rugged. The scientists designed bioreactors to search for their desired rugged enzyme.

They incubated their bioreactors with switchgrass feedstock for approximately one month. The conditions within these bioreactors can be precisely controlled and monitored, and are essentially a compost heap in the laboratory.

They varied the temperature in the bioreactors, over the course of a month, to simulate natural composting conditions. After initial microbial establishment within the bioreactors at 30°C (1 day), a slow initial heating to 54°C (2 days) was followed by constant heating at 54°C (7 days), which was then followed by an extended slow cooling period to 30°C (21 days).

Evaluating the bacteria.

The scientists found that carbon dioxide was emitted, and oxygen was taken up, most strongly during days 2 and 9. Heat-adapted bacteria are therefore probably responsible for the high sugar polymer breakdown rate.

The microbes within the bioreactor were still productive after this time, but their activity was greatly diminished. After it was all over, total solids within the bioreactor were reduced by 34%.

Specifically, 17% of lignin (a sugar polymer) was broken down. Between 27% and 33% of several other sugars were also broken down.

Identifying useful bacteria.

Which bacteria were responsible for breaking the sugars apart? The scientists observed a definite change in the microbial community over the course of the composting procedure.

The microbial community was dominated at the end of the procedure by a genus (group of species) of which one species possesses genes known to enable bacteria to break down cellulose and hemicellulose. These are sugar polymers found in plant cell walls.

This suggested to the scientists that they had found heat-adapted bacteria that break down plant cell walls, a rich source of sugar. They then partially sequenced the DNA of all the bacteria in their switchgrass-adapted microbial community, a procedure that enabled them to find the enzymes coded for by the DNA.

Identifying useful enzymes.

The scientists found that more than 0.5% of the microbial DNA codes for enzymes that are presumed to aid in the breakdown of cellulose and hemicellulose. Of these enzymes, almost 11% of the enzymes are those presumed to break down cellulose.

This percentage of cellulose-breakdown enzymes ("cellulases") is five times higher than the relative abundance of such enzymes in the microbial fermentation gland (rumen) of cows. It is half that present in termite hindguts.

Similarly to what is observed in cows, the switchgrass-adapted bacteria also had a high percentage of enzymes that break down the chemical appendages ("side chains") of hemicellulose. Thus, the bacteria possess enzymes that are on a par with those found in critters that are especially adapted to feed on plant matter.

Possible industrial utility of the enzymes.

The scientists identified one of the cellulases in their switchgrass-adapted bacterial community. Its optimum activity is at 50°C and pH 7 (neutral), but it retains over 50% of its function from 30°C to 55°C and pH 5.5 (mildly acidic) to 8 (mildly basic).

This is a wide range of conditions, and is in agreement with the observation that there was microbial activity within the bioreactors over the duration of the scientists' experiments. Its heat and pH tolerance also suggest that this enzyme shows promise for use in industrial applications.

Future directions.

Hugenholtz and coworkers have found conditions under which switchgrass can be readily broken down into useful molecules. Furthermore, they have identified the DNA sequence that codes for an enzyme which enables bacteria to do this job effectively under rugged conditions.

Recent biotechnology advances will enable other scientists to incorporate this DNA sequence into the genome of any bacteria, generating a microscopic army that is optimized to process switchgrass. This development will be very useful for making the most of switchgrass as an efficient, renewable carbon source.

for more information:
Allgaier, M., Reddy, A., Park, J. I., Ivanova, N., D'haeseleer, P., Lowry, S., Sapra, R., Hazen, T. C., Simmons, B. A., VanderGheynst, J. S., & Hugenholtz, P. (2010). Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008812