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Cutting-Edge Co-Culture

University of California-San Francisco researchers have genetically engineered a brewer's yeast and co-cultured it with a cellulose-eating bacterium. The symbiotic relationship of the two microbes has created a novel process to convert biomass into methyl halides.
By Anna Austin
the phrase "one man's trash is another man's treasure" couldn't possibly ring truer that it does in the biomass industry. In some cases, its application extends beyond unwanted lamps or old bottles. In one instance, it can be applied to a bacterium discovered in a garbage dump in France in the early 1980s.

According to Christopher Voigt, a University of California-San Francisco pharmaceutical chemistry associate professor, the bacteria was briefly considered for cellulosic ethanol production but was discarded because it was low yielding and produced too much acetate.

Today, the bacterium has been given a second chance. Voigt and his research team have utilized the bacteria Actinotalea fermentans in a co-culture along with genetically engineered yeast in a research project designed to discover a way to introduce flexibility into biorefining.

A Unique Symbiosis
Only a few papers about the bacterium have ever been produced based on research that was conducted in the early 1980s, according to Voigt. "It was actually misclassified for 25 years," he says. "Nothing was really known about it, but it's a very unique organism in the sense that when it was reclassified, there weren't many relatives out there."

Voigt says it was studied years ago as a biomass conversion organism, but was ruled out because it was capable of only producing a small amount of ethanol, and it accumulated acetate that inhibited the bacteria from thriving.

The enzymes genetically engineered into the yeast are the result of synthetic metagenomics, which Voigt explains is the identification and selection of specific DNA. "The ability to print DNA has exploded within the past couple of years," he says. "When we were first looking for the enzymes to do the process, we could go into a sequence database and print out every enzyme in there that looked like it might be what we needed."

Biomass Conversion Process

source: Christopher voigt

From his perspective, it has progressed into an easier task. "I send an e-mail with the desired DNA sequence in it, and nine days to a month later-they can send back the genes," he says. "In the plasmid, we want a complete clone. Today, there are a number of companies who do this. Biotechnology research has been accelerating tremendously."

The biomass conversion process is relatively simple. After the bacterium eats the feedstock, the yeast is able to transform the acetate byproduct into methyl halides. Therefore, the bacterium is dependent on the yeast for carbon and energy, and the yeast is dependent on the bacteria to metabolize toxic waste products. As a result of this unique symbiosis, the UCSF team has been able to efficiently convert multiple biomass feedstocks into methyl halides, which are typically used as agricultural fumigants and are precursor molecules that can be catalytically converted to chemicals and fuels such as gasoline, alcohols and soil fumigants.

New to Biotechnology
"Methyl halides are produced by nature in small quantities," Voigt says. Marine algae, fungi and halophytic plants, which thrive in salty soil, all naturally produce methyl halides.

"I don't think there has been a real appreciation that biology makes methyl halides," Voigt says. "There was quite a bit of work done in atmospheric chemistry, regarding the natural production of methyl chloride by rice-which is a major ozone depleter. People have tried to find the gene that is producing it and knock it out to create an environmentally friendly strain of rice, but it hadn't crossed into biotechnology. People are often surprised that methyl halides are so prevalent in nature."

The idea is new to the field of biotechnology, in that nobody has tried to overproduce it as a useful molecule, although methyl halides are well known in petrochemicals as a precursor for a number of different chemicals.

"The conversion of methyl halides into the other chemicals has been around since the 1970s," Voigt says. "It's pretty trivial and scalable, but what has not been tested yet is whether it is economical in large-scale quantities. It's been studied in the context of natural gas to liquid fuel conversion as an alternative to the Fischer-Tropsch process, and the catalysts (zeolite) have been used most prevalently in the petrochemical industry. There have been facilities that use methanol as an intermediate to gasoline for natural gas, and the same process can be used for methyl halides."

In the UCFS process, the methyl halides are converted into a vapor or gas. "Since it comes off in this form, it's quite easy to collect," Voigt says. "So that's a huge advantage of this-that you are able to collect the product as fermentation is occurring and you don't have to do any distillation afterward."

Flexible and Efficient
The researchers believe the significance of their work is two-fold. First, never before have a yeast and a bacterium been combined in fermentation to produce fuel from biomass.

Second, although methyl halides have been explored as intermediates in the conversion of natural gas to gasoline, they've been completely overlooked as a potential building block for biomass.

How might this impact the ethanol and biomass industries? "A characteristic of the current industry is that if you build a corn-to-ethanol plant, corn is your only feedstock and ethanol is your only product," Voigt says. "You can't switch on a dime. We have approached the feedstock and the product issue separately. The feedstock we did was based on the bacterium, which has the ability to eat a wide range of material. By picking the right building block chemical, it can then be converted into many different chemicals or fuels like gasoline." This would allow flexibility for the industry so producers wouldn't be locked into only two commodities such as sugar and ethanol. "It would allow you to switch your feedstock and your product based on market pricing," he says. "It's something that is done in petrochemicals, but it hasn't been integrated into biorefining yet." The yeast/bacterium combination would allow a producer to utilize the current best-priced feedstock.

Voigt says his research team has successfully converted sugarcane bagasse, corn stover, switchgrass and poplar into methyl halides, all without any pretreatment. "In the case of switchgrass, we use material sent to us by the U.S. Department of Agriculture as-is-it is sort of chopped so the blades are 10 to 15 millimeters." Wood chips are dumped into a household blender to break them down before processing.

The fact that the biomass feedstock doesn't need to be pretreated indicates that the UCFS process may prove to be quite economical. On top of that, the bacterium's optimum growth occurs at 30 degrees Celsius (86 degrees Fahrenheit) so a significant energy savings may also be realized. "You don't have to heat up the bioreactor like you would with other cellulosic organisms that require very high temperatures," Voigt says.

The Future of the Pairing
So what's the next step to advance the process? "From a scientific perspective, we need to improve our conversion rates," Voigt says. "It's about 40-fold slower than sugar-to-ethanol, and we need to get to that point through engineering the yeasts. Once we do that, the fermentation aspects become economical."

Voigt says the team will work to show that the two processes-the fermentation process and the chemical catalysis-can couple to each other in a scalable way that would be required to demonstrate feasibility. The group has started a company called Biomex Inc. to do that. Biomex will help with the commercialization process as the UCFS team's work progresses.

In the meantime, Voigt says the process of successfully identifying molecules that will be most valuable as intermediates would benefit from more communication between the biotechnology and petrochemical industries. "It's an issue, because it is a molecule that the petroleum industry appreciates for its conversion into longer-chain molecules," Voigt says.

As always the common goal of all new fuel and chemical technologies is to achieve greater yields. "Although theoretical yields are sufficient for the whole process to be economical, they must still be improved," Voigt says. "It's just a matter of going in and monkeying with the pathways."

Voigt and his team wrote a paper titled "Synthesis of Methyl Halides from Biomass Using Engineered Microbes," that was published online April 20 in the Journal of the American Chemical Society.

Anna Austin is a Biomass Magazine associate editor. Reach her at aaustin@bbiinternational.com or (701) 738-4968.
 

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