Researchers at the U.S. Department of Energy Joint Genome Institute (JGI) and the Energy Biosciences Institute (EBI) have found important microbes located in cow forestomachs that could lead to the mass production of biofuels.  

Eddy Rubin, study leader and Director of the JGI, along with postdoctoral fellows Matthias Hess and Alex Sczyrba, have studied microbes in the cow’s forestomach, or the rumen, in order to understand how cell wall materials in plants are broken down into sugars, which is a vital step in producing biofuels. 

It's not uncommon to drive past acres of farmland and see cows grazing in the fields all day. This grass diet consists mainly of cellulose and hemicellulose, which are tough plant cell wall materials that do not contain any nutritional value for most animals. But cows have microbes in their rumen that are capable of turning these tough materials into small sugar molecules, which makes this fibrous diet beneficial for the cow. 

"Microbes have evolved over millions of years to efficiently degrade recalcitrant biomass," said Rubin. "Communities of these organisms can be found in diverse ecosystems, such as the rumen of cows, the guts of termites, in compost piles, as well as covering the forest floor. Microbes have solved this challenge, overcoming the plant's protective armor to secure nutrients, the rich energy source that enables them and the cow to thrive."

Knowing that rumen microbes have evolved to create enzymes capable of turning these tough plant materials into sugars, researchers were eager to learn more about them in an effort to mimic their processes for the sake of producing biofuels. The problem is that only one percent of microbial species on Earth can be grown in the laboratory and those residing in cows is not within that one percentile. Instead, Rubin and his team had to use metagenomics, which is the recovery of genetic material directly from environmental samples.  

To do this, Hess and Sczyrba inserted a tube into the rumen of a fistulated cow model. They used switchgrass, which is the "most promising" bioenergy crop, as a means for the rumen to degrade. But instead of feeding the switchgrass to the cow, they placed it directly into the rumen in nylon bags for it to be digested. After 72 hours, the bags were taken away and the microbes' DNA was then isolated and sequenced.

Researchers generated the data, which amounted to 270 billion letters of the DNA code. According to the researchers, this is 100-fold greater than the number of letters in the human genome. But this wasn't even the most difficult part of the process. 

"The real challenge was to analyze the vast amount of data for which no reference genome was available and to identify and produce full-size functional enzymes based solely on information obtained from billions and billions of short snippets of DNA sequences," said Hess.

Using computational resources, the researchers created a genome assembly strategy in order to analyze the data. Then, by using different filters, Sczyrba managed to eliminate several genes that did not match the category of genes that could degrade the plant polysaccharides into sugars. Through the process of elimination, the researchers were left with 27,755 genes that did match this ability. Out of the 27,755 genes, 90 of the most promising "candidates" were tested for functionality, and 50 percent of these had the ability to degrade cellulose while 20 percent were able to degrade the switchgrass. 

With these results, the researchers were pretty sure that most of the 27,755 genes were capable of breaking down plant cellulose and hemicellulose, which are promising results. But their work didn't end there. These researchers took the study a step further by not only assembling genes, but the entire genomes of these microbes found in the rumen. To do this, the team utilized single cell genomics as an alternative to having to culture the microbial species in the lab. Single cell genomics study's the genome of a single uncultured microbial cell by isolating it with a cell sorter and then utilizing sequencing technology. This resulted in 15 assembled genomes that were unlike any genomes configured before. Also, 98 percent of the data "matched to one single genome that had been assembled in silico." 

"The single cell data made us confident that what we saw was real," said Hess. "Otherwise we'd have computational data only, which would have made our work much, much less convincing."

This study is published in Science.