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The ever important role of thermophiles in biofuel production

Laura Edwards NCSU Biochemistry graduate student

Laura EdwardsI'm on ScienceSeeker-Microscope

Well, it’s no secret that there are some major issues with our current dependency on fossil fuels. First of all, they don’t last forever, so at some point we’re going to run out (don’t worry, not any time soon). Second of all, when they are burned they emit greenhouse gases that are bad for the environment… not to mention the environmental damage done trying to get fossil fuels out of the ground. Lastly, the cost of gas for the American consumer has dramatically increased in the past years (it’s definitely hurting my wallet). These and other factors have prompted the research and development of alternative, renewable fuel sources such as biofuel (using organic matter for energy). Plants are a great source of energy, and the concept of using that energy for biofuel production has been around for decades. So why can’t we just stuff a stalk of corn in our gas tanks and call it day? Well, several reasons… plants are really good at converting solar energy and storing it as cell wall polymers, but the challenge comes from extracting out that energy and converting it into usable fuel, such as ethanol.

Biofuel Production

Biofuel Production

In the general biofuel process, solar energy is collected by plants via photosynthesis and is stored as polymers in the cell wall (includes cellulose, hemicellulose and lignin). Break-down of these cell wall polymers into simple sugars is achieved by pre-treatment and exposure to enzymes from microorganisms. These microorganisms are a key component to all of this and are required to withstand harsh pre-treatment conditions, including high temperatures. Next, the simple sugars are fermented by S. cerevisiae or other microorganisms that can convert sugars to ethanol. S. cerevisiae is not able to withstand ethanol concentrations over 25%; therefore, a distillation step must be performed.

As you may have gathered, this is a pretty labor intensive process, but it can be done. In fact, most all of the gas sold in the United States is blended with 10% ethanol, though at the moment the workflow and cost of this process is not efficient enough to increase ethanol use, so I’ll go over some of the issues this industry is facing.  Currently the main source of biomass (that is, plant materials) used in biofuel production is corn, which is not ideal since it is a consumer crop, and using it for food and biofuel could increase its price and limit its availability. The ideal biomass is one that grows quickly, takes up a small area and is easy for farmers to maintain, such as switchgrass. The problem with switchgrass, however, is that its cell wall contains a lot of lignin which is very difficult to break down, raising the importance of finding an optimal microorganism that can efficiently degrade cell wall components.

To address these concerns related to efficiency and cost, researchers have recently developed a process to minimize steps and improve the overall biofuel production workflow. Consolidated bioprocessing (CBP) integrates enzyme production, saccharification and fermentation into one step. The most important aspect of this process is finding/engineering a microorganism to do all of those things. Bob Kelly’s group here at NC State University, as well as several other groups, is working with members of the genus Caldicellulosiruptor. This genus of gram positive bacteria is extremely thermophilic and can withstand the harsh pre-treatment conditions necessary for biofuel production. A couple of other great features are that most members can efficiently chew up cell wall components, and can produce ethanol.

One of the most recent inductees into the Caldicellulosiruptor genus, C. obsenisis, was isolated from Obsidian Pool in the Yellowstone National Park in 2010. It exhibits characteristics similar to other members of Caldicellulosiruptor.

Obsidian Pool

Obsidian Pool, www.nps.gov)

Its optimal temperature range was found to be between 55-85 C, its pH range is 5-8, and its ethanol tolerance is 1%. To test how well it can degrade cell wall components, the bacterium was grown on avicel, switchgrass, filter paper and other model substrates. C. obsidansis was able to grow relatively fast on these media and could produce lactate, acetate and small amounts of ethanol. After its discovery, C. obsidansis has been studied along with the rest of the known members of this genus, to determine what makes them good at what they do.

In 2012, the Kelly Lab conducted a comparative analysis between several members of Caldicellulosiruptor to determine the origin of its cellulolytic (ability to hydrolyze cellulose) capabilities. First, the genomes of 8 Caldicellulosiruptor species were compared and a phylogenetic tree was made; however, this metric shows no correlation with phenotypes (especially cellulolytic capability) within the genus.

Phylogenic tree of the genus Caldicellulosiruptor

Phylogenic tree of the genus Caldicellulosiruptor

Next, the level of cellulolytic activity of each species was determined by growing them on different types of biomass. This experiment allowed the researchers to categorize the species into groups of weakly and strongly (Cbes, Calkro, Csac, COB47 (mentioned above, C. obsidiansis)) cellulolytic activity. The core genome, meaning genes that are common to all species, was determined in order to identify clusters of genes. Functional characterization indicated that translation and amino acid transport, and carbohydrate metabolism and transport families are enriched in the core genome (which is not surprising). By focusing on the carbohydrate transporter genes within the pangenome (which includes the core, genes present in two or more species and genes unique to single species), it was evident that a species’ glyociside hydrolase (GH) inventory reflects the capacity for cellulolytic activity. It was found that a specific GH (GH48) domain with a carbohydrate binding molecule (CBM3) module was the absolute determinant for cellulose hydrolysis; however, the sole presence of a GH48 domain is not enough to promote a strong cellulolytic phenotype. Additional research was done using LC/MS/MS to examine other determinants that might exist beyond the GH family containing enzymes. It was found that two type IV pilus-associated adhesins were enriched in the strong cellulolytic species.  These results have sparked new research in determining other features that make certain Caldicellulosiruptor species strongly cellulolytic, which could ultimately help engineer a CBP microorganism.

In conclusion, not only did writing this blog increase my knowledge of biofuel research being done here at NCSU and elsewhere, but it also made me want to buy an ethanol-powered car and take a trip to Yellowstone National Park. But in all seriousness, the research in biofuel production is very exciting and it’s cool to think that it could have a major impact on our day to day lives hopefully in the near future.

References:

1. Rubin E.M. (2008). Genomics of cellulosic biofuels, Nature, 454 (7206) 841-845. DOI:

2. Blumer-Schuette S.E., Kataeva I., Westpheling J., Adams M.W. & Kelly R.M. (2008). Extremely thermophilic microorganisms for biomass conversion: status and prospects, Current Opinion in Biotechnology, 19 (3) 210-217. DOI:

3. Hamilton-Brehm S.D., Mosher J.J., Vishnivetskaya T., Podar M., Carroll S., Allman S., Phelps T.J., Keller M. & Elkins J.G. (2010). Caldicellulosiruptor obsidiansis sp. nov., an Anaerobic, Extremely Thermophilic, Cellulolytic Bacterium Isolated from Obsidian Pool, Yellowstone National Park, Applied and Environmental Microbiology, 76 (4) 1014-1020. DOI:

4. Blumer-Schuette S.E., Giannone R.J., Zurawski J.V., Ozdemir I., Ma Q., Yin Y., Xu Y., Kataeva I., Poole F.L. & Adams M.W.W. & (2012). Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass, Journal of Bacteriology, 194 (15) 4015-4028. DOI: