Clostridium thermocellum - saving the world with a bacteria that can create biofuel

  

Original Article: Papanek, B., Biswas, R., Rydzak, T., & Guss, A. (2015). Elimination of metabolic pathways to all traditionalfermentation products increases ethanol yields in Clostridium thermocellum. Metabolic Engineering, 32, 49-54.

Post By: Lissette Deleon, Simo Nkomboni, and Chelsea Terrell

It is easy to see why politicians disagree when it comes to climate change. It’s not like one side has an overwhelmingly large amount of evidence to back up its claim while the other has zilch. One environmental issue that that we can agree on is that we can’t continue to depend on foreign oil to the extent that we currently do. Instead, it is important to find other sources of fuel to run our cars, power electric plants and withhold from cold college students freezing in their dorms.

          For a long time, that option was hugely subsidized corn. Producers would grow corn and then convert it into fuel. At the time, it seemed brilliant as we can grow it in the United States. However, recently there have been many concerns surrounding the use of corn as a biofuel source. This has led scientists to try and find other sources for sustainable options for biofuel. One of these options, is using a lignocellulosic crop. This would be a good solution since it would not require the crops to be grown just for fuel, for example, “in Taiwan, rice straw is the major agricultural waste, and is suitable for use as the feedstock for the production of bioenergy”. How brilliant is that! Using actual waste to make fuel.

           However, much like everything else ever, there is a problem. We can’t just take the rice straw and stick it into your tank. The stuff that we want (fermentable sugars) is hidden. We want to liberate it so we can take advantage of it. Once the sugar finds its way out of the shell it is made to ferment and a product of fermentation is ethanol. One option to help the sugars escape from their prison is consolidated bioprocessing (CBP). This process uses an organism that degrades plant biomass, which allows us access to the sugar. A possible microbe that can serve this function, is Clostridium thermocellum(see photo). This microbe is a thermophilic, which means that it loves heat. Don’t bother trying to grow it outside as it is anaerobic and does not use oxygen as a final electron acceptor. Lastly, it is a cellulolytic bacterium. For the paper we are reviewing, the most important characteristic is the last, the fact that this microbe includes cellulosomes. Cellulosomes gives it the ability to convert lignocellulosic biomass into fermentable sugars. Once that is complete, it subsequently converts the sugar to ethanol and other products.

          There are benefits and consequences to using Clostridium thermocellum. A huge benefit, is that it can be genetically engineered. This allows us to do what we wish with the genes.  However, like everything else ever, there is a problem. This microbe also makes other products, such as lactate. What these baaad products do is make the environment toxic. This will halt growth and ultimately produce less ethanol for us to use. Since we want to get the most use out of each bit of the crop, scientists are trying to figure out a way to keep the microbe from producing these toxic products so ethanol can continue to be made.

A way to do this, is to eliminate the pathways that make these toxic products. That is where Papanek et al. comes in. The researchers are trying to make it so those pathways are no longer an option so more ethanol must be produced instead. There have been studies that have tried to address this in the past, but toxic products were still produced, just a smaller amount. This study uses a new idea to try and eliminate that problem. Instead of manipulating one gene they decide to combine gene deletions and hopefully eliminate the uncooperative pathways. Doing this would generate the wanted results aka: more ethanol.

Papanek et al. attempted to force the microbe to produce more ethanol by making all of the following gene deletions: ΔhydG, Δpfl, Δpta-ack, and Δldh. The strain with the deletions was called C. thermocellum AG533.To create this strain, they grew E.coli and C. thermocellum cells in appropriate media. Plasmid DNA was isolated from Escherichia coli cells and electroporated into C. thermocellum, with genes of interest deleted using standard methods. The C. thermocellum cells were fermented in a Coy anaerobic chamber and fermentation products (lactate, acetate, formate and ethanol) were measured by High Performance Liquid Chromatography. Hydrogen production was measured using Gas Chromatography. A microplate reader was used to collect growth curve data at OD600 inside the Coy anaerobic chamber.

Each of the ΔhydG, Δpfl, Δpta-ack, and Δldh gene deletions resulted in elimination of pathways that lead to the production of hydrogen, formate, acetate and lactate respectively. The last three are organic acid byproducts that biofuel producers do not want to produce with ethanol, therefore eliminating them is ideal. The AG553 strain produced two-fold more ethanol than the wild type strain when grown in defined medium with 5g/L of cellobiose; shown by the red bar on the figure below with wild-type on the left and strain AG553 on the right on both graphs.

The bacterium produced three times more ethanol than the wild type on model crystalline cellulose Avicel PH105, a renewable and inexpensive model substrate. With the crystalline cellulose Avicel PH105, the wild type strain produced equal amounts of acetate and ethanol.

Dilute acid, pretreated poplar and switchgrass biomass were used to test the AG553 strain’s ability to convert complex plant biomass to ethanol. Control fermenters were set up with an equal amount of crystalline cellulose as in the pretreated poplar and switchgrass biomass for direct comparison. Strain AG553 produced 65.5% and 62.6% of the theoretical yield of glucan to ethanol with Avicel and pretreated poplar biomass respectively. In comparison, wild type had a theoretical yield of 34.2% on Avicel and 35.3% on poplar. In conclusion, strain AG553 produced more ethanol than wild type strain on Avicel and switchgrass. Papanek et al. hypothesized that the lack of organic acid production will allow for higher titers ethanol production and indeed, they observed an increase in ethanol titer in C. thermocellum AG553 using higher cellulose loadings of up to 50g/L in serum bottles with defined medium.

No other organism has been shown to produce such high levels of ethanol in the past together  with decreased medium acidification. C. thermocellum AG553 produced less hydrogen due to the deletion of the gene that codes for FeFe hydrogenase, maturase hydG. The strain also had similar level of yield for model substrates such as Avicel, showing that it is not inhibited by complexities of real-world biomass. All these findings by Papanek et al. show that C. thermocellum AG533 is a good candidate organism for genetic engineering and process optimization for consolidated bioprocessing of lignocellulose to fuels and chemicals.

This study opens the door of manipulating these bacteria for producing large amount of these biofuels within the same amount of time as the wild type. The paper discusses that the deletion of a gene is what causes this bacteria to this, but the authors do not include the disadvantages that can be presented by doing this, as well as including the consequences that can arise by doing this. For example, it might have deleted the pathway that produces ATP. Considering the climate changes occurring, this is a great opportunity to use these bacteria to produce alternative fossil fuels. Even though it is well on its way, to actually find a way to produce these biofuels in mass amounts without having negative consequences will be useful in keeping the planet healthy. Although showing great promise, there are some technical kinks that still need to be worked through. These include low volumetric production rates as well as these organisms having low product tolerance.  Once these problems are worked out then there will be successful commercialization of cellulosic hydrogen production. In other words, once these issues are resolved, these bacteria can be used for good, and stop the dependence on fossil fuels. This will present another option of biofuels to slow down climate change.