An experimental evolution-inspired step towards saving our planet

Do you every look up into the night sky of a big city in the hope to see stars, only to be disappointed by bleak smoggy darkness? Fossil fuel emissions are not pretty, and they certainly are not good for our environment. Annual Carbon Dioxide (CO2) emissions from fossil fuels have increased dramatically in the past decade and continue to rise rapidly. This has obvious negative health and environmental impacts such as accelerating global warming and deforestation.

Image URL: https://www.cbsnews.com/pictures/top-10-smoggiest-cities-in-us/

Therefore, we need to find alternative, more sustainable sources of energy so that we can reduce our carbon footprint for future generations. Of such, bioethanol is a renewable biofuel energy source that could meet our requirements as well as being environmentally sustainable [4]. Bioethanol is currently the most highly produced biofuel, meaning demand is high [4]. The process of bioethanol generation, called second generation bioethanol production, involves utilising cellulose and hemicellulose (plant biomass); the structural components of plants, into a renewable energy source called bioethanol [1].

So, you may be wondering why bioethanol, derived from plants, is so much better than fossil fuels? Well, in addition to being renewable and highly abundant, bioethanol has much lower CO2 emissions in comparison to fossil fuels. Also, the little amount of carbon dioxide that is released into the air by bioethanol use can be absorbed and utilised by the plants themselves, so that we can grow more plants and generate more biofuel. Hence the word “renewable”, It is an endless cycle of give and take, which is better for both us, our planet, and our environment.

The general process of second-generation bioethanol production involves pre-treating the plant biomass to weaken their strong structure provided by cellulose. This is followed by hydrolysis (breaking apart) of these cellulose structures into individual sugars [2]. Next, these sugars are fermented into bioethanol, which is assisted by a fermentable agent or microbe such as the commonly used yeast (S. cerevisiae) [2]. However, a problem arises when the hydrolysis step exceedingly releases acetic acid as a by-product. Yeast do not like acetic acid, it stops them from growing and being able to ferment ☹ [1]. In the big picture, this is bad because it means that bioethanol production yield will be reduced!

Expanding on this problem, experimental evolution has been an attractive approach for researchers to improve the efficacy of second-generation bioethanol production. Experimental evolution means that scientists can manipulate an environment experimentally and explore how this affects the evolution of a certain species [3]. In saying this, what if we could utilise experimental evolution to improve the efficacy of second-generation bioethanol production? A study that I will discuss in this blog post did just this, and the results were impressive!

González‑Ramos et al. (2016) used an experimental evolution approach to modify yeast to evolve a lasting tolerance to acetic acid, meaning these yeasts could thrive and survive in higher concentrations of acetic acid. This would allow the fermentation step of bioethanol production to be much more efficient and also improve the outcome of bioethanol production.

As a starting point, the researchers first tested whether prior exposure to acetic acid helps yeast to acquire tolerance. They did this by growing yeast either in the presence or absence of acetic acid (of a tolerable concentration), and then testing how the pre-adapted yeast cells grow, in comparison to non-adapted yeast cells, in acetic acid. To quantify these results, the researchers measured Final OD600 as an indicator of yeast biomass yield and were also able to determine specific growth rates (see Figure 1). So, as expected, both pre-adapted and non-adapted yeast cells were not very tolerant at all, as shown in Figure 1. There was a significant negative correlation between acetic acid concentration and growth of both pre-adapted and non-adapted yeasts. In saying this, the “pre-adapted” yeasts were slightly more resistant in concentrations greater than 10g/L, where they sustained slightly more survival (see Figure 1). Also, this tolerance of pre-adapted yeasts was lost after one exposure to non-stressed (low acetic acid concentration) conditions. Considering that the concentration of acetic acid can be more than 10g/L during bioethanol production, we want them to thrive in higher concentrations than this, and in a lasting manner, so this yeast has a fair way to go yet!

Figure 1: Growth and survival of either pre-adapted (grey lines) or non-adapted (black line) yeasts in gradually increasing acetic acid concentrations.

So here is where experimental evolution comes in…the researchers wanted to find a way to make these yeast able to thrive in and tolerate higher concentrations of acetic acid. To combat this, yeast cells were UV mutagenized (irradiating the cells with UV light to introduce mutations) and grown being transferred between conditions alternating from the absence and presence of acetic acid (of gradually increasing concentration); a technique called serial microaerobic transfer. Just like we train at the gym to build fitness, the yeast cells were training in the lab to build acetic acid tolerance. Over time, and approximately 50 transfers back and forth, these yeast cells were finally able to thrive in the higher acetic acid concentrations! They could even survive in 18g/L acetic acid, nearly twice the initial concentration that most yeasts could not survive in (Figure 2). Check out Figure 2 to see an example of the progress of one of these experimentally evolved yeasts with improved tolerance to acetic acid.

Figure 2: Acquired acetic acid tolerance of experimentally evolved yeast over serial microaerobic batch transfer. Acetic acid concentration is indicated by the grey line. Black dots are final OD600 measurements in the absence of acetic acid, while grey squares are final OD600 measurements in the presence of acetic acid.

 So, they experimentally evolved this yeast to be able to survive and grow in higher acetic acid concentrations, which is pretty cool (for a science nerd like me, anyways…), but the questions remain; what is going on? How is this happening? To approach this, researchers turned to genetics in the hopes to answer their questions. The research group used fancy scientific techniques allowing them to get a big archive of all the genes in these tolerant yeasts and compared these to the original non-tolerant yeast. This helped them to pinpoint a few culprits, mutated genes, for this acquired tolerance to acetic acid. They observed that many of these tolerant yeasts contained similar specific mutated genes (or, in ‘sciency’ terms; single nucleotide polymorphisms).

But what if this was just a crazy coincidence, and these mutated genes actually had nothing at all to do with this acquired acetic acid tolerance? To prove that this was not just a coincidence, and that these specific mutated genes were causal of this tolerance to acetic acid, the researchers carried out more fancy scientific techniques. They introduced each of these special mutated genes, individually, back into the non-tolerant yeast and… voila, these non-tolerant yeasts became tolerant to higher acetic acid concentrations! What is also cool is that the more of these mutated genes that were introduced back into the yeast, the more tolerant they became to high acetic acid concentrations (see Figure 3).

Figure 3: Growth of yeast with different variations of the identified mutant genes for acquired acetic acid tolerance.

In summary, using an experimental evolution approach, González‑Ramos et al. (2016) was able to genetically engineer yeast cells there were able to constitutively survive higher concentrations of acetic acid, which normally impairs yeast productivity and stops yeast from growing. This has beneficial implications in improving the efficacy of second-generation bioethanol production.

I found this study interesting because of the potential applications that it has. This study struck out to me as it shed some light on the clever way that we can apply scientific techniques like experimental evolution to biotechnology for the benefit of our environment and our future. I hope that you found this study as interesting as I did. Hopefully with more applicable studies like this, our cities will look less like *first picture* and more like *bottom picture*.

Image URL: https://www.azocleantech.com/article.aspx?ArticleID=618

Thanks for reading,

Bianca

Here are the papers I referred to:

  1. González-Ramos, D., Gorter de Vries, A. R., Grijseels, S. S., van Berkum, M. C., Swinnen, S., van den Broek, M., . . . van Maris, A. J. A. (2016). A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations. Biotechnology for Biofuels, 9(1), 173. 10.1186/s13068-016-0583-1
  2. Rastogi, M., Shrivastava, S. J. R., & Reviews, S. E. (2017). Recent advances in second generation bioethanol production: an insight to pretreatment, saccharification and fermentation processes. 80, 330-340.
  3. Kawecki, T. J., Lenski, R. E., Ebert, D., Hollis, B., Olivieri, I., Whitlock, M. C. J. T. i. e., & evolution. (2012). Experimental evolution. 27(10), 547-560.
  4. Branco, R. H., Serafim, L. S., & Xavier, A. M. J. F. (2019). Second generation bioethanol production: on the use of pulp and paper industry wastes as feedstock. 5(1), 4.
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