How does continuously culturing microbes facilitate evolution ? In the real world, microbes have spent millennia naturally evolving to their environment. Wild microbes are supremely adapted for their native environment, which is usually subject to continuous, and often extreme, fluctuations in variables such as light intensity, temperature, pressure, moisture, etc. and the microbes must be prepared for and be able to thrive under these fluctuations. These fluctuations, and the need for microbes to carry the required genetic material to adapt to them, slows down the rate of natural evolution. After all, microbes in a fluctuating environment spend less time trying to adapt to each variable they encounter and, therefore, face decreased selective pressure to adapt to any one particular variable. On the other hand, when one or many variables are kept constant, microbes evolve more rapidly. This is the benefit of experimental evolution via continuous cultures—the environmental conditions and be strictly controlled and either kept constant or changed very slowly to facilitate the emergence of adaptive mutations. As continuous cultures are periodically, grown, diluted and re-grown, the fittest variants and fastest growers under these culture conditions take over the population. The result is a strain that has been growth optimized for your conditions of interest. When using microbes for industrial purposes, environmental conditions must often be as uniform as possible to optimize biocatalysis and it is essential for the economic viability of a particular industrial process that the microbial biocatalyst be able can optimally under these steady conditions. Thus, experimental evolution via continuous culture is an ideal way of optimizing wild microbes for the steady conditions found in industry.

What is done with spent culture ? Spent microbial cultures are simply destroyed according to the appropriate waste material regulations, with respect to each related material.

Continuous culture is not new and has been known for decades to be an ineffective way of changing the properties of microorganisms. What would make it work now ? The use of serial or continuous culture to experimentally evolve microorganisms is not a new idea. Indeed, the first example of this approach was published in 1878 and continuous culture had its heyday in the middle of the 1900’s, culminating in the invention of the chemostat by Szilard and Monod. Unfortunately, continuous culture never reached its potential and has failed to deliver effective microbes for industrial applications. Both serial cultures and chemostats are limited by technical difficulties that either slow down the rate at which experimental evolution occurs (as in serial transfer) or prevents it completely (as in chemostats). Thus, companies that wish to develop industrial strains through experimental evolution are limited to methods that are largely ineffective. Not surprisingly, interest in experimental evolution has steadily waned in the last 30 years or so. We have developed new culture technology that circumvents the traditional problems associated with continuously culturing microbes allowing for rapid and robust experimental evolution. Improving natural micro-organisms through experimental evolution is now practical and our technology has reopened a wide field of applications for green chemistry.

Will the strains you adapt revert over time ? Clearly, microbes have spent millions of years adapting to be the way they are. If selective pressure to change is removed from a microbe we have adapted for a particular set of conditions and the microbe is returned to its original environment, it will experience pressure to revert back to its original state. However, long term experimental evolution results in the accumulation of many different genetic changes and their rapid reversion is highly improbable. Thus, it will take time for our microbes to alter phenotype once they are removed from the environment to which we have adapted them. That said, it is important for us to continuously apply selective pressure on our microbes, otherwise we run the risk of undoing the good work we have done.

When you adapt a microbe to a different optimal growth temperature, does it lose its ability to grow at its initial optimal growth temperature ? Does it narrow its range of temperature ? In evolutionary biology, there is a concept called antagonistic pleiotropy which postulates that the longer a microbe spends adapting to a certain set of conditions, the less robust it will be under other conditions. So the short answer is yes, if the difference between starting and final growth temperature is large enough and if the microbe spends enough time learning to be a specialist at a particular temperature, it is possible that a microbe will lose the ability to grow at the initial temperature. We have not pushed the limits of thermal adaptation and the strains we have produced are still capable of growing at both temperatures, although we do see a drop in robustness at the initial temperature.

Is it possible to evolve consortia of microbes in a same experiment ? If the microbes in this consortium are interdependent for growth, that is to say that the growth of one is required for the growth of another, then it is fairly straightforward. Selecting for growth rate improves the growth of the rate-limiting microbe. If the microbes are not interdependent, then this is one of the most difficult configurations to work on. In this latter case, the microbes that are to be cultured together must either have compatible replication rates or we will systematically wash out the slowest growing species. Under these circumstances, it is still possible to evolve consortia, however, it is important to run the experiment in what we call "chemostat" mode where the microbes are cultured for as long as possible prior to dilution. Under these conditions, different microbes will assume different niches in the culture chamber (e.g. eating different food sources) and become adapted to the new conditions this way.

Is it possible to predict the time it takes to achieve some specific goal ? Unlike some traditional engineering, IT, or building developments, the accumulation of biological mutations is a stochastic process. There is no way to predict when an adaptive mutation will occur, nor is it possible to predict what type of adaptive mutation might occur. Evolution is generally characterized by a series of high-probability mutational events that usually have only modest adaptive effects. However, it is our experience that the adaptation process also occasionally hits "metabolic walls" where it takes a significantly longer time for an adaptive variant to sweep through the population. At these "walls" it is likely that low-probability mutagenic events are required to continue the adaptation process. It is impossible to predict where these "walls" might occur or how long it might take to break through them. Progressing in the field of experimental evolution is like exploring a wild country beyond a new frontier.

What makes your technique better than genetic engineering ? The Evolugate technology and its ability to facilitate robust experimental evolution confers several competitive advantages over genetic engineering : • First, microorganisms have spent millions of years evolving exquisitely complex metabolic pathways, making it exceedingly difficult to properly re-engineer new functionality. We simply do not know enough about some characteristics to be able to predict what types of changes need to engineered. Moreover, even high-throughput recombinant methods of genetic engineering, such as genome shuffling, have difficulty altering complex traits, which may require the acquisition of multiple low probability mutagenic events. Experimental evolution is blind to these concerns. With enough selective pressure and time, experimental evolution can easily access the genetic diversity needed to alter complex and poorly understood traits. • Second, genetic engineering requires the ability to insert or remove genetic material. For many microbes, the tools required to make this work simply do not yet exist, requiring significant investment in the development of the appropriate molecular biology methods. There is no such limitation for experimental evolution, which only requires the ability to culture cells. • Third, tinkering with one trait via genetic engineering often comes at the expense of other traits, such as growth rate and even successful examples of genetic engineering tend to produce strains that are less robust than the parent strain. To make an analogy, it is a bit like adding or deleting some lines in a piece of software when you did not write the program and are not a master of all the code. Thus, engineered microbes may be able to perform a certain industrial task, but do so too slowly to be an effective biocatalyst from an economic standpoint. Strains produced by experimental evolution are simultaneously optimized for both the desired trait as well as growth rate. • Fourth, strains produced by experimental evolution are naturally occurring genetic variants of the original parent strain—no foreign DNA has been inserted and no endogenous DNA has been deliberately removed. As such, these strains are not considered "genetically-modified organisms" (GMOs). This is an important consideration for certain environmentally sensitive projects where the resulting microbe may be released into the biosphere. This said, our technique can be highly complementary with genetic engineering.

How is your technology integrated in the industry ? Our purpose is to produce variants of industrially important microbes that can grow robustly under the conditions that prevail in industry. To achieve this result our growth conditions are designed to be as close as possible to the actual conditions a microbe would experience in the real world. As a result, we are not making microbes that work in a lab, we will be able to more easily incorporate them into a functioning biorefinery.

When you evolve microbes, does it produce GMOs ? Strains produced by experimental evolution are naturally occurring genetic variants of the original parent strain—no foreign DNA has been inserted and no endogenous DNA has been deliberately removed. As such, these strains are not considered "genetically-modified organisms" (GMOs). This is an important consideration for certain environmentally sensitive projects where the resulting microbe may be released into the biosphere.

Is your technology harmless for the environment ? Many of the microbes we develop are destined for use in confined fermentors for bioproduct production at industrial scale, thus there risk to the environment is minimal. However, when the industry requires that a particular microbial biocatalyst be released into the biosphere, there is always a risk, however small, that that microbe can have a deleterious effect on the environment. There are two ways in which our technology minimizes this risk and makes our microbes the greenest solution to this problem. First, through experimental evolution, microbes become hyper-specialists at thriving under the conditions to which we adapted them. They do so at the expense of being able to thrive when those conditions change. Thus, once they are out in the wild they are at a distinct disadvantage once the conditions change from what they were designed for. This means that the most likely fate for our microbes after they "escape" is extinction. When they have nothing left to eat they just starve and die, resulting in microbial biomass that is highly and quickly biodegradable.