Robust growth at non-optimal temperatures, pH or aeration rates that are often encountered in real-world fermentations
Very often microbial cellular factories work beautifully under the optimal conditions in the lab, however, more often than not, these microbes never make it out of the lab and into production. This is because the conditions in the lab are usually ideal in terms of temperature, pressure, pH control and aeration rates. Yet, on the industrial scale, it can often be impossible to control these factors from batch to batch, or even spatially within a single batch due to the large size of industrial fermenters. BioTork’s evolutionary optimization technology is an effective tool for adapting cellular factories to the unique conditions associated with industrial fermentations. This technology allows microbes to perform at suboptimal temperatures and pH values as well as to tolerate more or less aeration as the situation requires. The result is a seamless and rapid scale up from the lab scale to the industrial scale.
Higher tolerance to product inhibition or to toxic compounds
Nearly everything can be toxic when it accumulates to high-enough levels. This is one reason why cellular factories often stop producing the desired end product when it accumulates above a certain threshold. The product ends up stopping its own production, either by inhibiting the enzymes that make it or by killing the cellular factory outright. Another factor is that the diversion of nutrients away from essential cellular functions towards the generation of the desired product can wreak havoc on a cells ability to perform. In addition, many cells also drastically change their environment as they grow by producing metabolites such as acids, bases or alcohols and some even produce antibiotics or other secondary metabolites with growth inhibitory properties. These “off-target” metabolites can also inhibit performance. Finally, inhibitory compounds can also be introduced into industrial fermentation via the feedstock used to grow the cellular factory. The use of highly purified substrates, like refined sugar or glycerol, is often too expensive, causing producers to switch to less pure “real-world” feedstocks. In many cases, the impurities in these cheaper feedstocks reduce the performance of the microbes. BioTork’s evolutionary optimization technology has been effectively used to adapt a variety of cellular factories to an array of toxic compounds. The resulting microbes can perform equally well in the presence or absence of toxins allowing producers to make more of a toxic end product, to make more of a desired end product in the presence of inhibitory compounds and to reduce the cost of goods by switching to cheaper impure feedstocks.
Higher growth rate, metabolic flux and productivity
Intimately linked to the concept of titer is the notion of productivity, which is a measure of how much product a microbe makes per volume per unit time. There are two fundamental contributors to productivity and they are growth rate and metabolic flux. Obviously, it is the cellular factory and the enzymes contained therein that make the desired end product. Thus, the faster the cellular factory grows and makes the required enzymes the faster it will make product. Furthermore, the flux of carbon source from feedstock to product can be improved in such a way that productivity can be increased without increasing growth rate. The functional implications of improved productivity are profound. A 50% improvement in productivity means that a biorefinery can make 2 times more product without installing any new capital equipment. BioTork’s evolutionary optimization technology is ideal for improving growth rates and metabolic flux, thereby improving productivity.
Titer is a term for how much product a cellular factory makes per fermentation volume. Titer is affected by a number of factors, including product inhibition, biomass yield of the cellular factory, optimal growth conditions and nutrient bioavailability. For obvious reasons, increasing titer is one of the quickest ways to increase the efficiency of a process. The use of evolutionary optimization to improve titer is ideal, particularly when the synthesis of the desired end product is tied to growth rate in some manner. Often, however, when the central factor limiting product titer is not the product itself, simply improving the robustness of the microorganism under industrial conditions can significantly improve titer. In the case where the product limits its own production through some type of inhibition or toxicity, evolutionary adaptation can be effectively used to alleviate this inhibition and increase titer. Even small increases in titer can have profound impacts on an industrial process, particularly in industries where large volumes of product are made annually.
Higher culture densities
Cellular factories are essentially agglomerations of enzymatic catalysts that convert substrate into product. In theory, increasing the production of cellular biomass increases the amount of biocatalyst. And when the biocatalyst has a finite lifetime, as is often the case with enzymes under industrial conditions, it is critical to make as much catalyst (biomass) as possible so to increase titer and productivity. In other situations, the cellular biomass is itself the desired end product, meaning increased culture densities, by definition, increase product. There is another consideration as well, often industrial processes are limited by the presence of toxins, whose toxicity is a function of how much toxin is present per cell biomass. Often, toxins are less growth inhibitory in dense cultures due mass action effects. Thus, the ability to produce dense cultures can positively affect things like production in metabolite inhibition. BioTork’s evolutionary optimization technology is ideally suited for the selection of increased cell density.
Greater product yield
Product yield, also known as stoichiometric yield, is a measure of the efficiency of conversion of feedstock to product. A 50% conversion means that for every metric ton of feedstock used, 500 kg of product are produced. For every product and feedstock there is a theoretical maximum yield. For example, for the conversion of sugar to ethanol the theoretical maximum yield is 51%, however, most industrial ethanol producers get a yield of 45-48%. This is due to inefficiencies in the process and the production of off-target products like biomass and other metabolites. The increase in flux to the desired end product will increase product yield and, subsequently, titer and productivity. Evolutionary optimization is an excellent way to improve stoichiometric yield, especially when product synthesis is tied to growth.
Utilization of non-optimal carbon or nutrient sources
Many microbes that are being investigated for industrial purposes perform well in the lab under conditions of optimal nutrition, yet fail to work upon scale up to industrial conditions. Moreover, they often work well on defined media that contain some nutrient sources but not on others. For example, many microbes grow well with a rich nitrogen source like yeast extract, but not with something like urea or ammonium sulfate. This “pickiness” with respect to nutrient sources can drastically increase the cost of production by requiring expensive inputs. The use of evolutionary optimization has been routinely used by BioTork to adapt microbes to perform as well on suboptimal nutrient sources as they do on the optimal sources, thereby reducing the cost of goods.