Biomass-based Feeding

The use case

Fed-batch processes are often advantageous in comparison to batch cultures, because they avoid a high initial substrate concentration, which imposes osmotic stress and can lead to a so-called overflow metabolism (cells quickly metabolizing a bulk of substrate tend to produce by-products which in turn can acidify the medium and negatively influence cell vitality). A controlled, reoccurring supply of substrate, on the other hand, offers just the right amount of substrate to the cell, minimizing the maximal growth rate but potentially increasing overall biomass production.

While commonly used in larger scale fermentations in bioreactors, fed-batch fermentations in shake flasks are rare due to the lack of technology. When designing experiments for e.g., protein production, it is essential to test different conditions or production strains in smaller (shake flask) scales, using similar feeding profiles as in the final (larger scale) experiments. 

Why biomass-based feeding for fed-batch cultures in shake flasks?

Starting a fed-batch process after a certain time requires good knowledge of the process. Have the cells used up most of the substrate after the given time already? Did the biomass increase sufficiently so that the added substrate would not cause osmotic or metabolic stress to the cells? These questions might especially be hard to answer in early shake flask-scale tests that mainly aim to obtain information. Letting the biomass threshold dictate the start of the feeding process makes sure the right time point is chosen without having to know the exact growth curve progression of the respective organism in the respective conditions.

Additionally, biomass-induced fed-batch cultivations of precultures can improve their quality by making sure they are in the same metabolic state when being transferred to the main culture. Read our blog post on this topic to learn more.

The use case

Promotors – the DNA sequences upstream of genes that initiate the genes’ expression – play a huge role in many bioprocesses. When producing recombinant proteins in microbial cells, inducible promotors are often used which allow the cells to maintain in an “expression-off mode” during cell growth, minimizing the additional metabolic burden. This is even more valuable if the expressed protein has toxic effects on the cell. With non-inducible promotors, the selection pressure is in favor of non-expressing mutant cells which would lead to lower overall protein levels. Substances that activate gene expression are called inducers and are small molecules. Depending on the promotor, sugars like IPTG (isopropylthio-β-galactoside), allolactose, arabinose, or alcohols like methanol can serve as inducers. By adjusting the inducer concentration, expression levels can furthermore be controlled. The right inducer concentration or induction time point for individual bioprocesses can be determined via induction screenings – a setup of experiments in which different induction conditions are tested.

However, inducing at the right time point is usually very time-consuming and necesitates intensive sampling and testing. And still, with unexact data points, the ideal induction time point is often missed.

Why biomass-based feeding for promotor induction?

Choosing the right time point for promotor induction is crucial, especially in processes that aim to produce high yields of recombinant proteins. The difficulty here is that the perfect time point is dependent on the cell growth – a parameter that is hard to reproduce and will therefore differ among cultures, even if the same conditions are tried to be maintained. Have the cell growth automatically define the start of induction is therefore a convenient solution for this challenge. Also, when applied in small vessels like shake flasks, numerous different starting points (at different cell growth levels) could be tested in induction screenings to figure out which is the most favorable one for the specific bioprocess.

The use case

Quite frequently, certain compounds in the medium or metabolic products can have toxic effects on microbial cells. It is often desirable to quantify the toxicity of specific compounds to e.g., estimate effects on the cell’s metabolism, draw conclusions about the best media compositions, or calculate the highest possible yields of a specific product that becomes toxic at certain concentrations. Therefore, tests determining the effect of different compound concentrations on cell growth are a common method applied in laboratories.

Why biomass-based feeding for toxicity tests?

Usually, the medium with different concentrations of the respective compound is provided in shake flasks and cell growth is tightly monitored. Naturally, many toxic compounds would prolong the initial lag phase in which no cell growth is visible. However, in many cases the situation might be more complicated and necessitate advanced analysis. If the compound in question has an effect, for example, on proteins that are expressed from genes on plasmids leading to a lower growth rate, the cell might react to this by enhancing the expression level of plasmids to counteract the effect. In these cases, it is beneficial to let the cells grow under the same (media) conditions until a certain growth rate is reached and start feeding the substance only then, observing the immediate substance-induced drop of growth rate. This can offer more in-depth insights into the metabolic effects since effects on protein expression would not be visible so quickly.

The use case

In the complex world of bioprocesses there is always room for improvements, even in the first steps, like the inoculation of the medium with cells. It is possible to optimize the inoculum by preparing the cells to the bioprocess conditions, that is: The temperature, shaking speed and most of all, the media compositions. Without the addition of carbon source (yet), cell growth in this preconditioning phase is still prohibited. This slow adaptation to the media compositions with LIS as an automated feeding technique and a preprogrammed shaker has been shown to reduce the lag phase and introduce an additional point of control to your bioprocess, while reducing manual work. The adjusted metabolic state of cells improves the quality of precultures and enhances reproducibility of main cultures. LIS can also be programmed to start, e.g., on weekends, using times that were previously not used effectively, and providing freshly grown cultures on Monday mornings. Learn more about this application by downloading the application note here. 

Why biomass-based feeding for inoculation of cultures?

Besides the slow adaptation of cells to the medium components by keeping them in the LIS reservoir, automated feeding can also be used for co-cultivations. Two types of cells are sometimes cultivated in the same medium for diverse purposes, e.g., for an improved availability of carbon sources or enzymes. In many cases, the growth behaviour of the two cell types are not identical and it might be useful to start cultivating one (the slower growing) cell type first and only add the second one, when a predefined biomass of the first cell type has been reached.

The use case

There are many reasons why researchers would want to inhibit specific proteins or enzymes. An enzyme might degrade the desired product, for example, or the experiment aims to determine the specific metabolic situation without a certain enzyme activity. Testing substrates for enzyme inhibiting effects can also serve as pre-clinical drug test and help to understand the detailed mode of action of inhibitors. Different forms of inhibition exist. An inhibitor can bind to the substrate binding site, also called active site, and thereby be in competition with the actual substrate. Or it can bind to a so-called allosteric site, apart from the substrate cavity and change its overall structure, slowing down or eliminating enzyme activity.

Why biomass-based feeding for enzyme inhibition?

Microbial cultures undergo different growth phases that can be clearly distinguished when looking at the biomass concentrations or the resulting growth curves. What is not visible at first sight is that changes do not only occur on a cell growth level but also inside cells. The cells’ metabolic profiles shift in the different phases. Certain enzymes are more or only expressed in certain phases. Connecting the addition of inhibitor with a certain growth rate can therefore be advisable.

The use case

Different organisms and strains will show different growth behaviors and some growth patterns are typical for certain organism groups. A typical growth pattern of yeast is the diauxic shift or diphasic growth form. When plenty of glucose is present in the medium, the yeast cells will use it as its preferred carbon source, resulting not only in rapid growth but also in the formation of ethanol. Once glucose becomes limiting, the cells will shift their substrate use from glucose to ethanol. This shift is characterized by a lag phase (in which the cells initiate the expression of enzymes needed for the metabolism of ethanol). It is followed by a slower growth in which ethanol is aerobically respired. Depending on the industrial purpose, this growth pattern can be accepted or even wanted. However, in certain cases, it is beneficial to circumvent this metabolic shift, e.g., if the desired product is ethanol.

Why biomass-based feeding for the alteration of growth behavior?

The metabolic shift of yeast cells or other cell specific growth patterns can be manipulated by feeding substrate at the critical time points. Providing more glucose by feeding once a reduced growth rate is detected with yeast cells could, for example, prevent the usage of ethanol and increase overall biomass and ethanol titers. The shift of metabolism has several other cellular effects that might influence bioprocesses, making this application an asset for many purposes.