Bacteria and other microorganisms are increasingly used to produce fuel and other chemicals useful in industry. So far, companies have used either aerobic or anaerobic fermentation in producing these chemicals, but a new study shows how genetically modified bacteria can carry out both aerobic and anaerobic fermentation in the same reactor at the same time.
Companies are increasing the biological production of industrially-useful chemicals, using bacteria to make biofuels, chemicals that can replace plastics and other chemicals.
Until now, companies have used one type of bacteria at a time with either aerobic or anaerobic fermentation for production, but a new study shows how to design bacteria that can carry out both aerobic and anaerobic fermentation in a reactor at the same time.
The dream of making a consortium of bacteria with different functions in biological production has existed for a long time, and the study shows how this can be done by using a new method.
Consortia of bacteria open the way for increasing the use of bacteria in industrially producing numerous chemicals.
“There can be several reasons for using different bacteria in the same production process. For example, different bacteria can use different types of substrates to make the same product, or different bacteria can complement each other in production by using each other’s by-products to make something that is useful to industry. We found a way to have both aerobic and anaerobic fermentation in the same reactor,” explains a researcher behind the study, Sheila Ingemann Jensen, Senior Researcher, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby.
The research has been published in Nature Communications.
Using bacteria to make poly(methyl methacrylate)
The researchers used non-pathogenic coliform bacteria to produce xylitol and isobutyl acid, which can be used to make various types of plastic, including poly(methyl methacrylate), a transparent and rigid plastic.
The challenge is that bioproduction must become more efficient if producing these substances more sustainably is to become economically attractive.
The researchers therefore focused on the possibility of having both aerobic and anaerobic respiration in one reactor, so that two fermentation processes at the same time can make two substances and with the same yield, which would reduce both capital expenditure and operating costs.
Today, researchers can already meet much of this challenge but not all of it by using CRISPR-Cas9 genetic scissors.
“Natural fermentation genes can be removed, but you cannot have anaerobic fermentation and remove the fermentation genes at the same time, because then the bacteria cannot grow without replacing the fermentation pathways with something else. If you do not remove the natural fermentation genes, you will instead get by-products such as lactic acid, and bacteria will expend much of their energy on growing rather than producing useful substances,” says Sheila Ingemann Jensen.
Using CRISPR interference to silence the last remaining gene
Sheila Ingemann Jensen and colleagues cut and pasted the genetics of two coliform bacteria to get them to cooperate in a new way with the aim of obtaining more efficient production of chemicals.
They constructed a xylitol-producing Escherichia coli strain and removed two of the three respiratory genes that enable the bacteria to use oxygen to grow, so the coliform bacteria could still grow by using the remaining respiratory gene.
The researchers then further used CRISPR interference to switch off the last remaining gene by adding a substance to the liquid in which the bacteria live – making the bacteria grow to a certain volume and then turning off respiration, and then the bacteria use all their energy to make xylitol without having to use more oxygen.
The researchers also showed how to inhibit the growth of the bacteria for a longer period of time so that the bacteria do not suddenly start growing again.
“However, the challenge is that this bacterium continues to produce acetic acid as a by-product, and it needs to be removed from the liquid, but this is a poor substrate under anaerobic conditions. For that, oxygen must be used,” explains Sheila Ingemann Jensen.
Combining two bacteria with different properties
The second bacterium the researchers designed requires acetic acid to grow, and it needs oxygen to use glucose and acetic acid to grow and produce isobutyric acid.
When the first bacterium produces acetic acid (and xylitol), it creates a substrate for the second bacterium without using the oxygen that the second bacterium needs to grow and produce isobutyric acid.
Overall, this means that the two bacteria support each other’s production of the desired products without obstructing each other by using the same substrates.
Further, the researchers showed that assembling the bacteria in the same liquid produces the same yield as when they are separate.
“This is the basic idea behind what we did. We showed that we can induce a consortium of bacteria to cooperate to produce chemicals useful in industry and that this does not reduce the yield. The idea of having both aerobic and anaerobic physiology in the same reactor without biofilm is new and does not exist in nature. Finally, we also showed that we can control the growth of the bacteria. We can stop the growth without the bacteria being able to avoid this with new mutations, and we can switch growth back on to increase the number of bacteria when needed. This clears the path for examining how to produce chemicals useful in industry with bacteria in a completely new way,” concludes Sheila Ingemann Jensen.
