The bacteria tester: the growth and productivity of individual cells can be examined with the microfluidic chip developed by Dietrich Kohlheyer.
They are important helpers in the production of fuel and medicines: bacteria. When cultivated in bioreactors, however, some cells work harder than others. Microfluidic chips with tiny channels and chambers can help make such differences visible.
Whether in brewing beer, making cheese or fermenting cabbage, humans have taken advantage of the work of tiny microorganisms for thousands of years. Today, they even do so in big factories: bacteria, yeasts and other fungi do important jobs in industrial biotechnology. The small helpers produce vitamins, protein building blocks, fuels, detergents and pharmaceutical agents such as insulin or antibiotics.
“For this, the microorganisms are cultivated in bioreactors,” explains Prof. Dietrich Kohlheyer from the Institute of Bio- and Geosciences’ Biotechnology section (IBG-1). “These are huge vessels holding several cubic meters of nutrient solution.” However, the experts do not always succeed in transferring a new process from the laboratory to industrial scale. While the capabilities of the microorganisms are initially tested and optimised on a small scale in the laboratory, the fungi and bacteria sometimes behave quite differently in the large bioreactor than in the small volume of the Erlenmeyer flask. In such cases, it can happen that the yield of the target substance falls short of expectations.
Picture above: The bacteria tester: the growth and productivity of individual cells can be examined with the microfluidic chip developed by Dietrich Kohlheyer.
Mechatronic engineer Dietrich Kohlheyer heads the Helmholtz Young Investigators Group “Microscale Bioengineering”.
Dietrich Kohlheyer aims at finding out the reasons for this in order to improve biotechnological processes and thus increase the yield. It is becoming more and more widely accepted that microorganisms do not necessarily behave as uniformly as textbooks describe them, he says. For example, his research group looks at bacteria that are genetically completely identical. “One could assume that they would then work similarly well, but that’s not the case. There are excellent and rather bad producers among them,” says Kohlheyer. “Moreover, if the bad producers grow better, the efficiency of the bioreactor will drop.”
Furthermore, in a large reaction vessel, these tiny little guys do not encounter the same conditions everywhere. Even if the liquid inside is constantly mixed thoroughly, zones can form that differ from each other – be it in nutrient content, pH value or the availability of oxygen. Some of the microorganisms react to these local fluctuations by reducing their metabolism, thus producing less of the desired substance.
“We want to observe as precisely as possible how the cells behave under different conditions,” explains Dietrich Kohlheyer. “When do they thrive particularly well? What happens when you put them under stress?” For this purpose, the mechatronic engineer has developed specific components with which he can take a close look at individual cells: so-called microfluidic chips. They are about the size of a thumbnail and consist of a transparent piece of silicone rubber. Inside them are four extremely narrow channels. To the left and right of each channel, innumerable small chambers open up that are just large enough for the microorganisms to fit in. They are typically only a few micrometers in size, that is, about twenty times smaller than the diameter of a human hair.
Do microbiologists have to rewrite their textbooks? A visit with Dietrich Kohlheyer.
The microorganisms to be investigated can be flushed into the chips through the channels. Ideally, no more than one single cell enters a chamber. It won’t be alone there for long, however, as bacteria tend to multiply. Soon a small colony of cells will develop. A microscope scans the chambers at regular intervals and takes pictures of the colonies. From these, a time-lapse film of bacterial growth can be assembled.
The team from Jülich was able to show that the cells of soil bacteria of the Streptomyces genus reproduce in a reproducible and stable way if the nutrient supply in the tiny chambers of the chip does not change too much. These microorganisms are important sources and producers of antibiotics and proteins. However, since the filamentous cells grow into complex networks, biotechnologists consider them difficult to cultivate. Kohlheyer: “Our observations show that it is crucial for this industrially important bacterial species to adjust the conditions in a bioreactor well in order to control the complex shape of the cell networks.”
The scientists are not only able to monitor the growth of bacteria with the chips, however, but also the metabolism. Microorganisms of the Corynebacterium genus produce glutamic acid and other protein building blocks in bioreactors. In order to identify the good producers, the researchers modified their genetic material in such a way that the bacteria produce a fluorescent dye when they activate certain metabolic processes. This way, it is easy to see under ultraviolet light which bacteria work well in the colony and which do not – and how the pattern changes when, for example, the supply of nutrients changes. Dietrich Kohlheyer: “We aim to identify strains that are particularly robust and that provide maximum yield even under fluctuating conditions.”
The microfluidic chips are produced by the Biotechnology section’s team itself. The plans for the complex architecture are created on the computer. On the basis of this data, a mould made of silicon is then produced – a three-dimensional negative of the microscopically small channels and chambers. “This is done here at Jülich in a clean room, the Helmholtz Nanoelectronic Facility. We use technologies such as those employed in semiconductor technology to manufacture silicon chips for computers,” says Prof. Dietrich Kohlheyer. These moulds are filled with a viscous silicone compound, which is then cured by heating to form a rubber-like material. A thin glass pane covers the system of channels and chambers.
Initially, companies were sceptical as to whether the observations from the small cell clusters in the microfluidic chips could be transferred to the large reactors in industry – after all, the volume of a reactor is about one quadrillion times larger. Meanwhile, however, the interest of the companies’ biotechnologists has been aroused: “We were very quickly able to present results that show how stable a certain bacterial species grows under different conditions. Let it be understood that this is still basic research. The next step is to apply our findings to industry in order to improve bacterial strains and processes,” says Kohlheyer.
Photos: Forschungszentrum Jülich/Kirsten Bräker, Audio: Forschungszentrum Jülich/Arndt Reuning, Video1: Forschungszentrum Jülich, Video2: N. Mustafi et. al., Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains. Plos One 2014, DOI: 10.1371/journal.pone.0085731