The German Federal Ministry of Education and Research (BMBF) regards systems biology as a basic life sciences discipline that bridges the gap between laboratory experiments and mathematical modelling and therefore attaches great importance to supporting research in this field. The BMBF therefore launched the “e:Bio-Innovation Competition Systems Biology” programme in 2010. A group of scientists from Stuttgart and Tübingen who submitted a proposal on the scale-up of microbial production processes were awarded funding totalling 2.1 million euros for a period of three years.

The cooperative project was started in early 2013 and is coordinated by Prof. Dr.-Ing. Ralf Takors from the Institute for Bioprocess Engineering (IBVT) at the University of Stuttgart. He explains the importance of the project for the biotech industry: “Laboratory research is always carried out on the small scale using standard laboratory equipment. Typically, we cultivate microorganisms in laboratory reactors. These reactors usually are less than a litre in volume, sometimes we use reactors with capacities of a few litres, but never more. However, the fermenters used in industrial production processes have a capacity of 500,000 to 700,000 litres or more. As we are aiming to use our research results to improve bioproduction processes, we have to keep in mind that simply transferring laboratory results one to one into large-scale production processes is impossible. If we aim at a 500,000 to million-fold increase in production scale, a whole range of biological, chemical and physical factors need to be taken into account. We want to use our project to investigate the effects on the cells and for the first time ever we will formulate biologically motivated criteria required for a successful scale-up.”

From the perspective of the cells, a lot changes when transferring the cell culture conditions from the limited dimensions of a laboratory reactor to an industrial production reactor. “In a reactor ten metres high and seven to eight metres in diameter, the cells will always be unequally distributed and have to withstand heterogeneous culture conditions for a prolonged period of time. This happens even when the culture is stirred,” says Takors. “Simply stirring the culture more vigorously does not work, in fact this is not even possible for practical reasons as the energy and technology required cannot be integrated into the system.”

The laws of physics also favour the presence of heterogeneous conditions in large tanks. The solubility of gases changes as a result of pressure, which increases from top to bottom. Larger amounts of oxygen and carbon dioxide are dissolved in the lower reactor areas. In addition, a thermal gradient also exists. In relation to its volume, a large reactor has a smaller surface area. This is why it is not possible to dissipate so much heat through the reactor’s outer shell and why it is considerably warmer inside the reactor than close to the reactor shell. All this affects the life of the microorganisms and hence their production performance. The e:Bio-Innovation project “RecogNice” (Modelling the regulation of carbon, oxygen and nitrogen in large-scale processes with E. coli) is specifically focused on three compounds that are of major importance for the metabolism of the living miniature factories, i.e. dissolved carbon, oxygen and nitrogen. The scientists are investigating how the unequal distribution of these compounds in the reactor affects the cells. The researchers have chosen E. coli bacteria for their investigations because these bacteria are widely used for the production of medically active substances and materials (e.g. fine chemicals).

“Here at the IBVT, we are specifically focused on the regulatory responses of the cells to carbon and nitrogen gradients and the effects of these gradients on the bacteria’s production performance,” says Takors. Takors’ research partners in Prof. Dr.-Ing. Oliver Sawodny’s research group at the Institute of Systems Dynamics (ISYS) in Stuttgart are investigating the bacteria’s regulatory responses to dissolved oxygen gradients. The scientists hope that the results will provide them with the information they need to improve the transfer of laboratory conditions to industrial production conditions, optimise the production processes and hence economic yield. “We need to study the cells very closely as well as build a quantitative understanding of the regulatory processes involved,” says Takors.

This is where the project partners from Tübingen come in. Prof. Dr. Michael Bonin and his team at the Institute of Medical Genetics and Applied Genomics at the University Hospital of Tübingen receive the samples from their colleagues in Stuttgart and continue their work by analysing the conditions under which the cells produce which kind of proteins and in what quantities. This information is derived from the mRNA transcripts of the DNA in all cells of a system under inestigation. These transcripts contain the genetic information that is required for the production of cellular proteins. The more frequently a certain gene is transcribed, the more mRNA molecules and proteins of a particular type are produced. To obtain information on the gene expression status, the scientists use next-generation sequencing, which are highly efficient high-throughput technologies for sequencing millions and even billions of DNA strands in parallel. These analyses provide the scientists with information about the presence and quantity of mRNA at a given moment in time. 

Next-generation sequencing technologies lead to a huge amount of data and it goes without saying that the data need to be processed further. The latter is the job of the project’s bioinformaticians: “We use a commercial platform as well as a publicly available public domain software for analyising the data,” says Takors. Once analysed and interpreted, the scientists can use the information for carrying out experimental simulations of large-scale production processes. For this purpose, a special testing system was established at the IBVT, including a laboratory reactor to which a bypass can be connected that is used to model the different periods the cells prevail in the reactor and the different gradients. This in turn generates research data that can be used to optimise bioproduction. 

Prof. Dr. Georg Sprenger’s group from the Institute of Microbiology in Stuttgart is tasked with the qualitative and quantitative determination of the effects observed during the experimental simulations. The microbiologists have extensive know-how in reporter genes, which can be attached to bacterial genes of interest. They are expressed along with the bacterial genes and lead to fluorescent proteins, which can then easily be identified. 

“We have a clearly detectable signal that is produced and also degraded quickly,” says Takors. The whole process can be controlled so that only particularly interesting regulatory proteins are labelled, for example proteins involved in critical regulatory mechanisms. Regulatory mechanisms that might lead to lower production rates can subsequently be eliminated or at least specifically modified using biotechnological tools.

Further information:

Prof. Dr.-Ing. Ralf Takors
Institute for Bioprocess Engineering IBVT
University of Stuttgart
Allmandring 31
70569 Stuttgart
Tel.: +49 (0)711/685 64574
E-mail: takors(at)ibvt.uni-stuttgart.de