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Research in Biberach – does the bioeconomy have a purple future?

What should be done with carbon dioxide? Following attempts to capture and deposit CO2 in places where it will not enter the atmosphere, the greenhouse gas is transforming from climate killer to raw material that can be used to produce valuable goods. Chemical processes require a great deal of energy to turn this inert gas into fuel, cement and plastics. Energy suppliers are also testing the feasibility of biotechnological methods for growing algae with CO2-containing flue gas. Microbiologist Hartmut Grammel from Biberach University of Applied Sciences and colleagues from the Magdeburg-based Max Planck Institute (MPI) for Dynamics of Complex Technical Systems are looking at another way to capture carbon dioxide with the distant objective of producing organic materials with bacterial, CO2-consuming enzymes in a cell-free environment that requires only small quantities of energy.

“Rhodospirillum rubrum”, a veritable quick-change artist, plays a pivotal role in the project “Analysis and design of bacterial enzyme cascades for the recycling of CO2”, a challenging project funded by the German Ministry of Education and Research (BMBF). Hartmut Grammel, who was recently appointed professor of industrial microbiology at Biberach University of Applied Sciences, has high hopes of this bacterium.

Unicellular quick-change artist

Hartmut Grammel, professor of industrial microbiology at Biberach University of Applied Sciences since 2012. © HS Biberach

The name ‘purple bacteria’ is derived from the bacteria’s pronounced purple colour which comes from the carotenoids that are part of the bacteria’s photosynthetic apparatus. Like plants and algae, these non-sulphur freshwater bacteria can use light for growth. However, Rhodospirillum rubrum bacteria do not use water as electron donor. Consequently, they produce cellular energy without oxygen as by-product (anoxygenic photosynthesis).

Rhodospirillum rubrum bacteria that are grown in the light but in the absence of oxygen form intracellular protrusions (known as intracytoplasmic membranes, ICMs), which take up almost the entire cell volume. ICMs contain the photosynthetic apparatus, including the reaction centre and light-harvesting complexes. When exposed to light, the pigment complexes become excited by the absorption of light and pass on electrons along an electrochemical gradient until the energy is eventually stored in the biochemical currency ATP. However, Rhodospirillum rubrum bacteria can also grow in the dark; this they do by switching from photosynthesis to cellular respiration on an organic carbon source, a phenomenon that hugely fascinates Grammel.

Biotechnologists have a long-standing interest in the bacteria

Oxygen, light and substrate influence the metabolism and phenotype of Rhodospirillum rubrum. The bottom half of the photo shows the colour change of the bacteria when grown on different substrates: bacteria grown in the dark on fructose (F), succinate (S) and on a succinate/fructose mixture (S/F). © Hartmut Grammel

Rhodospirillum rubrum bacteria have long attracted the interest of biotechnologists due to their ability to produce large quantities of pigments (carotenoids and chlorophyll). Grammel and his colleagues have already studied the bacteria’s suitability for the production of vitamins, membrane proteins, vaccines and biohydrogen and found that they can be produced in large amounts by the photosynthetic type of purple bacteria. This does not work when the bacteria are grown under aerobic conditions as they do not synthesise ICMs and are thus, according to Grammel, of marginal interest for technical purposes. 

The project, which the BMBF will initially fund for a period of five years (maximum funding period: eight years) under the “Biotechnology 2020+ - Basic technologies for the next generation of biotechnological procedures” is based on research Grammel carried out before he moved from the MPI in Magdeburg to Biberach. 

The microbiologist was a major driver of the MPI’s strongly systems biology oriented research. A cooperative project with Professor Robin Ghosh from the Department of Bioenergetics at the University of Stuttgart fired Grammel’s interest in Rhodospirillum rubrum. Since 2007, Grammel has been investigating the bacterium’s unusual metabolic characteristics and phenomena. Ghosh once made the following observation: Rhodospirillum rubrum bacteria that are enriched in the dark and ‘fed’ with organic acid (succinate) grow relatively quickly in small shake flasks. At some stage, when the bacteria have used up all the oxygen they turn pink. From this it can be assumed that oxygen inhibits the synthesis of carotenoids in the light-harvesting photosynthetic complexes. In the absence of oxygen, the bacteria also synthesise photosynthetic membrane complexes in the dark, albeit fewer than when grown in the light. 

A nutrient mix leads to unexpected results

What Ghosh discovered by pure chance when he added fructose to the growth medium amazed both him and Grammel: the cells had developed maximal levels of ICMs and were of a purple colour which is usually only the case when the bacteria are grown in the light. The fascinating thing was that the bacteria had synthesised maximal levels of light-harvesting membrane complexes although they were kept in the dark, something that at first sight did not really make biological sense. However, Ghosh found out that this behaviour was due to the bacteria being grown in the presence of fructose and succinate, rather than on fructose alone. The puzzle is not yet completely solved, but it has become clear that the presence of both fructose and succinate triggers the biosynthesis of photosynthetic genes at maximal level. Such activation of photosynthetic genes was previously only seen when the bacteria were grown in the light.

This phenomenon has considerable implications for the industrial application of purple bacteria. The ability of Rhodospirillum rubrum to synthesise high levels of photosynthetic complexes in the dark may overcome the major drawback of biotechnological production using algae. The cultivation of algae cannot be carried out in huge fermenters as is usually the case in industrial biotechnology, but requires complicated flat plate or tube cultivation systems in order to satisfy the algae’s need for light as energy source. This makes the biotechnological production of algae rather difficult.

Bioprocess does not need light

As part of his work at the Magdeburg-based MPI, Grammel developed a procedure that enables Rhodospirillum rubrum to grow without light. He successfully managed to grow the bacteria with maximal levels of photosynthetic membranes in 10-litre stainless steel fermenters in complete darkness. Based on a computer model, Grammel was able to increase biomass yield to around 60 g/L in laboratory fermenters, which was much higher than the yields produced by photosynthetic microalgae grown in the light.

Grammel is certain that Rhodospirillum rubrum bacteria have obvious advantages for the industrial production of carotene, which is presently done with algae or genetically engineered yeasts or E. coli bacteria. Carotenes are fat-soluble pigments and normally only exist in membranes, which are relatively rare in these microbes. In contrast, purple bacteria, which have a large number of intracytoplasmic membranes, are therefore excellent carotene producers.

Grammel has not tried this process with quantities of over 100 litres, but is sure that it would not be a problem. He has already tested the bacteria’s feasibility for industrial production. He produced bacterial chlorophyll for testing its suitability and efficiency for the photodynamic treatment of cancer patients. This clinical phase I trial was carried out in cooperation with industrial partners.

Biochemical mimicry

Grammel now knows that “certain cellular signals switch on the bacteria’s photosynthetic genes and hence the synthesis of ICMs and that the chosen culture conditions are able to biochemically mimic the activation of photosynthetic genes and ICM synthesis through light. Grammel and his colleague Steffen Klamt from the MPI in Magdeburg have investigated this phenomenon using theoretical tools. Using Klamt’s metabolic network model (CellNetAnalyzer), the metabolic pathways of a particular organism and their respective reactions and enzymes can be simulated in presence of specific nutrients and the outcome (i.e. expression of genes) can be monitored in the cells. Klamt and Grammel have already applied metabolic network modelling to biohydrogen production in purple bacteria and succeeded in optimising the process and increasing the biohydrogen yield.

Rhodospirillum does not grow without carbon dioxide

During his time at the MPI in Magdeburg, Grammel and his colleagues also analysed the central metabolism of Rhodospirillum rubrum and discovered a further phenomenon that was difficult to explain: Rhodospirillum rubrum bacteria do not grow on fructose as substrate in the absence of CO2. However, increasing concentrations of CO2 enhance bacterial growth. Moreover, in contrast to related non-sulphur purple bacteria, Rhodospirillum rubrum is also able to grow under certain conditions when the key enzyme of the Calvin cycle, ribulose-1,5-bisphosphate-carboxylase/-oxygenase (abbreviated to RuBisCo), is genetically deleted. In plants and bacteria, RuBisCo is involved in the first major step of carbon fixation.

This observation led Grammel to look in detail at the CO2 metabolism of Rhodospirillum rubrum. Looking for alternative CO2-fixing reactions – in the meantime, six different metabolic pathways that allow growth with CO2 as sole carbon source have become known in bacteria – Grammel and his colleagues looked at all theoretically possible reactions, but found that some had never been studied in great depth with regard to their characteristics, capacities and suitability for technical application. The characterisation of these reactions constitutes the first step of the BMBF-funded project.

Which enzymes consume the highest amounts of CO2?

Grammel will initially focus on the investigation and isolation of unusual carbon dioxide fixing enzymes and subsequently use them in a cell-free electrochemical system to produce organic materials. This is the ambitious, highly risky though very attractive objective of the project being carried out with engineers from the MPI in Magdeburg. The engineers, who have expert knowledge in the field of electrochemistry, have already succeeded in coupling other enzymes to an electrode, i.e. using them in a cell-free environment.

The unusually long funding period gives the researchers from Biberach and Magdeburg the time they need to crack some relatively hard ‘research nuts’. The researchers are confident that they will be able to do this, but at the same time they are aware that they have embarked on a highly ambitious project. The researchers from Biberach will initially investigate all CO2-dependent metabolic reactions in bacterial cells and cell extracts. The metabolites will be labelled with (stable) 13C isotopes and the reaction mechanisms will be analysed with mass spectrometry to identify the metabolic pathways that consume the largest quantity of CO2.

Vision of cell-free biohydrogen production using an electrochemical system

Metabolic model for non-sulphur purple bacteria. Screenshot of the CellNetAnalyzer. The model shows calculable reaction rates (blue) for photoheterotrophic growth on succinate (green: predetermined flux). © Oliver Hädicke, MPI Magdeburg
At the beginning of such a long-term project, it is difficult to foresee how it will develop and many issues will need to be clarified along the way: Will it be necessary to genetically optimise the CO2-consuming enzymes identified? How can the enzymes be stabilised in a cell-free electrochemical system? How can CO2 be applied and what kind of CO2 sources can be used? Another issue that needs to be taken into account for future technical application is the fact that Rhodospirillum rubrum bacteria are really fond of succinate, a substrate that is far from cheap. Grammel highlighted that older scientific papers used dairy waste and sulphite lyes from the paper industry for the same purpose. While the researchers in Biberach are specifically focussed on identifying and characterising those CO2-consuming enzymes that are best suited for technical application, the colleagues in Magdeburg led by Professor Klamt will extend the existing metabolic network in silico with all known CO2-fixing metabolic pathways. At present, the metabolic network involves the metabolic pathways of three bacterial species, but it can already be used to combine them arbitrarily and calculate the most efficient fluxes. Basically, the researchers’ project focusses on combining computer simulation and experimental approaches to identify those enzyme combinations that are the most promising for technical application.

Finally an alternative to RuBisCo?

It will take years to complete the simulations and begin the technical implementation of the cell-free production system. The fact that the BMBF funds projects like this one with public funds is due to the appeal of the approach: it would be the first time an alternative to the relatively inefficient plant enzyme RuBisCo would be available, an alternative that is not only feasible without the growth-limiting factor light, but which would also enable the production of carbon-dioxide based products with relatively little energy consumption. If the researchers from Biberach and Magdeburg are successful, then a small part of an as yet abstract idea of a knowledge-based bioeconomy would be turned into reality.

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