SmartBioH₂-BW – Biohydrogen from Industrial Wastewater and Residual Streams as a Platform for Versatile Biosynthetic Routes

Our approach

The biorefinery is based on two interlinked biotechnological hydrogen production processes:

• Hydrogen (H₂) and other products such as carotenoids are produced in a closed bioreactor using purple bacteria. Carbon dioxide (CO₂) is generated as a by-product.
• Carbon dioxide is fed into the connected microalgae plant. There it is bound in the algae biomass – releasing further hydrogen as well as other products such as starch. In addition to binding the carbon dioxide, the process serves to increase the hydrogen yield and expand the biorefinery's product range.

The biorefinery is to be analyzed and optimized according to ecological, economic and social criteria throughout the entire planning and development process. The project partners are developing a holistic evaluation system that can be transferred to other "biofactories" and that can be used to record the relevant environmental impacts and economic relationships.

On 3 August 2024, the demonstration plant of the SmartBioH₂-BW project was inaugurated by Dr. Andre Baumann, State Secretary in the Baden-Württemberg Ministry of the Environment, as part of his summer tour. Numerous politicians from Rheinfelden, the district of Lörrach and several members of the state parliament also took the opportunity to visit the biorefinery plant. Test operations are now starting under real conditions.

Hydrogen (H₂) is considered a key element in the energy transition. Processes using photosynthetically growing purple bacteria or microalgae to produce hydrogen have been studied for a long time. However, these have not yet been implemented on a larger scale. This is where the SmartBioH₂-BW project comes in. As part of the initiative, a biorefinery was developed based on two processes of biotechnological hydrogen production, which were interconnected in a demonstration plant at the Evonik Operations GmbH site in Rheinfelden.

Initially, purple bacteria are cultivated in a bioreactor under controlled microaerobic conditions. This process generates not only hydrogen from ethanol-containing rinsing waters but also usable carotenoids, the biopolymer polyhydroxyalkanoate (PHA), and CO₂ as a by-product.

The second step is the so-called "Direct Photolysis": here, microalgae produce both hydrogen and oxygen from water using light energy. To maintain this reaction, IGB utilizes a new type of photobioreactor that can remove the oxygen produced during hydrogen production.

To minimize CO₂ emissions from hydrogen production with purple bacteria, the CO₂ is fed into a coupled microalgae system, which produces usable products such as starch from CO₂, light, and the residual material stream ammonium chloride. The microalgae system is a compact photobioreactor illuminated by LEDs. Its special feature is its modular design, a high degree of automation, and a significant volume on a small footprint.

Bacterial hydrogen production: from laboratory to demonstration scale 

With the help of the first process step, hydrogen production using bacteria, a previously unutilized ethanol-containing material stream could be tapped. To accomplish this, a fermentation was carried out in a 75-liter bioreactor in which the purple bacterium Rhodospirillum rubrum metabolized ethanol – with the help of sugars such as fructose – and produced hydrogen under so-called microaerobic conditions. Microaerobic conditions mean that by controlling the aeration and stirring within a bioreactor, the production of oxygen is limited so that the bacteria “work” exactly at the threshold between respiration and anaerobic fermentation.

To achieve the project goals, the first step in the actual implementation was to analyze the material stream generated at Evonik and evaluate it using growth trials and toxicity tests. This was to clarify in which concentrations the substrate could be used, as ethanol at high concentrations has a growth-inhibiting effect even for these bacteria. An optimal compromise between the maximum consumption of ethanol and still high growth rates turned out to be a substrate combination of ethanol and fructose, each at 15 g/L. Subsequently, the composition of the nutrient solution was optimized to further increase growth.

Building on this basis, the bacterial cultivation could then be transferred from the flask to a small laboratory-scale bioreactor. The laboratory reactor offered the advantage that growth and hydrogen production could be controlled by regulating various cultivation parameters, thus already showing parallels to scaling up to the demonstration scale.

In the bioreactor, the project team divided the process into two phases. Initially, the bioreactor was aerated with air, i.e., operated aerobically, because respiration in bacteria is more efficient in this case, achieving higher growth rates and cell densities. This, in turn, resulted in a more efficient process with higher hydrogen production rates. Subsequently, the air supply was limited, creating conditions under which the bacteria could produce hydrogen through microaerobic dark fermentation.

Since more bacteria also produce a larger amount of hydrogen, a feeding process was developed. During fermentation, new nutrients are gradually supplied to maintain a constant growth of the bacteria. It is important for the feeding solution that carbon sources like ethanol and fructose, nitrogen sources, trace elements, and vitamins are in the right ratio to prevent nutrient deficiencies, which could inhibit further growth. After comparing various concepts, the IGB researchers established a pO₂-dependent feeding profile. This involves measuring the partial pressure of oxygen (pO₂) in the fermentation medium. When the cells respire, oxygen is consumed and the partial pressure decreases. When ethanol and fructose are completely consumed, metabolism slows down, and less oxygen is consumed by the bacteria – recognizable by an increase in partial pressure, the so-called “hunger peak. ” Based on this hunger signal, researchers can recognize that substrate is lacking and can supply fresh nutrient solution so that the bacteria continue to grow. In the laboratory scale, a cell density of up to 34 g/L of bacterial dry mass was achieved.

In the microaerobic phase, the so-called microaerobic dark fermentation takes place, producing hydrogen. In respiration, oxygen is transferred as a so-called terminal electron acceptor. This allows the bacterium to regenerate central molecules for the respiratory chain and maintain respiration. However, if this oxygen is limited at that point, respiration is at risk of stopping. As a survival mechanism, purple bacteria then produce hydrogen. In this case, electrons can be transferred to protons and excreted as gas from the bacteria. To implement this functional principle in the bioreactor, the project first established reliable hydrogen analytics. Subsequently, it was assessed how different oxygen concentrations in the fermentation medium affect hydrogen yield. The hydrogen yield was maximized by adding additional nitrogen to the aeration air.

After establishing and optimizing both phases of fermentation at the laboratory scale, the next step was to implement the process into a biorefinery concept. During the transfer to the demonstration scale at Evonik in Rheinfelden, it was demonstrated that the construction of a biorefinery for climate-neutral hydrogen production is technically feasible through the successful coupling to the algae reactor and the hydrogen production from ethanol-containing material streams.

Through the establishment of the feeding process and the scale-up, hydrogen production could be increased by approximately five times compared to the laboratory scale. The result: Extrapolated over a month, up to 3400 L of hydrogen gas could be produced with fermentation at this scale. Particularly exciting was the finding that during hydrogen production, polyhydroxyalkanoates (PHA) were also formed, which made up to 54 percent of the bacterial mass. PHA are biopolymers and can be purified and used as biodegradable bioplastics for packaging and films. This opens an exciting perspective for obtaining even more valuable products within this biorefinery and making the process more profitable in the future.

Microalgae bind by-product CO₂

The goal was to establish a biorefinery that utilizes residual streams and CO₂ from the chemical industry, thereby connecting circular economy and hydrogen production. For this purpose, the project team selected the following microalgae strains: Chlorella sorokiniana and Chlamydomonas reinhardtii. Both can produce hydrogen under suitable conditions and accumulate starch as a storage product, thus serving as a potential fermentation substrate. Additionally, Chlorella sorokiniana is characterized by a high lutein content. However, due to current legal regulations, lutein obtained from residual streams of chemical production cannot be used in food or animal feed.

The project also investigated the toxicity of contaminants from chemical production in residual streams of ethanol-containing rinsing waters and ammonium chloride, which could affect the growth of the microalgae strains. It was found that both algae strains can utilize the residual streams. However, ethanol provides only a small additional biomass increase. Ammonium chloride can replace previous nitrogen sources, but additional pH control is required.

Another residual stream – CO₂ produced during the fermentative hydrogen production with Rhodospirillum rubrum – can be efficiently converted into starch-rich algal biomass by Chlorella sorokiniana. At the same time, ammonium chloride can be used for growth. The utilization of CO₂ and ammonium chloride was initially transferred to 6-L laboratory photobioreactors. In this scale, researchers investigated the key parameters of algal production: light input and biomass concentration. Depending on these two factors, biomass productivity in the reactor increases. The light yield, or the efficiency of converting light into biomass, remains high only up to a specific strain-dependent biomass concentration and light input and then decreases.

After establishing and optimizing this residual stream utilization at the laboratory scale, the next step was to transfer the process to the demonstration scale. The SmartBioH₂ demonstration plant that was set up for this purpose is a compact and modular photobioreactor system, illuminated with LEDs and characterized by a high degree of automation.

The conclusion: For the establishment of the biorefinery concept at the demonstration scale at Evonik Industries in Rheinfelden, it was demonstrated through the successful coupling of the algae reactor to the R. rubrum fermentation that residual streams and fermentation offgas streams can be utilized, and that the construction of a biorefinery for climate-neutral hydrogen production is technically feasible.

Hydrogen production via direct photolysis with microalgae

For hydrogen production using microalgae in the second process, a novel concept based on direct photolysis was chosen. By immobilizing the algae and using a special reactor setup, the oxygen produced as a by-product along with hydrogen by the algae is to be efficiently separated to a partial pressure of less than 100 ppm in the reactor gas volume. The continuous removal of oxygen to a very low oxygen partial pressure in the reactor is crucial for the functioning of the process. If the oxygen content in the system is too high, the extremely sensitive enzymes needed for hydrogen production are being oxidized.

After setting up the experiment in the laboratory, hydrogen was successfully measured in short-term experiments. However, the system has not yet been optimized. In particular, the method for immobilizing the microalgae still needs to be further developed. The chosen concept for hydrogen production with microalgae can be easily scaled up and is planned to be applied on a pilot scale.

In a second laboratory setup, the separation of oxygen from the gas phase was also successfully tested, using a known oxygen separation method. With this setup, it was possible to reduce the oxygen partial pressure to below the critical threshold for hydrogen production within a short time.