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Biosurfactants - effective fat solvents made by bacteria

In early 2015, a company called Biotensidon GmbH from Karlsruhe established a white biotechnology subsidiary to develop a fermenter prototype for producing rhamnolipids, which are excellent bacterial surfactants. The project, which was carried out in cooperation with scientists from the Science & Technology Center in Ukraine, means that traditional petroleum-based surfactants can now be replaced by biosurfactants. The latter are extremely versatile and 100% biodegradable.

Biotensidon GmbH was established by Rolf Hartmann (marketing director, on the right) and Jörg Joegel (technical management, on the left) at the end of 2011. © Biotensidon GmbH

World production of surfactants is estimated at 18 million tonnes per year. They are used almost everywhere from emulsifiers in face creams to solvents in dishwashing detergents. This versatility comes from the amphiphilic structure of the surfactant molecules. They have a hydrophilic head and a hydrophobic tail, which effectively links two different types of liquid together. They also reduce surface tension. This makes a drop of water much broader and causes it to lose its near-spherical shape.

Most conventional surfactants are made from petroleum and are often harmful to the environment. Although they meet the legally required level of primary degradation – even though this only refers to the loss of surface activity - the surfactants cannot usually be completely degraded. Alternatives are therefore being sought and a quarter of these surface-active substances are already produced chemically from renewable resources such as palm oil or coconut oil. However, microorganisms can also produce surfactants, and these so-called biosurfactants are considered 100% biodegradable," says Rolf Hartmann, marketing director of Karlsruhe-based Biotensidon GmbH, a company that has specialised in biological detergents for the past three years.

Production of biosurfactants has not been at all economically viable up until now

Pseudomonas aeruginosa rhamnolipids are biosurfactants that consist of different ratios of rhamnose entities and β-hydroxydecanoic acid. Mono- and di-rhamnolipids consist of one or two rhamnose molecules respectively. © SF/BIOPRO

Biosurfactants are therefore a class of surface-active molecules that are, inter alia, produced by bacteria and fungi. Bacteria release natural surfactants into their environment where they reduce the surface tension of the biofilm in which they evolve, thus facilitating the motility of bacteria. Other bacteria alter the properties of biosurfactants by incorporating them into their cell walls.

In addition to their broad spectrum of activity, most biosurfactants are not generally toxic, possess antimicrobial properties and have been shown to act against a number of bacteria. This qualifies them as additives in cosmetics and soaps and for pharmaceutical products. In addition, they are biodegradable, and can therefore be widely used in many industries such as agriculture, food production and chemistry.

Rhamnolipids (RL) are bacterial surfactants. They belong to the glycolipid class, as they contain at least one sugar moiety (in this case rhamnose). Around 60 different rhamnolipid types are known. A mixture of the four most common rhamnolipid representatives, RL1 to RL4, are produced by the bacterium Pseudomonas aeruginosa and released into the growth medium. Although rhamnolipids have excellent surfactant properties, they are rarely produced industrially because conventional methods are very costly. One of the highest concentrations of rhamnolipid (112 g/l) was produced using the strain P. aeruginosa in 1997. However, this highly productive strain is a classified risk 2 pathogen, which means that the bacteria can cause human/animal disease. Using P. aeruginosa for producing rhamnolipids is therefore an economic challenge both in terms of production processes and facilities. Production costs of biosurfactants tend to be around 40 times higher than those of synthetic surfactants.

The non-pathogenic Pseudomonas strain is cultivated in round-bottomed flasks and transferred to a fermenter when bacteria density is high enough. The brownish-coloured medium can be harvested after an incubation period of around 72 hours. This medium contains the rhamnolipids. © SF/BIOPRO

Researchers around the world have therefore been looking for a non-pathogenic production strain. However, the usual rhamnolipid concentrations in potentially suitable strains were found to be between 50 and 400 times lower than those of P. aeruginosa. It took a while for a suitable strain to be found. Biotensidon now owns a competitive, non-pathogenic, wild-type Pseudomonas aeruginosa strain. "This Pseudomonas strain has been shown to be non-pathogenic and can be used according to regular laboratory standards. Non-pathogenicity was also an important prerequisite for consumer acceptance," explains Dr. Alexandr Shulga, scientific director at Biotensidon. "This particular strain produces a mixture of RL3 and RL4 rhamnolipids. These rhamnolipids have properties that make them ideal alternatives to previously used surfactants, and increases the value of the product in which they are used."

Biosurfactant production – a challenge for the field of white biotechnology

"We were lucky to find premises in the city of Bruchsal that are ideal as a production site," says Hartmann. A Ukrainian research team from the Science & Technology Center in Ukraine (STCU) in Kiev was found to have the necessary scientific expertise for developing an economically feasible rhamnolipid production process. Terms were negotiated with the STCU, and Shulga's team, which has since grown to seven specialists, were able to start work.

(From left to right) Rolf Hartmann, Jörg Joegel, Dr. M. Pristai (microbiologist) and Dr. Alexandr Shulga (scientific project management) were some of the scientists involved in the project to develop a fermenter prototype. © SF/BIOPRO

"Laboratory-scale rhamnolipid production already worked quite well and we were able to transfer production to a 100l fermenter," says Jörg Joegel, the project's technical director. "From a technical point of view, increased foaming in the fermenter significantly disturbed rhamnolipid production, and was the major challenge we had to deal with," he continued. The team came up with a solution, which involved developing a fermenter with a completely new interior design that ensures minimal foaming.

"We were also able to avoid using chemical anti-foaming agents," says Joegel. Following this success, Shulga and his team were able to optimise the necessary process parameters as well as adapt the composition of the growth medium to overproduction. "In the laboratory, pseudomonal rhamnolipid production is stimulated by restricting the bacteria's access to nutrients. Unfortunately, this approach could not be transferred to the production fermenter. However, we managed to solve this problem in a different way," says Shulga who brings 20 years of experience in rhamnolipid production to the project.

"In early February, after two and a half years of R&D work, we were able to recover rhamnolipid-containing medium from the 100l fermenter. In addition, the 10g/l quantity of rhamnolipid concentration was quite promising. Fine-tuning the system paid off after a few weeks. The cell-free supernatant now has a rhamnolipid concentration of 15 g/l," said Joegel. Successful production now seems possible, but each new product represents another financial risk. Rhamnolipids were therefore initially produced by a newly established Biotensidon subsidiary. "Production is now up and running, and we are able to advance the project with the help of new investors. We are planning to install several 250l fermenters over the next few months in order to ensure continuous production. Our trump card is that Biotensidon, which is an established sales company for biological detergents, has a global distribution network that it can rely on. In order to be able to respond to initial demand, we are planning on purchasing a spray dryer that will then enable us to produce pure rhamnolipid powder," says Hartmann.

Website address: https://www.biooekonomie-bw.de/en/articles/news/biosurfactants-effective-fat-solvents-made-by-bacteria