Walk on the white side

Experts estimate, that by 2010 white biotechnology will account for about 20 percent of the global chemistry industry's annual turnover of about $300 billion. White biotechnology is acknowledged by the chemical and pharmaceutical industry as a key to make manufacturing processes more cost efficient and ecofriendly. Already, many organic basic and fine chemicals as well as end products like vitamins, drugs, polymers, agrochemicals, and enzymes are produced by biotechnology today. North Rhine-Westphalia (NRW) is particularly strong in developing and improving such processes. The state's research is highly diversified in this area and currently several issues crucial to implementing even more white biotechnology are being addressed.

A white biotechnology research and development program usually starts by identifying a process or product that needs improvement in terms of cost efficiency, speed, or yield. Then, organisms or enzymes able to perform the desired task are selected. These are improved by means of genetic engineering. Once the process is established in the laboratory, it is scaled up to become feasible on an industrial scale.

The first steps are well established in many research laboratories and even applied as an industrialized process in small and medium size enterprises. However, the process development for scaling up, analyzing and purifying still needs improvement.

Very often, upscaling is done empirically, step by step. "Developers of biotechnological downstream processes often do not sense the consequences of a decision for or against certain processing steps and their order," writes Gerhard Schermbecker, head of the Chair for Plant and Process Design at the Department of Biochemical and Chemical Engineering of Dortmund University. The same is true for the sensitivity of downstream processes to changes in reaction conditions such as pH value, salt concentrations, etc. "Process developers often focus on maximum yield of the single step they are working at," he adds, "and they do not take into account integration in the overall process. Even after the start of manufacturing, often there are no reliable data on mass and energy balance available so that a lot of potential for optimization is idle."

Superior recovery and purification technologies can have a considerable economic impact, Product recovery can account for up to 80 percent of manufacturing costs. These problems have come to the attention of public research institutions only in recent years, and NRW has pioneered this important field.

"In the last decades, university research was focused on the screening for biocatalysts, that is, enzymes suited for industrial application, and their characterization and optimization," says Eiden. "Only a few research groups, such as those at the Jülich Research Center were dealing with process development, integrating biological, chemical and engineering approaches, and industrial applications were confined to technically well-defined reaction systems."

The Technical University of Dortmund, too, is a center of research that's into bioprocessing development, bioengineering, systems dynamics, process control and other aspects of developing and upscaling biotechnological syntheses. "It is the biggest standalone faculty for bio and chemical engineering in Europe," says Andreas Schmid, head of the faculty's Laboratory of Chemical Biotechnology.

Researchers in NRW recognized that research along the entire value chain—from identifying genes to selecting and optimizing production organisms to process development and manufacturing control need to be addressed in a concerted fashion.

"At present, designing a white biotechnology production process is done by developing individual solutions," Schmid adds. "A lot of companies have created considerable expertise in special areas, but there are no basic modules available [such as those] in traditional chemical process engineering. This is not to say that white biotechnology is a marginal technology, but investments are still higher because of the necessary individual solution."

Technical University Dortmund's curriculum therefore covers everything from designing a biocatalyst to product recovery and workup. "In addition, we identified matching activities and together with other universities in NRW created a graduate school combining aspects of genomics, enzymology, organic chemistry, analytics and microbiology," Schmid says. Moreover, researchers from both academia and industry have initiated networks and excellence centers where basic research, process developers and producers can exchange and test ideas, concepts, problems, and solutions.

"Solutions for some of the problems will come from integrating systems biology and bioprocessing technology," Schmid adds, "but we also need better interdisciplinary cooperation between engineers and scientists—chemists as well as biologists and mathematicians. Many of these problems are now addressed in the ChemBioTec network by basic research institutions and industry."

ChemBioTec is funded by the Deutsche Bundesstiftung Umwelt (DBU), a federal foundation promoting innovative environmental projects and by Dortmund University. It is initiating and coordinating the joint research and development efforts of academia and industry. ChemBioTec also coaches funding applicants, organizes symposia and workshops, and provides for co-funding via DBU. Since 2006, 17 ChemBioTec projects involving 47 partners have been funded to the tune of €14 million, of which €6.6 million has been provided by DBU.

The goal of one project is to develop useful products from the ten million metric tons of crop waste produced each year from Europe's olive oil farmers. It contains a wealth of phenolic substances that might be used as natural preservatives for food and cosmetics, substituting conventional preservatives chemically derived from fossil resources.

Another project deals with succinic acid, more than 15,000 metric tons of which is used every year for the organic synthesis of dyes, pharmaceuticals and polyester resins. Production of succinic acid needs heavy metal catalysts, organic solvents, high pressure and high temperature, using a huge amount of energy and producing toxic waste. The project is aiming to establish a biotech-based production process in yeast.

The electrical BioChip from Henkel allows the activity of marker genes to be measured after just one hour. Marker gene activity is a measure of how efficiently the bacteria produce laundry detergent enzymes.
Courtesy of Henkel International

"The vision of ChemBioTec," Eiden adds, "is to implement various novel bioprocesses in specialty and fine chemistry with production volume of up to 10,000 metric tons a year, to establish new products not accessible by chemical synthesis and to create a stable network between academia and industry with biocatalysis as a new research and teaching subject in at least three universities."

While ChemBiotec is an international network that focuses on addressing crucial bottlenecks of biotech production processes by academic and corporate research collaboration, it is also a member in a cluster called CLIB2021 which is aiming to promote technology transfer and setting up new entrepreneurial businesses in white biotechnology.

CLIB2021, which in 2007 was awarded €20 million in funding as winner of the nationwide government sponsored BioIndustry 2021 competition, promotes networking along entire value chains by linking members both along the R&D-oriented value chain (research-development-commercial application) and along the production-oriented value chain (raw material-intermediate-component-consumer product). The network initiates and accelerates market-oriented research projects so that they get to the market faster and with competitive advantages for all participants.

"There still is a lot to do," Eiden summarizes. "We still need better biocatalysts addressing the problems of stability, complexity and production of side products, but we also need better concepts for product purification and analysis." According to Eiden, better programs to model or simulate processes and improvements for scaling up processes developed in the lab are also on the wish list.

While the list of processes that need to be improved is long, there have already been several commercially successful projects in white biotechnology in NRW. One of the first companies addressing the industry's need for novel, improved biocatalysts is Direvo Biotech. The company was founded in 2000 in Cologne as a spinout of Hamburg-based Evotec, then a drug discovery service company, to apply screening-based technology of directed evolution for the development of better biopharmaceuticals and enzymes for industry, food and feed. In September last year, the biopharmaceutical business of Direvo was acquired by Bayer Healthcare, a subsidiary of Bayer Schering, for €230 million in cash, while its "white" biotechnology subsidiary was not subject to this transaction.

Within eight years Direvo had proven its technology and its development skills in various industry collaborations with international pharma and chemistry companies, such Pfizer, MedImmune, Danisco, Genencor, Novozymes, and others, for the development of industrial enzymes.

To optimize an enzyme, Direvo's scientists isolate the gene and alter its sequence by mutagenesis and recombination. The resulting variants are then expressed in cells and the performance of the proteins tested by automated high-throughput screening assays. The technology can test up to 100,000 variants a day. The best performers are selected and subjected to further rounds of evolution until a variant with the desired properties is obtained.

One example is engineering of a phytase, an enzyme that cleaves phytate, the principal storage molecule of phosphate in many plants; among them forage crops for farm animals. Swine and fowl cannot digest phytate so that feed for these animals must be supplemented with phosphate and other nutrients that are bound by phytate. Unabsorbed, the phytate passes through the gastrointestinal tract and elevates the amount of phosphorus in manure which can lead to environmental problems such as eutrophication.

Phytate can be processed by phytase, however there was no natural phytase available that could endure the acid and proteolytic environment of an animal stomach and the high temperatures used in processing feed. Direvo therefore engineered a phytase to improve thermostability by 20°C as well pepsin resistance and activity over a broad range of acidity. The enzyme now is used as feed additive.

Another example comes from Henkel, a global player in detergents, cosmetics, and adhesive technologies. The company has a long-standing expertise in using enzymes to improve the cleaning power of its washing agent consumer products. "We launched the first enzyme-containing washing agent, Fakt, in 1968," says Karl-Heinz Maurer, head of Henkel's Global R&D Chemistry Laundry & Home Care. Enzymes used are proteases against protein-containing stains, as well as amylases and lipases to break down starch and fat and cellulases to remove shopworn cotton fibers leading to fluff and pilling and to inhibit graying of the laundry. "Genetic engineering of enzymes at Henkel goes back to 1986," he adds. Research is done in-house as well as in cooperation with biotechs and academic partners, while enzyme production is performed in a joint venture with Sandoz called Biozym.

Henkel's research recently developed novel proteases able to remove protein-containing stains in laundry at low temperatures. Henkel researchers collected soil samples in chilly environments such as bat caves and penguin habitats to find microorganisms with proteases that operate at low-temperatures. To sample microorganisms that could not be cultivated in the lab, Henkel teamed up with the company Brain AG to transfer the protease genes that they discovered into cultivable bacteria for further testing. "More than 10,000 different bacteria were tested and finally a protease was identified which at about 20°C can remove grass and chocolate stains with unprecedented efficiency," says Maurer. "Washing at 20°C instead of 40°C saves more than 50 percent energy and carbon dioxide. Besides, improved enzymes help reducing the amount of detergent used, packaging material, and of persistent organic ingredients." These multiple benefits are what white biotechnology is all about.