The terms “sustainability” and “greenness” are omnipresent, whether in advertising for clothing or in project proposals for the funding of academic research. However, “greenwashing” and the careless use of these terms blurs our understanding and turns them into rather abstract concepts that are nonetheless necessary. In the following we will take a closer look at the meaning for biocatalysis and more specifically on the example of the Horizon 2020 funded EU project CARBAFIN.

The purport and history of “green chemistry” and “sustainability”

In chemical industry an increasing environmental awareness appeared in the 1980s. Waste prevention instead of waste remediation and pollution control by end-of-pipe solutions was the new credo. The U.S. Pollution Prevention Act of 1990 (1) not only focused on the need to reduce pollution, but also recognized that preventing waste at the source reduces its treatment costs and enhances economic competitiveness through a more efficient use of raw materials. This profitable side-effect of environmental protection most likely led to a fundamental shift in the strategy for developing new “greener” technologies, and the term “green chemistry” was born. Green chemistry is primarily defined as pollution and waste prevention rather than remediation during a chemical production process. The overall guiding element is “benign by design”, as described in the book Green Chemistry: Theory and Practice written by Anastas and Warner in 1998.(2) The term sustainability was introduced in 1987 with the publication of the Brundtland report, Our common future, by the World Commission on Environment and Development (3). The report emphasized the need for industrial and social development to keep up with a growing global population. In other words, a satisfactory quality of life for present generations should be as important as the ability of future generations to meet their own needs (3). Ideally, sustainable technologies must fulfill two conditions:

(a) natural resources are to be used at rates that do not deplete supplies in the long term, and
(b) the residues produced may not exceed assimilation rates of the natural environment (4).

Nevertheless, our economic system continues to rely on technologies that are largely based on non-renewable fossil resources. Especially, petroleum manufacturers consume natural resources at a much higher rate than they are produced by nature, generating carbon dioxide levels that nature can no longer absorb, leading to climate change. Hence, there is an urgent need to balance societal equity, environmental impact, and economic development, often referred as the three Ps – people, planet and profit (2). Summarized, the term “greenness” exclusively refers to environmental protection, while “sustainability” encompasses long-term social and economic developments that are only possible by protecting our planet.

What makes biocatalysis green and sustainable?

In general, enzymes are renewable catalysts, they work efficiently at mild and safe conditions, they are energy efficient, and waste formation is significantly reduced. Biocatalysis is furthermore characterized by a low toxicity and thanks to advances in biotechnology, it has become easier to implement biocatalytic concepts in industry (2). In the last two decades technologies, such as DNA sequencing have helped biocatalysis to conquer the chemical industry. More than 20,000 bacterial and fungal genomes have been sequenced and the data have become available in public domains. A target gene of a potential enzyme catalyst can easily be identified in online databases by „genome mining“. The conversion of the gene into the enzyme catalyst requires an appropriate, well developed host microorganism and takes roughly a week in a proven cell cultivation system. This in turn enables production on an industrial scale for acceptable prices (2).

Green and sustainable process development in CARBAFIN

In the course of the CARBAFIN project we developed a green and sustainable production process for functional glycosides, which can be used in food and feed, cosmetics and medicine. One among others is cellodextrin, a glucooligosaccharide and potential prebiotic. Its production requires the action of three different enzyme catalysts in a cascade reaction. Typically, each enzyme must be produced individually in a cell cultivation system and the actual catalyst production. costs and waste formation add up with each enzyme in the cascade. Therefore, we developed a so-called non-living whole-cell catalyst that produces all enzymes at once. The usage of a whole-cell catalyst, in general, reduces costs by 2- and even 10-fold compared to usage of cell-free lysate and isolated enzyme, respectively, this multiplies for each enzyme (5). Interestingly, in the early 60s bioconversions were mainly performed by growing whole-cell catalysts (so called fermentations) partly also due to insufficient enzyme purification techniques. Subsequently, fermentations were successively replaced by bioconversions with isolated enzymes, as unwanted side-reactions can be avoided (6). However, in the last decade non-living cell catalysts experienced a boom in biocatalysis due to the need of greener and more sustainable technologies.

Non-living whole cells – one-pot for a biocatalytic cascade

Non-living whole cells show numerous advantages compared to living whole-cell catalysts: The actual bioconversion can be separated from catalyst production. Consequently, the reaction medium can be less complex, which facilitates product purification afterwards. Besides that, less biomass waste and carbon dioxide are produced, especially when the non-living cell catalyst is recycled several times. The productivity can easily be increased by adding more cell catalyst, whereas in a fermentation product formation depends on the growth rate of the whole-cell catalyst (2,6). Nonetheless, also a non-living cell catalyst can experience low productivity as result of low enzyme expression yields in the cell or an imbalanced activity ratio, a pre-requisite in multi-step bioconversions. Overcoming this latter bottleneck is especially important in the synthesis of cellodextrin since an uncontrolled activity ratio would lead to product losses due to the formation of insoluble material. An increasingly sophisticated synthetic biology toolbox, which is the outcome of several years of research from all over the world, helped us to produce our target enzyme cascade in an optimal activity ratio and additionally in very high expression yields (namely, as much as of half of the cells natural protein). Consequently, the designed whole-cell catalyst synthesized also high yields of soluble cellodextrin in a product titer that is relevant for industrial production (100 g/L). Further investigations for recycling of the cell catalyst might even surpass industry benchmarks.

Measure sustainability

Finally, improvements in the sustainability of a production process need to be quantitatively measured. The CARBAFIN project, therefore, included a life cycle assessment (LCA). LCA specifically evaluates the environmental impact of a product in all stages of its “life”. The assessment is based on environmental impact indicators, such as energy usage, global warming, smog formation and so on (2). There are several methodologies for distinct applications, however, further details would go beyond the scope of this article. A separate article is planned that specifically addresses LCA strategies including techno-economic analysis. So, stay tuned for more information. In conclusion, it is more important than ever to raise awareness among funding agencies, policymakers and citizens of the urgency and benefits of basic and applied research. The realization of “truly” green and sustainable technologies would not be as far along as it is today, without the advances made in biotechnology and related fields. Industrial implementation of green and sustainable technologies is the main goal of projects like CARBAFIN and should be encouraged in every way possible.
Author: Katharina Schwaiger Picture Credits: Shutterstock

References
1. Summary of the Pollution Prevention Act | US EPA [Internet]. [cited 2021 Dec 22]. Available from: https://www.epa.gov/laws-regulations/summary-pollution-prevention-act 2. Sheldon RA, Woodley JM. Role of Biocatalysis in Sustainable Chemistry. Chem Rev. 2018;118(2):801–38. 3. Report of the World Commission on Environment and Development : [Internet]. [cited 2021 Dec 30]. Available from: https://digitallibrary.un.org/record/139811 4. Popa V, Volf I. Green chemistry and sustainable development. Environ Eng Manag J. 2006;5(4):545–58. 5. Tufvesson P, Lima-Ramos J, Nordblad M, Woodley JM. Guidelines and cost analysis for catalyst production in biocatalytic processes. Org Process Res Dev. 2011;15(1):266–74. 6. Woodley JM. Microbial Biocatalytic Processes and Their Development. Adv Appl Microbiol. 2006;60(06):1–15.

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This work is based on the project CARBAFIN: This project has received funding from the  European Union’s Horizon 2020 research and innovation programme under grant agreement No 761030.