Isolation and domestication of novel environmental bacteria for the valorisation of second-generation biomass

Lucas Nicolaas Ludovic van der Maas

Research output: Book/ReportPh.D. thesis

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Abstract

The current climatological and environmental problems created by our society’s reliance on fossil resources is one of the most complex challenges that has ever faced humanity. Industrial biotechnology can play a vital role in reducing our
dependency on fossil feedstocks for the production of chemicals and materials. Currently these biotechnological processes employ mostly model organisms and are based on so-called first generation feedstocks, comprising the edible fractions of agricultural crops like corn and sugarcane. This has the inherent downside that it competes with our own food supply and the availability of agricultural land, and therefore is suboptimal for large scale production from a cost and sustainability perspective. To alleviate these drawbacks, second-generation biomass, i.e. the non-edible parts of plants, should be used instead. Secondgeneration biomass has a high potential to be used for bioprocesses as it is renewable, abundantly available and does not compete with food supply. It consists of hexose and pentose sugars but during its processing, degradation products like furan and aromatic compounds derived from lignin are formed that
can inhibit the growth of microorganisms. The currently used model organisms like Escherichia coli and Bacillus subtilis can use both hexose and pentose sugars but have a low tolerance to the inhibitory degradation products. The tolerant cell factory strain Pseudomonas putida KT2440 can withstand the inhibitors but cannot naturally utilize pentose sugars.

In this thesis, two novel bacterial strains with a natural ability to utilize both pentose and hexose sugars and have tolerance to the inhibitors present in hemicellulosic hydrolysate derived from beech wood have been isolated from soil samples. Pseudomonas hemicellulosum is a novel Gram negative bacterium that is part of the well-known Pseudomonas genus. It can grow on a wide range of substrates including hexose and pentose sugars and some aromatic compounds
and is furthermore able to grow in acidic conditions at a pH of 4.0. Pantoea zelandia is also a novel Gram negative bacterium from the highly versatile but not well-studied Pantoea genus, with the ability to use several substrates such as
hexose and pentose sugars and some aromatic compounds as well, while being able to tolerate temperatures up to 42 °C.

To enable further use of these novel strains, a molecular toolbox has been developed for P. hemicellulosum and P. zelandia. The toolboxes include efficient electro transformation protocols in both stationary phase and exponential phase,
and compatibility with three (P. hemicellulosum) and 8 (P. zelandia) different origins of replication.

The characteristics of four constitutive promoters and five inducible promoters has been assessed for both strains as well at a single cell level. The basic molecular toolbox presented in this thesis provides a solid foundation for the further development of more advanced molecular tools. Pantoea zelandia and Pseudomonas hemicellulosum could alleviate some of the disadvantages of wellknown model organisms and could be turned into future cell factory chassis for the valorisation of second-generation biomass.
Original languageEnglish
PublisherTechnical University of Denmark
Number of pages148
Publication statusPublished - 2024

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