Rhodothermus marinus: a cell factory : Exploring products and cultivation technology

Abstract: The unsustainable human impact on ecology and ensuing climate change necessitates the reduction of greenhouse gas emissions. The largest contributing industrial sector is the chemical industry, due to its petrochemical dependency. For this reason, and for the finite nature of fossil fuels, the chemical industry is forced to shift to sustainable sources, such as biorefinery products. The biorefinery uses renewable biomass and converts it into fuel, energy, and chemicals through enzymatic catalysis and microbial conversion. Microbial production strains need both marketable metabolic products and good growth characteristics to be considered in the biorefinery. Genetically engineered microbial production strains have been termed cell factories. Most cell factories are mesophilic model organisms, but many biorefinery processes are improved by elevated temperatures. The use of thermophilic cell factories could improve biorefinery economy by decreasing costs associated with cooling. An interesting thermophilic candidate is Rhodothermus marinus. This organism was isolated from a submerged hot spring and has been a great source of thermostable glycoside hydrolases and is thus itself active on a broad range of substrates. Even though the bacterium has positive traits, the focus on its enzymes and its strictly aerobic metabolism has contributed in it being overlooked as a production organism.This doctoral thesis aims to explore the potential of R. marinus as a cell factory by development of cultivation technology and exploration of its products. Growth of R. marinus, in both shake flask and batch bioreactor cultivations, prematurely stagnates, before carbon and nitrogen sources are depleted. Feeding the initial nutrients did not alter this. When cultivated using sequential batch cultivation with cell recycling (SBCR), cell density tripled in comparison to normal batch cultivation. The reached cell density is the highest reported, so far, with R. marinus DSM 16675.Two metabolic products have been selected for study: exopolysaccharides (EPSs) and carotenoids. The EPSs monosaccharide composition of DSM 4252T and DSM 16675 differed slightly but consisted of xylose, arabinose and glucose, in descending order of concentration. The EPSs of strain DSM 16675 also consisted of mannose. Moreover, the EPSs composition, of both strains were affected by the type of growth medium and carbon source. FT-IR analysis detected sulfate bonds (O-S-O) and the presence of amino sugars. SBCR cultivation of strain DSM 16675 produced 19 mg/L of EPSs using Marine Broth 2216 spiked with 10 g/L maltose.The second product, carotenoids, were structurally analyzed using ultra high-performance supercritical fluid chromatography time-of-flight mass spectrometry. The previously elucidated Salinixanthin and its dehydroxylated variant, found in R. marinus DSM 4253, were also detected in strains DSM 4252T and DSM 16675. In addition, a non-ketolated Salinixanthin and dehydroxylated variant was detected in all strains. Measurement of antioxidant capacity tests (DPPH and TROLOX), in carotenoid-containing extracts, showed a 2-4-fold increase of antioxidant activity in comparison to a carotenoid pathway knock-out mutant. The native carotenoid acyl glycosides of R. marinus make comparisons with commercially successful production organisms difficult. Efforts were therefore made to genetically engineer R. marinus for the production of a marketable carotenoid. The study resulted in three mutants, of which a strain named TK-3 produced lycopene with a yield of 0.49 g/kg, as a sole product from the carotenoid biosynthetic pathway. Furthermore, analysis of the knock-out mutants gave insights of the carotenoid biosynthetic pathway of R. marinus. These studies lay the foundation for the development of R. marinus as a cell factory.