Quantitative bioprocess development with extreme halophilic microorganisms for waste to value applications

Abstract

Halophiles are capable of growing on a wide variety of carbon sources and can survive up to saturated NaCl concentrations that exhibit inherent cultivation selectivity. Extreme halophilic archaea are a yet unexploited source for the production of value added halophilic bioproducts such as carotenoids. Microbial carotenoids can offer a natural alternative to the commercially available synthetic carotenoid food colorants and antioxidant products. Hence, the capacity for cost-effective non-sterile cultivation can make haloarchaea potentially valuable microorganisms for biotechnological applications. Following a "waste to value" principle by coupling industrial streams and converting the organic by-products, which are contained in several kinds of waste streams, various valuable bioproducts can be produced. As sustainable process intensification solutions, this possibility opens up feasibilities for economic and ecological rationalizations of chemical and biotechnological processes with stream couplings. Although the ecology and physiology of haloarchaea are widely identified on the microbiological level; little emphasis has so far been laid on quantitative bioprocess development. At elevated salinities, however, material corrosivity issues occur and the performances of process analytical as well as quantification techniques are strongly affected. The main goal of this PhD thesis was to create a general quantitative bioprocessing platform of extreme halophilic archaea with also showing process-oriented and product-oriented applications of the platform. A) A corrosion resistant bioreactor setup was implemented with special attention to the bioprocess quantification approaches. With providing defined and controlled cultivation conditions in the bioreactor and therefore obtaining datasets of suitable quality, robust quantitative data evaluation methods for haloarchaea was developed. B) The physiological characterization of extreme halophiles in bioreactor was also assessed. Stoichiometry and the kinetics was investigated on various substrates, which are common organic residues in industrial waste streams, and the results of this study were interlinked with the published primary carbon metabolism of extreme halophilic archaea. C) In order to avoid growth-affecting medium limitations, a set of shake flask and bioreactor DoEs were carried out to develop a defined medium for extreme haloarchaea. Regarding the limited analytical facilities available in hypersaline medium, novel analytical solutions had to be implemented for monitoring nutritional limitations. D) Moreover, dynamic experiments for process parameter pH and Temperature optimization for accelerating bioprocess development with extreme halophilic archaea were introduced. E) To overcome the physiological limit of low biological activity and to achieve increased volumetric productivity, a bioreactor setup equipped with an external cell retention hollow fibre membrane unit was characterized and parameterized, applying the developed feeding and bleeding strategy. Focussing on maximizing the volumetric productivity, 10-fold productivity increase was achieved compared to chemostat continuous cultures. The two real medium examples encompass process-oriented applications of the general quantitative bioprocessing platform with extreme halophiles. On one hand, the first example describes a process intensification example, where chemical plant "A" is coupled to chemical plant "B" via the hypersaline effluent stream with a halophilic biological unit. On the other hand, the versatility of potential application areas for the bioprocessing platform with halophiles are also demonstrated by another biological example. During dark fermentative anaerobic biohydrogen production, various small metabolites are remaining in the liquid process effluent, which are - with NaCl addition to the effluent stream - potential C-sources for halophiles to produce value added bioproducts. The identification of the lipophilic bioproduct portfolio and downstream processing of bioreactor samples of extreme halophilic archaea was presented. The applied HPLC-MS/MS identification method using atmospheric pressure chemical ionization revealed, that, in contrast to literature on shake flask cultivations, only the seven geometric isomers of a C50 carotenoid, Bacterioruberin and no anhydrous analogues, as by-products of the carotenoid biosynthesis, were detected in bioreactor sample extracts of the extreme haloalkalophilic archaeon, Natronobacterium gregoryi. In case of bioreactor samples, not only was the product impurity beneficially affected, but also multiple-fold carotenoid content was measured. Furthermore, due to the lack of commercially available Bacterioruberin (BR) analytical standards, antioxidant assays were implemented to provide semi-quantitative information on the Bacterioruberin content of the sample extracts. Although the carotenoid production of extreme halophilic archaea is widely documented, less is known about the parameters that can influence carotenogenesis. Thus, for the product-oriented application example of the platform, various strategies were applied to identify growth and bioproduct carotenoid formation affecting process parameters. Among others, the effect of medium limitations with special focus on the nitrogen source in the medium was investigated. Additionally, along the medium limitations studies, it was demonstrated for the first time that the extreme haloalkalophilic archaeon, Natronobacterium gregoryi can also produce PHB.