Browsing by Person "Kiefer, Dirk"

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Bioprocess strategies for efficient microbial conversion of acetate as alternative biotechnological carbon source derived from lignocellulose streams
(2024) Kiefer, Dirk; Hausmann, Rudolf
In near future, humanity will be faced with the global challenges from climate change, fossil fuel depletion to food shortage. Our current economic system will thus need to make a transition into a sustainable bioeconomy which uses key technologies like biotechnology for conversion of renewable feedstocks. The majority of all biotechnological carbon sources is generated from food plants. In context to the global population growth coming along with food insecurity, competing use of these food plants for human and animal nutrition creates a substrate dilemma for future biotechnology. Future biorefineries will thus need to realize a paradigm shift to carbon sources generated from non-competing or waste resources. As such, especially lignocellulosic biomass is regarded as the central feedstock for a bio-based industry. Among the different substrate streams obtained by lignocellulose refining strategies, the C2 carboxylic acid acetic acid (from now on referred to as acetate) is one of the most common occurring components. In contrast to its high economic role as building block in the chemical industry, acetate shows low relevance as biotechnological carbon source yet. This is mainly related to its inhibitory characteristic, the reason for why acetate has been used in food preservation for thousands of years. However, most industrial platform organisms are also capable to grow on acetate as sole carbon source. It also shows several attractive characteristics for bioprocessing which is why research studies on microbial acetate conversion have significantly increased in the past years. Together with the potential routes to generate non-fossil bio-acetate in large quantities from non-food competing resources like lignocellulose and biomass-derived/waste C1 gas streams, acetate might become a next-generation platform substrate for a future bio-industry. In order to make acetate-based bioprocesses competitive to those using conventional sugar-based carbon sources, adapted process strategies for efficient acetate conversion need to be established. The present thesis is aimed to address efficient bioprocess strategies for biotechnological conversion of lignocellulosic acetate into microbial biomass and growth-coupled products under fed-batch culture conditions. The experimental studies presented in this work were divided into two parts: In Part I, the potential of acetate as carbon source for high cell density cultivation of industrial platform bacterium Corynebacterium glutamicum was evaluated. Preliminary growth studies with different initial acetate concentrations revealed a high natural acetate tolerance for wild-type ATCC 13032 with growth at high acetate levels up to 60 g/L. In addition, it was shown that maximum growth rates (μmax) of 0.47 h-1 for acetate concentrations below 10 g/L are competitive to that on D-glucose as common carbon source. By implementation of an online feeding control which enables automated supply of pure acetic acid (HAc) via pH control, high cell density cultivation on lignocellulosic acetate was demonstrated for the first time in a 42 L stirred-tank bioreactor. Optimization of the molar carbon-to-nitrogen feeding ratio resulted in a highly efficient bioprocess yielding cell densities up to 80.2 g/L CDW after 28.9 h with biomass yields (YXIS) of 0.35 g/g and space-time yields (STY) of 66.6 g/L·d. Part II focused on the potential of acetate as carbon source for integrated protein production in C. glutamicum. Batch cultures of genome-reduced strains MB001/MB001(DE3) demonstrated that acetate clearly shows no inhibitory effect on protein production of eYFP as model protein using C. glutamicum as production host. Interestingly, comparative expression studies with C. glutamicum T7 expression system indicated an up to 83 % higher biomass-specific production on acetate compared to D-glucose as carbon source. By transferring the implemented pH-coupled online feeding control for high cell density cultivation of strain MB001(DE3) pMKEX2-eyfp in a 42 L stirred-tank bioreactor, this study showed efficient protein accumulation on lignocellulosic acetate yielding final protein titers of 2.7 g/L after 27 h with product yields (YPIX) of 40 mg/g and volumetric productivities (PV) of 0.10 g/L·h. In conclusion, the presented results demonstrate the high biotechnological potential of acetate as alternative carbon source. In contrast to most other studies before, it is shown that a suitable process strategy based on pH-coupled online feeding control allows efficient microbial acetate conversion under industry-relevant fed-batch culture conditions. This work provides exemplary proof-of-concept bioprocesses for high cell density cultivation on lignocellulosic acetate and its growth-coupled conversion into protein using industrial platform organism C. glutamicum. Therefore, the presented thesis contributes to the development of efficient concepts for microbial conversion of next-generation platform substrates like lignocellulosic acetate.