Investigations on the use of defined co-cultures for the consolidated bioprocessing of cellulose to itaconic acid

Schlembach, Ivan Bernd Maria; Agler-Rosenbaum, Miriam (Thesis advisor); Blank, Lars Mathias (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019


Lignocellulosic biomass is the most abundant renewable resource on earth and holds the potential to replace fossil resources as sustainable substrate for the production of fuels, various polymers and other chemicals. An important intermediate step for the valorisation of lignocellulose in this context, is the conversion into platform chemicals, which serve as precursors for the production of a broad range of final products. However, the major barrier thereby is the high recalcitrance of lignocellulose towards chemical and biological transformation. One concept is the enzymatic saccharification of lignocellulosic polysaccharide constituents into monomer sugars, which can then be used as substrates for various fermentative transformations. Although technically possible, the economic feasibility is affected by the high costs associated with the execution of three separate processes; the production of cellulolytic enzymes, the saccharification of the lignocellulosic feedstock and the fermentation of the resulting sugars into the target product. Therefore, the combination of these three sub-processes into a single consolidated bioprocess (CBP) is expected to be a key technological breakthrough to make lignocellulose valorisation economically viable. The aim of this thesis was the development of a consolidated bioprocess for the direct conversion of cellulose to the platform chemical itaconic acid. Because no organism is available that can efficiently produce cellulolytic enzymes and itaconic acid at once, the development of a mixed culture consisting of a specialized itaconic acid producer and a specialized cellulase producer was targeted. After an initial assessment of available itaconic acid producing organisms, the currently industrially applied fungus Aspergillus terreus was selected. However, it is known that A. terreus only produces itaconic acid when the prerequisites of a strict manganese deficiency, a low fermentation pH, a high initial sugar concentration and a non-interrupted oxygen supply are fulfilled. As a first step to elaborate the most feasible process setup, the challenges associated with the fulfilment of these prerequisites in a CBP scenario were investigated and the possibilities to overcome these challenges were explored.It was found that the extreme sensitivity towards manganese precludes the use on non-purified substrates. Manganese concentrations as low as 22 µg/L reduced the itaconic acid yield by more than 90% and laboratory grade α-cellulose pulp was shown to contain enough manganese to completely inhibit itaconic acid production. Itaconic acid production in presence of cellulose was only possibly after extensive washing of the pulp using dilute sulfuric acid. As the optimum itaconic acid production pH of 3.4 is far more acid than the optimum cellulose hydrolysis pH of 4.5 used with commercial cellulase enzymes, alternative cellulase producers that were better adapted to the low pH conditions were screened. Penicillium verruculosum was thereby identified as promising alternative to the best publicly availably cellulase producer Trichoderma reesei Rut-C30, showing higher sugar release rates, faster enzyme induction and improved cellobiase activity under the targeted process conditions. However, for induction of itaconic acid production, the A. terreus culture has to experience a pH below 2 during the initial growth and it was found that such low pH irreversibly inactivates the cellulase enzymes of both T. reesei and P. verruculosum. Furthermore, the need for sugar concentrations above 120 g/L to induce high yield itaconic acid production was found incompatible with CBP. New methods were developed to predict and measure the sugar release during CBP. By systematically analysing the influence of cellulose concentration and enzyme/substrate ratio, it was found that the sugar release rate was directly proportional to the cellulose concentration but only proportional to the logarithm of the enzyme/substrate ratio. Therefore, the sugar release rate can be most effectively increased by increasing the cellulose concentration in the process. The practically usable cellulose concentration is however limited by the hydro-mechanical properties of the cellulose slurry. With increasing solids concentration, the slurry becomes hard to mix and aerate. This especially impedes the fulfilment of a non-interrupted oxygen supply, which can block itaconic acid production within a few minutes. Furthermore, the importance of cellulose digestibility was highlighted as a third factor to increase the sugar release rate and a drastic decrease in cellulose digestibility over the cultivation time was identified as central barrier for continuous cellulose hydrolysis at a high rate. Therefore, even under optimistic assumptions and application of extreme enzyme loadings of more than 250 FPU/g, an accumulation of high sugar concentrations in unlikely.It was attempted to partially solve the described challenges by performing the targeted co-culture in a sequential manner. First, the cellulase producer was cultivated in fed-batch mode to accumulate a high cellulase titre, then large amounts of cellulose were added to the culture and the fermentation temperature was increased to achieve an accumulation of sugars. Finally, a pre-induced pre-culture of A. terreus, that was grown at a starting glucose concentration of 125 g/L and that experienced low pH phase was introduced. This strategy was performed for both a T. reesei and a P. verruculosum based co-culture with A. terreus. However, despite reaching high cellulase titres of more than 11 FPU/mL and using cellulose concentrations as high as 120 g/L, the maximum achieved sugar accumulation was only 22.5 g/L and only 0.3 g/L of itaconic acid were produced in case of the T. reesei based co-culture. The P. verruculosum co-culture produced no itaconic acid.Besides the general production capabilities of the single mixed culture organisms, also their interaction pattern is of critical importance. Different methods to independently quantify the population dynamics between a model co-culture consisting of A. terreus and T. reesei were developed and compared. Both organisms were genetically engineered to express different fluorescence proteins. Especially fluorescence microscopic image analysis and online fluorescence measurements were found suitable to study co-culture interactions in a cheap and fast manner. The gathered results were validated using an independent qPCR based method. By investigating the population dynamics between A. terreus and the cellulolytic mixed culture partners T. reesei or P. verruculosum, it was found that neither of the two combinations evolves into a stable mixed culture, instead the more dominant organism displaces the inferior partner. Thereby, A. terreus was found more dominant than T. reesei, while P. verruculosum was found more dominant than A. terreus.The presented results clearly point out, that unless all of the described challenges will be overcome in future, an A. terreus based consolidated bioprocess will not be feasible. Instead, other organisms should be investigated whose itaconic acid production conditions do not contradict the principles of CPB. In this regard, promising results were gathered in a simultaneous saccharification and fermentation (SSF) setup using an engineered strain of the alternative itaconic acid producer Ustilago maydis. More than 18 g/L of itaconic acid could be directly produced from both amorphous Sigmacell and more crystalline α-cellulose, which is the highest reported titre ever achieved in an SSF setup.Although the initial goal of establishing an effective mixed culture CBP for the direct production of itaconic acid from cellulose could not be fulfilled yet, important new methods for the general setup and characterization of mixed culture based consolidated bioprocesses were developed that can be applied in future for the development of other CBP processes.