Abstract
BackgroundPectin is a rather complex and highly branched polysaccharide strengthening the plant cell wall. Thus, many different pectinases are required for an efficient microbial conversion of biomass waste streams with a high pectin content like citrus peel, apple pomace or sugar beet pulp. The screening and optimization of strains growing on pectic substrates requires both, quantification of the residual substrate and an accurate determination of the enzymatic activity. Galacturonic acid, the main sugar unit of pectin, is an uncommon substrate for microbial fermentations. Thus, growth and enzyme production of the applied strain has to be characterized in detail to understand the microbial system. An essential step to reach this goal is the development of online monitoring tools.ResultsIn this study, a method for the online determination of residual substrate was developed for the growth of the plant pathogenic fungus Ustilago maydis on pectic substrates such as galacturonic acid. To this end, an U. maydis strain was used that expressed a heterologous exo-polygalacturonase for growth on polygalacturonic acid. The growth behavior on galacturonic acid was analyzed by online measurement of the respiration activity. A method for the online prediction of the residual galacturonic acid concentration during the cultivation, based on the overall oxygen consumption, was developed and verified by offline sampling. This sensitive method was extended towards polygalacturonic acid, which is challenging to quantify via offline measurements. Finally, the enzymatic activity in the culture supernatant was calculated and the enzyme stability during the course of the cultivation was confirmed.ConclusionThe introduced method can reliably predict the residual (poly)galacturonic acid concentration based on the overall oxygen consumption. Based on this method, the enzymatic activity of the culture broth of an U. maydis strain expressing a heterologous exo-polygalacturonase could be calculated. It was demonstrated that the method is especially advantageous for determination of low enzymatic activities. In future, it will be applied to U. maydis strains in which the number of produced hydrolytic enzymes is increased for more efficient degradation.
Highlights
Pectin is a rather complex and highly branched polysaccharide strengthening the plant cell wall
If pectinases are available to degrade the polymeric substrate to monomers, monomeric galacturonic acid is consumed by U. maydis
The resulting method for prediction of the residual substrate concentration was applied for monomeric galacturonic acid but, more importantly, for polygalacturonic acid that cannot be quantified by offline measurement
Summary
Pectin is a rather complex and highly branched polysaccharide strengthening the plant cell wall. Many different pectinases are required for an efficient microbial conversion of biomass waste streams with a high pectin content like citrus peel, apple pomace or sugar beet pulp. The sugars required as a carbon source during the fermentation of agricultural waste streams usually originate from lignocellulosic biomass that is, e.g., thermochemically pretreated and enzymatically hydrolyzed [1,2,3]. U. maydis has the capability of unicellular and non-filamentous growth with a high resistance to hydromechanical stress. This makes U. maydis favorable over established filamentous strains, as hydromechanical stress is usually elevated in stirred tank reactors [3, 18]
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