Trichoderma harzianum P49P11 was selected among several other microorganisms at the Biorenewables National Laboratory (LNBR, Brazil) and it was considered a promising strain to produce cellulase. Here in this project, a mathematical model and simulation platforms were developed as potential tools to be used for cellulase maximization using fed-batch mode (Chapter 2). Feeding strategies were simulated to maximize cellulase production, at first, only using cellulose as the substrate, and then using glycerol for cell growth and cellulose for cellulase production. Although the mathematical model and simulation platforms were built up for a wild type strain, these tools help to predict data and they can be adapted for optimized strains.
Chapter 3 evaluates the continuous production of enzymes using different carbon sources under carbon-limited conditions. It was found that glucose has a positive influence on the production of enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (PNPGase). Sucrose and fructose seem to inhibit PNPGase synthesis; however, these substrates could also have a positive influence on the synthesis of other enzymes not evaluated in this project. Cells can uptake glucose without the need to synthesize extracellular enzymes like PNPGase. The increase in the production of PNPGase during the continuous culture using glucose as the carbon source indicates the presence of inducers. It was also discovered in this project that polysaccharides were present in the supernatant of all conditions using glucose, fructose/glucose and sucrose (Chapter 4 and Chapter 5). This suggests that the possible inducers could have come from fragments of the extracellular polysaccharides.
Sugar analysis showed the presence of sugar with the same retention time as gentiobiose in the supernatant of the conditions using glucose as the carbon source, which could be a fragment from polymers released from the cell wall. Gentiobiose could be acting as an inducer of enzymes. In addition, a mechanism was also proposed for continuous PNPGase production under glucose-limited conditions assuming that PNPGase includes beta-glucosidase (Chapter 4).
The carbon sources used under carbon-limited conditions influenced the PNPGase productivity and possibly the whole enzymatic cocktail secreted by the fungus. For this reason, shotgun proteomics and SDS-PAGE analysis were performed for the proteins present in the supernatant of the conditions using glucose, fructose/glucose and sucrose (Chapter 4). The shotgun proteomics analysis suggested that the different carbon sources used provided the production of different extracellular proteins including several uncharacterized proteins, which can also include different enzymes. This brings the possibility of creating a hypothesis that different carbon sources easily assimilated by the cells could lead to the synthesis of different inducers (fragments of extracellular polysaccharides), which could induce the synthesis of different enzymes under carbon-limited conditions.
Extracellular polysaccharides were the by-products discovered in this project during the production of enzymes under carbon-limited conditions. The behaviour of intracellular metabolites (glycolysis, citric acid cycle, pentose phosphate pathway and nucleotides) was evaluated under four different conditions in duplicate during the production of extracellular polysaccharides by Trichoderma harzianum under carbon-limited conditions (Chapter 5). This chapter has provided the first step for the optimization of the production of extracellular polysaccharides and the information about the behaviour of intracellular metabolites using this wild type strain is essential to the development of optimal strains.