Solid-state fermentation of the microalgae Scenedesmus sp. for improved conservation and protein digestibility

English. As part of a research grant aimed at producing chicken feed from the novel microalgae Scenedesmus sp., called the ReMAPP project, this thesis investigates opportunities to increase the conservation potential and protein digestibility utilizing solid-state lactic acid fermentation and commercial enzyme additives. The lactic acid fermentation mimics the traditional agricultural ensiling process, where crops are anaerobically fermented at room temperature for upwards of a year. The ensiling process conserves the material by using lactic acid bacteria to convert water soluble carbohydrates in the biomass to mainly lactic acid, lowering the pH and preventing growth of pathogens. This approach to microalgae conservation has not been attempted previously. Enzyme additives are hypothesized to degrade cell wall polymers to monosaccharides and are used to increase the substrate for lactic acid production, further decreasing the pH and thus improving the silage quality of the microalgae. At the same time, the protein digestibility of the specific microalgae species is hypothesized to be limited by bioavailability as the incalcitrant cell wall of Scenedesmus could prevent access to intracellular proteins, rendering them indigestible. The same enzyme additives used to improve fermentation could also affect the protein digestibility, since degradation of cell wall polymers could release intracellular proteins. To investigate the fermentation properties and protein digestibility, lab scale fermentations are carried out where Lactoplantibacillus plantarum and a variety of enzyme additives are mixed with Scenedesmus sp. biomass and left in vacuum-packed bags to ferment for 21 days. The results show bacterial inoculation by itself to be effective in decreasing pH to desirable levels but fermentation with β-glucanases, which seem to degrade polyglucans to glucose which are consumed by the lactic acid bacteria, proved the most effective. Only fermentations with β-glucanases achieved pH < 4.0, a benchmark for product safety. The effect on protein digestibility did however seem to be largely negligible. Lactic acid fermentation by itself did increase the protein digestibility, measured in PDCAAS, from 0.79 to 0.83 which is a relevant increase but still below the gold standard set by soybean. Using additional enzymes had only a marginal effect on increasing protein digestibility, and as such the hypothesis of degrading the cell wall to increase protein digestibility cannot be confirmed by this study. Endoproteases increased protein digestibility the most, the do likely not increase protein digestibility by degrading the cell wall but rather by hydrolyzing peptides. The most successful non-protease in terms of protein digestibility was a pectinase which was unexpected since no galacturonic acid, the main monomer of pectin, could be detected in the sample. To determine the cause of increase in protein digestibility and the effect pectinases have on the biomass, in-depth characterization on the biochemical composition of Scenedesmus sp. would be required. In conclusion, solid-state fermentation of Scenedesmus sp. biomass using lactic acid bacteria and enzyme additive was successful and could potentially be a useful conversation method for green microalgae in general. While some enzymes tested did yield an increase in protein digestibility where a pectinase and an endoprotease proved most effective, the hypothesis of releasing intracellular proteins to increase digestibility in combination with fermentation could not be confirmed.

English. Microalgae are, as the name implies, microscopic individual algae cells which are present in almost every natural water source in the world. Thousands of different types of microalgae exist, where a few have been found to both be easy to grow in reactors and contain a lot of protein. One recently discovered kind, called Scenedesmus sp., is up to 55% protein and grows quickly which makes it an interesting candidate for creating cheap and sustainable chicken feed. The science of using microalgae in food and feed is very new, and a lot of research is required to make production cheaper and competitive with already existing feed alternatives like soybeans. Two of many important questions to answer to create a successful product is how to store the algae without it rotting and how to draw out protein out of the algae cells. The first question is applicable to many foods. If the algae are left for too long dangerous bacteria will grow on it, making in unsuitable to eat. How can we prevent this? The second question is more specific to microalgae. The small cells have thick cell walls, which can be thought of as a shell and prevents the animals eating the algae from absorbing the protein since the protein is inside the shell. Can breaking the cell wall increase the amount of protein which the animals can absorb? This project attempts to answer both questions using the same method: fermentation with both lactic acid bacteria and enzyme mixtures. Lactic acid fermentation is a very popular method in many foods, like when creating sauerkraut and kimchi. Essentially, safe bacteria (in this case one called Lactoplantibacillus plantarum) consume the sugars in the algae and create lactic acid, which makes the whole material very acidic. Other dangerous bacteria which cause rotting cannot grow in high acidity, and as a result the algae are preserved. To improve the fermentation, enzymes which break down the cell wall into smaller parts can be used. The cell wall is in large part composed of cellulose, which is a long molecule composed of many individual sugar molecules. The enzymes release the individual sugars from the cellulose, which the lactic acid bacteria can eat and create more acid to make the material even more inhospitable to dangerous bacteria. At the same time, it is hypothesized that the cell wall being broken down by the enzymes could also release proteins from the inside of the cell. The same enzymes which improve the fermentation quality could as a result also be found to increase the protein digestibility of the microalgae. The study did find the fermentation part of the project to be successful. The acidity of the algae did increase significantly when only fermenting with the added safe bacteria. When also adding the cell wall degrading enzymes called β-glucanases, the algae became even more acidic, to the point where it is acidic enough deny any growth of dangerous bacteria. It was also found that a lot more of the sugar in the algae had been eaten when the enzymes were added, which indicates that the hypothesis was correct. However, no such clear confirmation could be found in increasing the protein digestibility. Even though the cell wall seemed to be damaged, almost no difference in protein digestibility was detected. Some enzymes did have an effect. Endoproteases increased protein digestibility, although they break down proteins into smaller parts which are more easily digested which means they work in a different way than what was hypothesized to increase the protein digestibility. Another enzyme, pectinase, was found to be somewhat successful in increasing the digestibility. However, pectinases only break down a substance called pectin, which could not be found in the algae. It is therefore unknown why it worked at all. To conclude, while fermentation seems to be a good way to conserve algae, it could not be said to have a meaningful effect on increasing the protein digestibility.