With the use of a feed additive that minimizes the ammonia secretion from cattle and capturing left-over ammonia from pig and poultry barns with a Metal-Organic-Framework (MOF), our project BYE-MONIA is aimed to be an innovative, safe and circulair solution that tackles the (ammonia part of the) nitrogen crisis at the source. To create a full circle solution, we developed several proposed implementations that can be used by farmers to help tackle the Dutch nitrogen crisis together with other possible solutions. The ultimate goal is to deliver a feed additive to the farmers which is ready to use in cattle feed, next to that a MOF can be placed in the barn to be collected when saturated so that there is no additional work for the farmer. Our (integrated) Human Practices work helped us realize that it is unlikely that BYE-MONIA on its own is enough to protect all of Dutch nature, which suffers from more things than just excessive ammonia deposition, and that much additional research is needed before BYE-MONIA is ready to be proven effective and safe. However, we are convinced that, even though more still needs to be done, BYE-MONIA offers a set-up for a solution that, in combination with complementary solutions, has potential to help towards reducing both the burden on nature and the burden on farmers.

In order to increase the yield of enzymatic feed additive alpha-amylase, large scale cultures of our engineered Saccharomyces spp. are needed. To achieve this bioreactors can be used, in which large amounts of Saccharomyces spp. can be cultured in liquid media. The alpha-amylase produced in Saccharomyces spp. is present intra- and extracellular, which we saw after testing the break-down of starch. In order to have a higher yield of alpha-amylase it would therefore be needed to not only use the supernatant of the Saccharomyces spp. cultures, but also the intracellular fluids. In order to achieve this, it would be needed to lysate the cells. During our experimental phase we used a bead beater to lysate the cells. To enlarge the scale of lysating Saccharomyces spp., cooled grinding with glass beads can be used in which large volumes of Saccharomyces spp. cultures can be lysed without denaturation of the enzyme ^[1].

To enhance the functionality of BYE-MONIA, more scientific research should be conducted. The main research phrase that remains is the functionality of the feed additive alpha-amylase. Current research shows the effect of alpha-amylase on the milk production of cattle[5]. However, no (extensive) research was performed on the effect of alpha-amylase on the emission of ammonia. In parallel to that, other gas emissions need to be observed. For instance, the change in the digestional tract could lead to an increase in CO2 and methane production. The effect of alpha-amylase on the digestive system of the cow is explained on the description page. During one of the stakeholder interviews for (integrated) human practises, the increase of these two gasses was mentioned and should therefore be taken into account when performing further research. Lastly, the change of digestion by the addition of alpha-amylase could have an effect on the microbiome of the cow. Not only can deposition of bacteria change the functionality of interaction between the microbiome and the alpha-amylase, it could also have an effect on the complete digestional tract of the cow as was discussed during one of the stakeholder interviews for (integrated) human practises. These effects differ per cow breed since the microbiome could differ greatly per individual. Therefore, extensive research needs to be conducted on the effect of alpha-amylase on the microbiome of the cow. 

After the experimental wet lab phase and processing the quantitative data from the alpha-amylase assay kit, the ART recommended testing several combinations of promoters, signal sequences and genes. For further development of BYE-MONIA, these recommended combinations should be tested prior to large scale production of alpha-amylase to find the ideal combination.