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.

Scheme of how ammonia would be implemented. 1. The engineered Saccharomyces spp. is cultivated in a bioreactor in the lab. 2. Alpha amylase produced by this GMO would be used as feed additive in cattle. 3. Leftover ammonia produced in the farm would be captured by a MOF (Metal Organic Framework) 4. When full, the MOF would be transported to the lab . 5. In the lab, the MOF would be heated up, causing the release of ammonia, which will be used as the nitrogen source for our yeast.

Increasing yield of alpha-amylase

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 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]. Moreover, even though we propose to first gather more data on the combinations that the explorative recommendations from the ART, based on the exploitative recommendations from the ART, we would for now produce alpha-amylase by cloning the B. subtilis alpha-amylase behind the αMF signal sequence and either the pPGK1, pTDH3 or pRNR2 promoter. Seeing as the ART does not give a clear preference for Saccharomyces spp. strain, we suggest cloning the construct in the strain that has the shortest and most consistent doubling time among replicates under several different ammonia concentrations: sYB76 (NCYC3597, S. cerevisiae). Seeing that we could not observe a clear tendency that would suggest the optimal ammonia concentration for growing the cells, the concentration of this nutrient doesn't necessarily need to be kept constant, which allows for more flexibility in ammonia concentration of the cell culture and thus more flexibility in how much ammonia the MOF should be able to capture.

Purification and admission of alpha-amylase

Since the feed additive is produced in Saccharomyces spp., it is necessary to extract the alpha-amylase from the cell lysate and supernatant. Ion Exchange Chromatography (IEC) is a widely used method to separate enzymes from different fluids. In the IEC, the stationary phase contains charged functional groups[2]. In the purification of enzymes, modern IEC columns with optimized separation conditions are known to give a high yield. Due to the similar molecular weight of the isoforms of enzymes (isoenzymes), it is impossible to purify enzymes from fluids by gel filtration. However, due to small differences in charge properties as a result from altered amino acid composition, purification of enzymes by IEC is possible[3]. To purify large scale produced alpha-amylase, the use of IEC would be ideal due to its large capacity.

As described in our project description, once alpha-amylase is purified and added to the cow feed, it will help break down the carbohydrates in the feed, thus reducing the formation of urea and ammonia and enhancing the growth and milk production of the cow. Therefore, it is not needed to further process the alpha-amylase and powdered alpha-amylase can be added to the cow feed making it accessible for farmers to use.

Capture and use of excess ammonia

The leftover ammonia from the pig and poultry barns will be captured by MOFs. These materials are characterised by exceptional adsorbance capabilities. However, up until recently, ammonia capture by MOFs seemed to be infeasible due to the corrosive nature of ammonia. In 2016, Rieth et al.[4] reported a class of MOFs that showed unprecedented stability upon ammonia adsorption and remained intact after 3 adsorption-desorption cycles. Since then, several materials[5,6] from the same class have been reported with improved stability and ammonia capacity. These results led us to believe that MOFs could become the most efficient way of ammonia capturing. Although the ammonia capturing by MOFs might currently be financially infeasible, we believe that further research in MOFs will lower their production costs and allow for wide industrial applications.

Once the MOF is saturated, pure ammonia can be yielded. The addition of pure ammonia to the nitrogen depleted culture media of the Saccharomyces spp. has a positive effect on the global Saccharomyces expression patterns[7]. This could potentially lead to a higher expression of alpha-amylase and therefore a higher yield. Secondly, with the addition of ammonia to the nitrogen depleted culture media no changes within the growth curve was observed and therefore it can be concluded that the addition of ammonia to the media has no harmto the 4 Saccharomyces spp. that we tested. 

Additionally, extracted ammonia from the MOFs that was not used in the bioreactor for the growth of the Saccharomyces spp. can be sold as pure ammonia. Currently ammonia factories are used to produce ammonia for chemical purposes, however, with our application pure ammonia would be harvested in a sustainable manner.

Future research prospects

To enhance the functionality and safety of BYE-MONIA, more scientific research should be conducted before BYE-MONIA is actually ready to be used “in real life” and before farmers make (possibly) costly investments. 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[8]. 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 enhanced fermentation of carbohydrates by ruminal microbes might lead to an increase in CO2 and methane production. The effect of alpha-amylase on the fermentation of carbohydrates by ruminal microbes 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 addition of alpha-amylase could have an effect on the microbiome of the cow. Moreover, the bacterial species composition of the microbiome could influence the functionality of interaction between the microbiome and the alpha-amylase, 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.


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