Team:GenetiX Tec CCM/Design
So what is denitrification?
The denitrification process starts with several metalloproteinase catalyzing the transformation of nitrites to nitrates; these nitrates are then transformed to nitric oxide; which in turn are converted into nitrous oxide; and finally into molecular nitrogen.
Within the nitrogen cycle, there are two processes complementary to each other: nitrification and denitrification. Both are conducted by different kinds of bacteria. The first one, nitrification, is an aerobic process, which transforms ammonia (NH+4) to nitrates (NO-3). On the other hand, denitrification, is an anaerobic process, which “continues” the process of nitrification, and which can be summarized with the following reactions’ schematic:
Due to the nature of nitrates and nitrites as potential eutrophication factors, the nitrification-denitrification process is often exploited by governments to reduce the amount of NO-3 and NO-2 present in water where wastewater is disposed, transforming them into N2. As denitrification is a process entirely carried out by bacteria. We see an opportunity to create a biological system which is capable of carrying out this process for the same purpose, but only within regulated conditions or parameters that we are capable of controlling and establishing beforehand.
In the figure 1 the denitrification process is explained. Denitrification encases many reductions of nitrogen and oxygen bonds, for example the NO-2, NO-3 and NO. This is a multistage process and so the diagram explains it broadly. The first cluster to take action in the chain is the Nar cluster, in here the NarH, NarG and NarL reductases take Nitrate (NO-3) and transform it into NO-2, leaving Oxygen behind. After this the next stage is taken by the Nir cluster. In this cluster the NirQ protein starts to encode for Nitrites in the environment, and so the Nitrites are further reduced to Nitric Oxide (NO).
The next process starts when the bacteria bonds the nitrogen dissolved into the Nitric Oxide by the means of the Nor Cluster to form Nitrous Oxide (N2O) , effectively lowering levels of Nitrogen in the water. The last part of the process involves reducing further the newly formed Nitrous Oxide into its basic components, Molecular Nitrogen and Oxygen. This final part of the process is achieved by the Nos cluster; after it is decomposed into both the Nitrogen and Oxygen the Nitrogen is released as gaseous element and the Oxygen is dissolved in the water. As a result the oxygen levels rise, reducing the anoxia.
Previous Work
Last year our team participated in the high school division with a project that started the development of a biosensor using an oxygen (BBa_K258005) and iron (BBa_I765000) promoter, this activates a reporter. We took the GFP (BBa_E1010) and mRFP (BBa_J04650) as indicators. The system was created in order to develop a precise and economical way to measure low levels of oxygen dissolved in water. Iron promoter was used to exemplify as a method to quantify heavy metals as Hg or Pb. Creating the biosensor helps to report the actual conditions of the water in Xochimilco. Ultimately this information would reach the citizens, council and governmental institutions. For further information about our last project click here
Why pseudomonas stutzeri
Pseudomonas stutzeri is a non-fluorescent denitrifying bacteria, from which some strains have been classified as opportunistic pathogens for humans. For the past 15 years, P. stutzeri has been studied due to its particular metabolic properties which consists in the study of their strains. Several of its strains have natural transformation properties, this stands that the bacteria is mainly able to do by itself are:
Fix dinitrogen
Degradation of pollutants or interactionwith toxic metals
Denitrifying
P. Stutzeri is one of the few organisms who have a high effectivity in completing the whole cycle of denitrifying, it has even been recognized as a model system for denitrification process with 95% of effectivity. This is crucial for our experiment due to the danger in the denitrifying process. Danger can start when the denitrifying strain activates and suddenly stops in nitrous oxide or nitric oxide which are highly poisonous gases. This happens because the denitrifying cluster has a low effectivity rank in most of pseudomonas.(Lalucat, Bennasar, Bosch, García-Valdés, and Palleroni, 2006).
Click here to see the data base of the cluster and genes of the pseudomonas stutzeri
How does the denitrification process work in the pseudomonas stutzeri
Denitrification process is a complex biological system (Figure 1). It is a multistage procedure that encompases four nitrogen reductions. The first step is Nitrate (NO-3) reduction, which is performed by the proteins encoded by the Nar gene cluster: NarH, NarG, and NarL reductases. It ends with the formation of Nitrite (NO-2), which becomes the reactant for the next reaction, also a reduction. This next step is in accomplished by the Nir cluster. This gene set functions after NirQ is activated by the presence of nitrites and further reduce them to Nitric Oxide (NO). Then, the Nor cluster-encoded proteins bond dissolved nitrogen to form Nitrous Oxide (N2O). The last part of the system is achieved by the Nos gene cluster. These proteins decompose Nitrous Oxide into its main components: nitrogen and oxygen. The first one is released in its molecular formed, as a gas, to the atmosphere, while the second one remains dissolved in the water. As a result, oxygen levels rise, reducing anoxic conditions.
What do we plan doing with the biosensor and the denitrification processes?
Our aim for the future is to combine our biosensor and the denitrification process into a single interconnected system. This would work based on the output of the Oxygen promoter (BioBrick Part BBa_K258005). If it detects an oxygen level lower to 2% in the water, it will permit the beginning of the denitrification process, by allowing the replication of the gene clusters (Nar, Nir, Nor) which start the reduction of nitrates and nitrites.
After the denitrification process has been active for an extended period of time, our hypothesis is that the Nymphaea specimens in the lake will diminish due to the decrease in Nitrates and Nitrites, since they are nutrients for water lilies. Therefore, and also as a direct consequence of the biological pathway, O2 levels in the water should normalize, making our system behave differently. After the Oxygen promoter detects normalization in the lake’s O2 levels, the bacteria will stop denitrifying the ambience and will report a green glow with the GFP reporter (BioBrick Part BBa_E1010). This will indicate the process has been successfully completed and the O2 levels have been increased to optimal ones.
The regulation is necessary as we cannot completely remove the NO-3 & NO-2 from the lake; doing so would result in affectation to all vascular plants and other photosynthetic organisms. This, in turn, would end up in a new unbalance of the ecosystem. By modifying the behaviour of the system accordingly to the Oxygen levels we can ensure that we do not damage the dynamic of the lake once we reach the desired results.
The method to achieve this is constructing plasmids with which we will transform E. coli. The system inside the bacteria will work though positive feedback. By this, the product of one reaction will stimulate the expression of the genes that encode for the proteins in charge of the next process. In the very end, we aim to have a bacterium that contains the biological system, able to respond to its environment.
In order to create this we will study the Nir and Nor cluster in order to implement the previews statement about the system. We figured out that the NirS, NirK, NirT,B,M are the key factor in producing proteins to carry out the process.