2023: Combating Nitrate Pollution with Synthetic Biology

2023: Combating Nitrate Pollution with Synthetic Biology

Every 6th groundwater sample in Germany shows an excess of the maximum allowable concentration of nitrates per liter of water. In 2018, the European Court of Justice even fined Germany for violating the rules of the nitrate code.(Umwelt Bundesamt, 2022) Nitrates enter the environment as a result of agricultural activities and other man-made factors, they harm the entire ecosystem and pose a health hazard, contributing to the development of various cancers, anemia, cardiovascular diseases, sepsis etc. (Singh et.al., 2022)

But What is Nitrate Pollution

Nitrate, the oxidized form of dissolved nitrogen, is an essential nutrient to plants for their growth. In nature, nitrogen is present in the form of nitrates, nitrates, and ammonia but as the latter two are unstable and easily converted to nitrates, most of the nitrogen present naturally is in the form of nitrates. (Yu et al., 2020) In nitrogen-containing fertilizers, besides nitrates, nitrogen can be applied in the forms of ammonium and amide which can then mineralize and produce nitrates through a quick process. (Majumdar et al., 2000)

These excessive amounts of nitrates present in the environment refers to nitrate pollution and when these sizable amounts of nitrates are present in the water, they can have an impact on the quality of water and can therefore have negative health and environmental effects.

And Where does it come from?

With the benefits nitrates provide to plant and crop growth, they are often used in fertilizers. When the soil containing nitrates due to these agriculture methods is subject to heavy precipitation, it can become prone to irrigation and leeching, if not taken up by plants or denitrified to nitrous oxide or nitrogen, since nitrates have a high solubility in water as well as low retention by soil particles. (Majumdar et al., 2000) This leads to nitrate entering groundwater which can then be highly contaminated with nitrates. (Kaur et al., 2020) Groundwater can be contaminated due to non-point sources such as fertilizers etc, as well as due to point sources which include industrial pollution etc. (Almasri, 2007) The nitrates contaminating the groundwater eventually end up in drinking water and these high amounts of nitrates prevent the water from being drinkable. (Abascal et al. 2022) Hence, major sources of nitrate pollution include irrigation water with fertilizers as well as improper waste disposal, animal waste, wastewater treatment plants and other industries, landfills, and septic tanks. (Abascal et al., 2022)

Nitrate is a natural part of the nitrogen cycle, but these activities create an imbalance in the cycle.

Picture retrevied from Beta Analytic

Why is it harmful?

Nitrate pollution has serious health and environmental impacts.

When consumed in a high concentration, nitrates can have a harmful impact on human health:

  • Excessive consumption of nitrates can cause methemoglobinemia, or baby blue syndrome, a severe blood disease. This can happen since nitrates, converted to nitrites, react with oxygen to oxidize many essential substances necessary for the functioning of a healthy human body. The disease itself occurs when hemoglobin is turned into methemoglobin, preventing it from transporting oxygen. The high methemoglobin levels further reduce the amount of oxygen traveling through the bloodstream and hence lower the overall oxygen levels in the body. (Miodovnik, 2009) Effects of methemoglobinemia include the skin turning a bluish color, decreased blood pressure, increased heart rate, dark brown coloration of blood, lethargy, headaches, stomach cramps, vomiting, and even death. This disease mostly affects infants and pregnant women with bottle fed babies under six months old having the highest risk. (Manassaram, 2010) A person having anemia, cardiovascular disease, lung disease, sepsis, glucose-6-phosphate-dehydrogenase deficiency, and other metabolic problems has a higher chance of developing nitrate induced methemoglobinemia.(Kaur et al., 2020) Having more than 2% of methemoglobin shows that the person has the disorder. (Miodovnik, 2009)
  • High levels of nitrates can also cause birth defects, one of which includes a neural tube defect in which the neural tube of an unborn child turns into the brain and spine. (Ward et al., 2018)
  • Nitrates can also increase the risk of thyroid disease by blocking the uptake of iodine. As the thyroid needs iodine to make hormones, the now low levels of the hormones can result in fatigue, weight gain, dry skin, hair loss, and goiters. (Ward et al., 2018)
  • Nitrate pollution can also potentially lead to cancers, especially gastric cancer, as nitrates can enhance the cancer potential of compounds. (Ward et al., 2018)

Nitrates also have serious environmental effects when present in large quantities:

  • Eutrophication can result if there is constant nitrate contamination in water bodies. It can occur due to the spread of invasive plants like algae and phytoplankton that feed off of nitrates and have access to large quantities of it. This leads to foul-smelling algal bloom in the water body, referring to the explosive growth of algae due to the runoff of nitrate rich soil into nearby rivers and streams caused by heavy precipitation. Some algal blooms are called harmful as they secrete toxins, poisoning the drinking water, fish, and marine birds and mammals feeding off of them resulting in so-called “dead zones” that prevent any life from surviving. (Murray et al., 2019) The algae can consume oxygen as they decay which leads to eutrophication: aging of the water body turning it into a swamp like environment. 
  • Nitrate water pollution can have negative effects on the biosphere. With the decreased levels of oxygen present in the bodies of water, many plants and freshwater animals such as fish, invertebrates and amphibians are subject to suffocation and could possibly die. Nitrate pollution is also correlated with the loss of diverse macrophytes since certain types (e.g. charophytes) only survive in clean, unpolluted water bodies. (Lambert et al., 2010) The nitrate toxicity level towards freshwater aquatic animals also increases with the increased nitrate concentrations since freshwater animals are particularly sensitive to pollutants. (Camargo, 2005) Therefore, if there is a constant long term exposure of nitrates in the environment, ecosystems can be destroyed and resulting in extinction of  certain species and general ecological damage. 

Other impacts of increased nitrate concentrations in the environment can affect lifestyles universally. Nitrogen fuelled algal blooms can limit the use of lakes, rivers, and other water bodies for swimming, boating, fishing etc. resulting in effects on economies, and tourism.

Why is it important? 

Nitrates are one of the main causes of water pollution in Europe and nitrate pollution is a serious issue all over the world.

Global map showing zones with high nitrate presence retrieved from IGARC

EU regulations have limited the maximum amount of allowable nitrate concentrations to be 50 mg/l and for nitrite, 0.5 mg/l in drinking and ground water in order to protect health. However, in order to achieve these standards, costly programmes must be implemented by public utilities and water companies in nitrate concentrated areas. This is even more challenging for private instances and it has been reported by WHO, that private well use is often related to recent methemoglobinemia cases. (Murray et al., 2019)

Combating Nitrate Pollution

In order to prevent nitrate pollution and lower the amount present in drinking water, there are several strategies in use. 

  • Limiting the amount of nitrogen applied: this can directly influence the amount of nitrates present and hence reduce nitrate leaching. This can be done by using slow-releasing nitrogen sources and avoiding over-irrigation.
  • Environmental protection policies and management practices: many governmental policies are addressing the reduction of nitrogen pollution which include the Nitrate Directive, Water Framework directive, Groundwater Directive, Ambient Air Quality, National Emissions Ceilings Directive, Urban Waste Water Treatment Directive in Europe. (Zhou, 2015)
  • Wastewater management can aid majorly in lowering nitrate pollution in the environment.
    • Septic tanks: these are underground household water treatment systems in which toxic materials, including nitrates, can be separated from the water before releasing it into the soil. However, these hold a huge disadvantage as the material collected overtime, may seep into the soil as well with time or if the septic tank malfunctions. As these contribute to a high amount of nitrate pollution in groundwater, this is not a satisfactory solution.
  • Drinking water standards: by establishing certain drinking water standards for the presence of nitrates (50 mg/l), WHO and other local environmental protection agencies are able to reduce the health and environmental risk posed by nitrates.
  • Agriculture related prevention techniques
    • Crop rotation: this method can increase the amount of Nitrogen present in the soil and hence prevent the excessive need for the application of nitrogen based fertilizers, hence minimizing the chances for nitrate leaching. (Al-Kaisi, 2001)
    • Slurrystore: this focuses on building stores for manure in farming areas with concrete to prevent leakage. However, these might pose a problem similar to that of septic tanks.
  • Water treatment: once the nitrate has already entered the water, there is a possibility of removing it. It can be done by the following methods:
    • Blending drinking water: this involves mixing contaminated water with clean water in order to decrease the overall concentration of nitrate. It is also called a non-treatment technique and is not safe for infants, although it reduces expenses greatly. Another disadvantage is that it can only be applied to nitrate concentration limited in specific areas. (Zhou, 2015)
    • Ion exchange: substances, ions, are exchanged with nitrate in water. A tank filled with resin beads charged with the substance to be exchanged has water pass over it, and through this the nitrate is substituted by the desired substance. 
    • Reverse osmosis: in this method water is moved under high pressure through the membrane and nitrate and other inorganic chemicals cannot pass through, separating them from the water. However, this method is relatively expensive.
    • Biological denitrification (bioremediation): this process involves the usage of denitrifying bacteria and microbes which can then convert nitrate ions back to nitrogen. A disadvantage of this method is the long time needed to start up. (Kaur et al., 2020)

Some general solutions include minimizing discharge from irrigated agricultural lands, implementing nitrate management programs, groundwater quality protection strategies, etc. (Abascal et al., 2022)

Our contribution to the solution: Nitranix

We focus on reducing the amount of nitrate present in the water through biological denitrification in which bacterial denitrifying enzymes are used for the purification of water. This can be done by modifying the enzymes through a direct electron transfer in a process known as enzymatic electrosynthesis. Specifically, enzymatic electrosynthesis is a bioelectrocatalytic process which uses electrons for enzymatic catalysis to produce desired products. (Wu et al., 2020) The main aim is to couple these processes in order to produce a more efficient method of water purification.

Learn More About Our Project Here

References

  • https://www.umweltbundesamt.de/en/data/environmental-indicators/indicator-nitrate-in-groundwater#environmental-importance (Accessed, Feb 2023)
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  • Murray, C., Reker, J., Anderson, J. H., Harvey, T. E., Prins, T., & Peterlin, M. (2019). Nutrient enrichment and eutrophication in Europe’s seas. https://doi.org/doi:10.2800/092643
  • Ward, M., Jones, R., Brender, J., de Kok, T., Weyer, P., Nolan, B., Villanueva, C., & van Breda, S. (2018). Drinking water nitrate and human health: An updated review. International Journal of Environmental Research and Public Health, 15(7), 1557. https://doi.org/10.3390/ijerph15071557
  • Wu, R., Ma, C., & Zhu, Z. (2020). Enzymatic electrosynthesis as an emerging electrochemical synthesis platform. Current Opinion in Electrochemistry, 19, 1–7. https://doi.org/10.1016/j.coelec.2019.08.004
  • Yu, G., Wang, J., Liu, L., Li, Y., Zhang, Y., & Wang, S. (2020). The analysis of groundwater nitrate pollution and health risk assessment in rural areas of Yantai, China. BMC Public Health, 20(1). https://doi.org/10.1186/s12889-020-08583-yZhou, Z. (2015). (rep.). (N. Ansems & P. Torfs, Eds.)A Global Assessment of Nitrate Contamination in Groundwater . igrac.

Nitrogen Cycle

Nitrogen is the fourth most abundant element in cellular biomass, and it comprises the majority of Earth’s atmosphere. (“The nitrogen cycle – ScienceDirect,” n.d.) We can find it in soils, plants, the water we drink and in the air we breathe. To say it is essential to life would be an understatement as nitrogen is also the key building block of DNA, which besides being crucial to determine our genetics, it is also essential for plant growth – when a plant lacks nitrogen they will present a stunted growth and produce smaller flowers and fruits (“THE NITROGEN CYCLE on JSTOR,” n.d.). In a way to fight against this and to increase crop growth, farmers add fertilizers, containing this element. However, too much of this type of fertilizer can contaminate groundwater, which will eventually contaminate drinking water. Therefore, it is important to understand how the nitrogen cycle works, so that healthy crops can still be grew, while caring for the environment and ourselves.

But what is the nitrogen cycle?
The nitrogen cycle is a biogeochemical cycle (1) by which nitrogen, N, is converted into different chemical forms as it moves through atmospheric, terrestrial, marine ecosystems and bacteria. In order for nitrogen to circulate between these environments, it must change its form. For example, in the atmosphere, nitrogen exists as N2, while in the soil it shows itself as nitrogen oxide (NO) and nitrogen dioxide (NO2). When used as a fertilizer, nitrogen displays itself as ammonia (NH3) or even ammonium nitrate (NH4NO3) (“An introduction to the global nitrogen cycle – Jenkinson – 1990 – Soil Use and Management – Wiley Online Library,” n.d.).
This cycle is basically divided into five different stages: fixation, ammonification, nitrification and denitrification.

Nitrogen fixation
In the first stage, nitrogen fixation, nitrogen moves from the atmosphere into the soil. However, N2 cannot be directly used by plants and therefore has to undergo a transformation called fixation.
Nitrogen can be fixed by two different ways: it can be fixed when light provides enough energy needed for the N2 to react with oxygen, producing nitrogen oxide (NO) and nitrogen dioxide (NO2); or it can be fixed through the industrial process that creates fertilizers. For the former process, both new forms of nitrogen enter soils through precipitation (rain or snow). For the latter, the fixation process occurs under high heat and pressure, during which atmospheric nitrogen, N2, is combined with hydrogen to form ammonia (NH3) or, if processed even further, to form ammonium nitrate (NH4NO3).
Most of the nitrogen fixation occurs naturally in the soil, by bacteria. Some bacteria attach to plant roots and simply have a symbiotic relationship -the bacteria get energy through photosynthesis, and, in return they fix the nitrogen in a form the plant needs. Other bacteria live soils and water and still fix nitrogen without the need of this relationship.

Ammonification
The second stage is ammonification, also known as mineralization, which takes place in the soil. Bacteria or fungi convert the organic nitrogen from manure, or a decomposing plant, to ammonia (NH3). This ammonia will then react with water in the soil to form ammonium (NH4). This new form of ammonium is held in the soils and available for use by plants that do not get it by the symbiotic relationship described previously.


Nitrification
The third stage also occurs in soils. In the first stage of nitrification, ammonia is oxidized by a special type of bacteria, Nitrosomonas, which produces nitrites (NO2-).


NH3 + O2 → NO2- + H+
Chemical reaction for the conversion of ammonia to nitrite

As plants cannot use nitrites, a second oxidation has to occur – another type of bacteria, Nitrobacter, will oxidize these nitrites and produce nitrates (NO3-). Both kinds of bacteria can act only in the presence of oxygen, O2.

2 NO2- + O2 → 2 NO3-
Chemical reaction for the conversion of nitrite to nitrate

Denitrification

In the last stage of the nitrogen cycle, the nitrates are reduced back into atmospheric nitrogen (N2). This is performed by bacterial species such as Pseudomonas and Paracoccus under anaerobic conditions. This stage will be further explained in the subsequent text.

As nitrates are reduced back into N2, the nitrogen cycle will repeat itself, over and over again, transforming to be infiltrated by the soil, taken up by the plants and moulding into the atmosphere. It might go as far as contaminating groundwater with the usage of fertilizers, making us get caught up in this cycle too.

(“Nitrogen Cycle – Definition, Steps, Importance with Diagram,” n.d.)

(1) Recycling of inorganic matter between living organisms and their non-living environment (“3.2 Biogeochemical Cycles – Environmental Biology,” n.d.)

Denitrification

Denitrification is the process of reducing nitrates into nitrogen gas with nitrite, nitric oxide and nitrous oxide being intermediate products. As shown, this is an essential part of the nitrogen cycle by being one of the few pathways that can produce atmospheric Nitrogen (“Structural aspects of denitrifying enzymes – ScienceDirect,” n.d.). This process is essential for lowering the amount of nitrates in the soil, lakes, rivers and oceans that can further have harmful effects on the environment and human health.

Denitrification can be done through ion exchange and reverse osmosis, as well as through bacteria. Bacterial denitrification is a natural process that is done mostly by facultative anaerobes. This process allows bacteria to use nitrogen oxides, like nitrate, as electron acceptors instead of oxygen. Denitrification can take place in anaerobic, microaerophilic, and at times, aerobic conditions (“Cell biology and molecular basis of denitrification | Microbiology and Molecular Biology Reviews,” n.d.).

Denitrifying bacteria are able to reduce nitrogen oxides as they respire using nitrogen as a terminal electron acceptor when there is minimal or no oxygen present (“Cell biology and molecular basis of denitrification | Microbiology and Molecular Biology Reviews,” n.d.). This process in which microorganisms are able to denitrify by replacing oxygen in the respiratory process under anoxic conditions with nitrate or nitrite is referred to as nitrate dissimilation. Due to this ability of using either nitrogen or oxygen, most denitrifying microorganisms are facultative heterotrophic (“Types of Bacteria Accomplishing Denitrification – Nitrogen Removal,” n.d.). 

Four electron transport pathways are required in order to convert nitrates into nitrogen. This process utilizes specific enzymes in its four main steps which can be summarized as following:

Step 1: Nitrate (NO3–) (+5) is reduced to form nitrite (NO2–) (+3) with the help of nitrate reductase.

Step 2: Nitrite is reduced to nitric oxide (NO) (+2) with the aid of nitrite reductase.

Step 3: Nitric oxide is then reduced to nitrous oxide (N2O) (+1) with the aid of nitric oxide reductase.

Step 4: Finally, nitrous oxide is reduced to dinitrogen (N2) (0) with the help of nitrous oxide reductase.

Image taken from (“Structural aspects of denitrifying enzymes – ScienceDirect,” n.d.)

As most denitrifiers also use aerobic respiration, they are able to repress the genes encoding denitrifying enzymes in the presence of oxygen. Furthermore, nitrous oxide reductase is sensitive to molecular oxygen as it alters the ligand structure in the enzyme (“Denitrification and N-Cycling in Forest Ecosystems – ScienceDirect,” n.d.). Besides the oxygen concentration, denitrification is also influenced by the availability of electrons in organic carbon compounds as this controls the heterotrophic character. The pH levels also affect denitrification efficiency as the optimum pH is typically 7 to 8 (“Denitrification. – PMC,” n.d.).

Bacteria

As mentioned, denitrifying microorganisms are able to use nitrogen oxides as terminal electron acceptors in place of oxygen. As they are a very diverse group of microorganisms, they can be classified in many ways. They are typically chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism. However, denitrification is not only limited to bacteria as this trait has been found in halophilic and hyperthermophilic archaea, as well as in the mitochondria of some fungi (“Cell biology and molecular basis of denitrification | Microbiology and Molecular Biology Reviews,” n.d.).

Majority are heterotrophic, using one-carbon compounds as their source of energy, while some are autotrophic, using H2, CO2, or reduced sulfur compounds as their source of energy (“Denitrification. – PMC,” n.d.). These microorganisms can also be photosynthetic such as Rhodopseudomonas palustris (“Characterization of denitrifying photosynthetic bacteria isolated from photosynthetic sludge – ScienceDirect,” n.d.). Some of these organisms can also be lacking in an enzyme used in the denitrification pathway. Those lacking nitrate reductases are called nitrite dependent. Some can lack nitrous oxide reductase and hence the terminal product is nitrous oxide instead of nitrogen. There are also some which possess nitrous oxide reductase but are not able to produce nitrous oxide with nitrate or nitrite. These denitrifying bacteria are common in sludge as it can be anaerobic or aerobic, either way not having much effect on the organisms as they are facultative, as described above (“Introduction to Denitrification – Nitrification,” n.d.).

Common examples of denitrifying bacteria include Pseudomonas Stutzeri and Paracoccus denitrificans. They are found abundantly in soil, freshwater, oceans, and animals (“The Denitrification Characteristics of Pseudomonas stutzeri SC221-M and Its Application to Water Quality Control in Grass Carp Aquaculture – PMC,” n.d.). Both are aerobic gram negative bacteria and are being used for bioremediation. Pseudomonas Stutzeri is also able to denitrify with high oxygen concentrations as well as perform nitrification and denitrification simultaneously (“The Denitrification Characteristics of Pseudomonas stutzeri SC221-M and Its Application to Water Quality Control in Grass Carp Aquaculture – PMC,” n.d.).

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