Root Of Our Problem

Background

Plants cannot directly take in nitrogen from the air like carbon dioxide and oxygen. Ammonia, a nitrogen-containing compound, has been used as a fertilizer ever since we figured out how to commercially mass produce it. Plants now directly absorb nitrogen from the soil instead, which has single-handedly doubled the number of people one acre of land can feed.

During and after fertilizer application, nitrogen does not remain in the soil in an unaltered state. Due to abundant nitrifying bacteria, nitrate molecules increase and co-exist with ammonium molecules.

Since clay is composed of negatively charged particles, negatively-charged nitrate molecules can adhere to them and participate in mass flow during erosion and weathering events. This phenomenon is known as nitrate leaching.

Unregulated fertilizer use in farmlands leads to accumulation of excess nitrate, which in turn causes greater leaching. In fact, several studies have shown that agricultural activities are a significant source of surface and ground water pollution due to long-term and excessive fertilizer use.

Agricultural Contribution to Non-Point Water Pollution by Country

75%

US

60%

Netherlands

94%

Denmark

This extensive pollution leads to the following problems:

• Degradation of soil quality due to excessive loss of nutrients.
• Groundwater pollution, with ions seeping into wells and reservoirs.
• Eutrophication resulting from algal blooms on the surface of water bodies.

The secondary problems of nitrate leaching prove even worse. Excessive nitrate in drinking water has been proven to cause methemoglobinemia (blue baby syndrome). Ingested nitrate is reduced to nitrite by bacteria in the mouth and in the infant stomach, which is less acidic than adults. Nitrite binds to hemoglobin to form methemoglobin, which interferes with the oxygen carrying capacity of the blood and causes breathing issues.

There are also studies linking high nitrate consumption to thyroid disease, colorectal cancer, and pregnancy complications.

In India, 90% of the rural populace uses groundwater as their primary source of drinking water. This is a huge chunk of people directly exposed to a concerning concentration of nitrate in their water. This problem had to be tackled, and tackled quickly.

Plan

To understand how we arrived at the current iteration of our project design, it is important to piece different aspects of the situation together and move forward with the insights. We begin, then, with ammonium.

We Need To Talk About Ammonium

Agricultural bioremediation presents complex challenges due to intricate anthropogenic and natural factors involved in the field. On that note, we must consider two situations; nitrate leaching during crop cycles, and nitrate leaching in between crop cycles.

In between crop cycles, nitrate and ammonium have nowhere to go because plants aren’t available to take them up. Residual nitrate molecules from past fertilizer application are, therefore, free to leach upon instances of rainfall and soil erosion. Ammonium molecules are retained in the soil due to their cationic state in these conditions.

During crop cycles, nitrate and ammonium are both utilized as nitrogen sources for plant growth and development alongside the simultaneous process of nitrate leaching. However, research has revealed a general higher growth response of terrestrial plants to ammonium-N than nitrate-N addition.

A separate study also found that leaf chlorophyll content increased upon treatment by ammonium or ammonium-nitrate, but not by nitrate alone. Furthermore, on an energetic level, ammonium uptake and assimilation is less costly to plants when compared to nitrate uptake and assimilation.

All these facts point us toward an obvious solution. If we could somehow modify the total nitrogen (TN) content of the soil to comprise of more ammonium-N than nitrate-N, we could accomplish the following outcomes:

• Reduction in nitrate leaching, and subsequently, higher retention of nitrogen in agricultural soil
• Increase in bioavailable nitrogen as ammonium for plant roots

The Nitrogen Cycle Family

To identify the natural processes that already deal with nitrogen, we turned to microbial metabolic pathways in the nitrogen cycle. A consolidated list contains the following major pathways:

Operon Investigation

Since our efforts were directed towards reducing nitrate molecules to ammonium, we focused on denitrification and DNRA.

DNRA and Denitrification Process

Denitrification is the more well-established, well-researched pathway of the two, which initially drew us to the possibility of using it to our advantage. The following points were kept in mind and assessed as we researched the situation.

• Denitrification is a four-step pathway.
• One of the intermediaries is nitrous oxide, which is a greenhouse gas. N2O is approximately 270 times more potent than carbon dioxide in terms of warming the planet, and is currently responsible for approximately 10% of net global warming since the Industrial Revolution.
• The final product of denitrification is N2 gas, meaning total nitrogen (TN) content is constantly being lost from the soil.

Dissimilatory Nitrate Leaching to Ammonium, the Forgotten Second Cousin

DNRA, on the other hand, piqued our interest because of the following pros:

• DNRA is a two-step pathway.
• DNRA does not produce any intermediates that could cause environmental harm.
• Total Nitrogen content remains constant in the soil. In fact, research has shown that DNRA could be a viable biological pathway to conserve terrestrial nitrogen and reduce nitrous oxide emissions.

This seemed absolutely perfect for our purpose. The catch? DNRA is comparatively highly understudied. It was a difficult decision, reinforced by our PI and advisors, to incorporate DNRA as such an integral part of our project, regardless of the fact that we had very limited information to go on.

Identified Operons

The nir and nrf operons perform the same function, but have subtle differences in their operation. The nir operon encodes a periplasmic nitrite reductase, while the nrf operon encodes a cytoplasmic nitrate reductase. Furthermore, there are two variations of the nrf operon that have been discovered across bacterial species.

This presented an exciting opportunity for us to test the efficacy of these operons in a bacterial chassis of our choice. We could therefore empirically understand which protein complex proved best for nitrite reduction as part of our project, which we have described in our experimentation section.

Blessed be the Biofilms

Having identified the operons and proteins we required to enact our objective, we moved on to choosing our bacterium. This process involved multiple overhauls based on results and feedback received across multiple DBTL cycles, which we have described in detail on our engineering page.

We finally chose Pseudomonas putida KT2440, a common and robust soil bacterium. In the meantime, on our advisor’s direction, we chose to demonstrate the proof of concept in Escherichia coli DH5-Alpha. Our chosen method of gene insertion is via a plasmid, so we identified plasmids with an origin of replication that would work for both E. coli and P. putida.

An exciting discovery was the fact that this bacterium forms biofilms around plant roots, and extensive research has proven that these biofilms promote plant growth and health. In fact, P. putida KT2440 is part of a group of microbes known as plant growth-promoting bacteria (PGPB).

These biofilms are evidence of microorganism colonization on plant roots. They have remarkable stress resilience, and they empower crops to thrive and yield even in harsh conditions. These conditions include drought, high salinity, and even fungal infections. Furthermore, they stimulate plant growth, increase yield, as well as reduce biotic or abiotic plant stress without conferring any pathogenicity.

Using P. putida KT2440 as our organism of choice gives us the added benefit of beneficial plant root colonization, and therefore increases the application of our final product in agriculture from just nitrogen retention to also being beneficial for plants.