The Alchemy of Nitrogen-Fixation

By Kevin Hancock

With an ever-increasing world population comes an increased demand for food, fuel, and fiber. Land, water, and energy resources are becoming scarcer. Nitrogen is abundant worldwide, and is needed for the growth of most plant species. The majority of the world’s nitrogen is in the gaseous form, which cannot be utilized by most plants. This means that most plants must rely on additions of synthetic fertilizers to supply the needed nutrients.

There are very few plant species that are capable of fixing atmospheric, N2 gas, converting it into a usable form like ammonia, and storing it in root tissue. These plants are referred to as nitrogen-fixing. Symbiotic nitrogen-fixation (snf), which occurs naturally in some leguminous crops, can play a vital role in transforming atmospheric nitrogen gas into ammonia that can be utilized by these plants.

Biological reduction to ammonia can only be performed by prokaryotes and is a highly oxygen-sensitive process. Symbiotic interactions between prokaryote partners occur in two groups of soil bacteria — rhizobia in symbioses in legumes and Frankia bacteria in actinorhizal symbioses.

Snf is highly important in the production of biofuel feedstocks. Many current plants which produce abundant amounts of biofuels such as oil palm, canola, and corn are not nitrogen-fixers and consequently they rely on inorganic nitrogen fertilizers. Every step in the production, delivery and application of nitrogen fertilizer requires fossil fuels. Even though the formation of fossil fuels occurs naturally through anerobic decomposition of plants and animals, they are not considered renewable sources of energy.  Decomposition takes millions of years to form large enough quantities of fossil fuels. Those reserves are being depleted at a much higher rate than they are being formed.

The problem with the use of synthetic fertilizers is that plants only absorb a small percentage of applied fertilizer at any one time. The remainder of the applied fertilizer (30-50%) is subject to runoff, volatilization, and are leached beyond the root zone or denitrified. In many areas this can create algae blooms and eutrophication – a condition of high concentration of nutrients, but low oxygen levels. For example, Lake Okeechobee in Florida is experiencing this due to nutrient runoff from adjacent croplands.

Snf reduces the plant’s dependence on inorganic nitrogen sources and can provide a substitute for nitrogen fertilizers, thus reducing costs and helping the environment at the same time. Biological nitrogen fixation has been estimated to produce approximately 200 million tons of nitrogen annually.

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Leguminous root nodules – PC: NMSU

It would be very beneficial to humanity as well as the environment if all agriculturally important plants were capable of fixing atmospheric nitrogen.  Although a lot is still unknown, a lot of work has been conducted to better understand the intricacies involved in symbiotic nitrogen fixation. Nitrogen-fixation is composed of 3 components; first, the formation of nodules which provide the correct environment for nitrogen-fixing bacteria; second, the regulation of nodule numbers by both internal and external factors, and third, the actual conversion of atmospheric nitrogen into ammonia by the invading bacteria using the nitrogenase enzyme complex.

Nitrogen-fixing plants are not capable of extracting N2 gas directly from the atmosphere, they work in concert with common soil bacteria called Rhizobium. Rhizobia attached to root hairs, induce a pronounced curling of root hair cells. The root hair becomes deformed and the bacteria enter the plant by a newly formed infection thread growing through it. At this same time, cortical root cells are mitotically activated giving rise to the nodule primordium. Infection threads will grow towards the primordium and the bacteria are released into the cytoplasm of the host cells. The bacteria become encapsulated in the small compartment formed by the curling. The bacteria enter the plant’s root system and form nodules along the root pathway. The plant supplies all the essential nutrients as well as energy to the bacteria. Within a week after infection, nodules will become visible by the naked eye. Under field studies, nodules appear within 2-3 weeks. The nodules allow the plant to absorb the N2 gas that is present in the soil, and the plant converts it into ammonia that enters into a biochemical pathway producing both organic and inorganic forms before reverting it to N2 gas. Nitrogen-fixing bacteria need high calcium levels to work efficiently. Three micro-metabolic elements, iron, molybdenum, and cobalt are essential for the nitrogen-fixing process in bacteria.

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Nodule Formation Cycle in Pea Plant – PC: Pearson Education

Most legumes form symbiotic relationships with a select few Rhizobium, however Pongamia pinnata is able to produce snf relationships with various strains of Rhizobia as well as Bradyrhizobium. In areas of India the results clearly demonstrate the major advantage of the leguminous nature of Pongamia when compared to the Jatropha tree, another plant feedstock being evaluated as a source of biofuel energy.

Since the presence of oxygen can inactivate the process of nitrogen-fixing, it is important to know that legumes can regulate the gas permeability in their nodules allowing enough oxygen to maintain respiration without deactivating the nitrogenase enzyme. Nodules contain a heme protein called leghemoglobin. Leghemoglobin is present in the cytoplasm of infected cells at high concentrations (700 uM in soybean nodules). This protein gives the nodule a pink color.

The mystery of the symbiotic relationship is that it only occurs through a complex exchange of signals between specific genes of the plant host and symbiont. Infection and nodule organogenesis occurs simultaneously during root nodule formulation. The symbiotic relationship between legume and bacteria is not obligatory. It is quite possible for a seedling to live out its life cycle without becoming associated with a symbiont.

Among many compelling characteristics, the reduction of dependence on commercial, nitrogen fertilizers, the reduction of runoff and minimizing other environmental concerns all show the benefits of the snf process inherent in Pongamia pinnata.

 

References:

Majda, W. (2014). How to increase the rate of biological nitrogen fixation. Retrieved from https://permaculturenews.org/2014/09/25/increase-rate-biological-nitrogen-fixation/

Meyer, S. B., Anderson, D. B., Bohning, R. H., & Fratianne, D. G. (1973). Introduction to plant physiology (2nd ed.). New York, NY: D. Van Nostrand Company.

Rhoades, H. (2017). Nitrogen nodules and nitrogen fixing plants. Retrieved from https://www.gardeningknowhow.com/garden-how-to/soil-fertilizers/nitrogen-nodules-and-nitrogen-fixing-plants.htm

Taiz, L., & Zeiger, E. (2002). Plant physiology (3rd ed.). Sunderland, MA: Sinauer Associates, Inc.

Wikipedia. (2017). Nitrogen fixation. Retrieved from https://en.wikipedia.org/wiki/Nitrogen_fixation

Flynn, R, & Idowu, J (2015) Guide A129 Nitrogen Fixation by Legumes. Retrieved from http://aces.nmsu.edu/pubs/_a/A129/

Fixing Nitrogen, Waste

By William Kusch

irina-sorokina-253176footprint grass

Figure 1: What is your nitrogen footprint?

You may be familiar with the concept of carbon footprint, but when was the last time you measured your nitrogen footprint? If you are like me, up until very recently, the answer to that question would be: “huh?”.

I got to thinking about the topic when I read an article[1] that National Public Radio (NPR) published, profiling research on life cycle analysis (LCA) of producing a loaf of bread. The article concluded that 66% of greenhouse gas emissions were not from transportation, or baking, but from growing the wheat itself.  Further, “of the environmental impacts … 40% is attributable just to the use of ammonium nitrate fertilizers alone.”

Intrigued, I read on, re-read my colleague’s excellent blog post on animal and livestock nutrition, then clicked my way to a related article[2], also on NPR that dove deeper than greenhouse gas emissions. This story looked specifically at the nitrogen pollution linked to agriculture, with an emphasis on meat production. This piece outlined some agricultural sources and forms of this significant pollutant:

  • Gaseous emissions of nitrogen oxides (NOx) from livestock
  • Release of N2O, and NOx from soil microbes
  • Runoff from excess fertilizer applied to farm fields.

Well, you may say, so what? Isn’t most of the air we breathe nitrogen anyway?  While it is true that a large majority of the atmosphere is nitrogen, it comes in the form of inert N2. N2 is far different from N2O and NOx , two recognized pollutants. Here are a couple of the potential implications from the release and accumulation of N2O and/or NOx:

  • WK gulf mexico

    Figure 2: Image depicting marine dead zone in Gulf of Mexico

    Marine dead zones, such as the famous one in the Gulf of Mexico, where most ocean life has died due to lack of oxygen[3]

  • If concentration is elevated in drinking water, can lead to potentially fatal blue baby syndrome, other negative health impacts[4]
  • Emissions of NOx can lead to the hazardous type of ozone that remains near ground level. This type of ozone can trigger health problems, especially for children and the elderly[5].

Given that agriculture is one of the biggest contributors to nitrogen pollution, and also that no one is going to stop eating in order to stop polluting, what can people do to reduce their nitrogen footprint? Fortunately there are some simple, and effective options to pare the amount of nitrogen pollution associated with our daily activities:

  • Average Americans “eat about 1.4 lbs of protein per week, 2/3 of which come from meat and dairy. …you could cut your nitrogen footprint by more than 40% just by reducing your total protein intake to 0.8 lbs, the amount recommended by the USDA and the National Academy of Sciences”.
  • Get creative with your spending power: think about ways you could change one meal a week from animal protein to one that is centered around plant protein such as that from chickpeas, or assorted beans.
  • Throw away less of your food: an estimate from Natural Resources Defense Council[6] indicates that America wastes ~40% of our food by throwing it in the garbage prematurely, or unnecessarily.
  • Encourage your legislators to support agricultural land conservation efforts, especially in areas where plants filter fertilizer runoff before it enters the local watershed.
  • Consider a more fuel efficient, or electric vehicle when choosing your next set of wheels: while agriculture is the largest source of N2O, transportation also accounts for a large share of NOx[7].
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Figure 3: Nitrogen-fixing pongamia trees in TerViva’s Hawaii orchard

At TerViva, we’re doing our part to mitigate this global nitrogen problem as well. We are growing orchards of pongamia: oilseed-producing trees that are legumes and harness the power of symbiotic bacteria to capture nitrogen from the atmosphere. This ability to provide nitrogen for itself allows pongamia to be cultivated using significantly fewer costly inputs relative to most conventional crops, like nitrogen fertilizers. After we harvest the seeds, we crush the crop in an oilseed press, yielding oil and seed cake. The oil serves as an excellent feedstock for biofuel. The seed cake is high in protein and we have discovered how to convert the pongamia protein into animal feed. In addition to feeding livestock, pongamia seed cake can also be used as a fertilizer[8]; we know this because people have been using pongamia cake as fertilizer in Southern and Southeast Asia for many hundreds of years. The reason this anecdote is relevant here, is that modern scientific techniques have recently been brought to bear, analyzing and quantifying the value of pongamia seed cake as fertilizer. In fact, in addition to demonstrating the value of pongamia products as fertilizer, recently published research shows that if pongamia seed cake is used as a fertilizer, there are compounds in the fertilizer that prevent nitrogen pollution from happening in the first place when farmers apply fertilizer to their fields [9].

Through this idea of considering our Nitrogen Footprint, we at TerViva are exploring ways that we can provide renewable, plant-based energy and protein to society, while at the same time preventing and mitigating some of the issues that arise from the modern lifestyles that afford us comfort and convenience.

References:

[1] http://www.npr.org/sections/thesalt/2017/02/27/517531611/whats-the-environmental-footprint-of-a-loaf-of-bread-now-we-know

[2] http://www.npr.org/sections/thesalt/2016/02/25/467962593/why-your-hamburger-might-be-leading-to-nitrogen-pollution

[3] http://www.noaanews.noaa.gov/stories2015/080415-gulf-of-mexico-dead-zone-above-average.html

[4] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1638204/

[5] https://www.epa.gov/ozone-pollution

[6] https://www.nrdc.org/sites/default/files/wasted-food-IP.pdf

[7] http://www.pnas.org/content/100/4/1505.full.pdf

[8] http://oar.icrisat.org/424/1/IndJourFer5_2_25-26_29-32_2009.pdf

[9] http://nopr.niscair.res.in/bitstream/123456789/5647/1/NPR%207(1)%2058-67.pdf