From useful ammonia to even more useful nitrate

I'm trying to answer the plant biology question: why does nitrogen make leaves? (Which is something my mum told me.) This led me to look at the nitrogen cycle and I started off investigating how nitrogen from the atmosphere is converted to ammonia (nitrogen fixation). This can be done industrially but in the biological world it's done by nitrogen fixing bacteria.

Nitrogen in the form of ammonia can be taken up by plant roots and as such is a bit useful to them, but not that useful. It gets even more useful by being converted into nitrate. This is a two step process and is called nitrification. 

• In the first step ammonia (NH3) is converted to nitrite (NO2-)
• In the second step nitrite is converted to nitrate (NO3-)

Both of these reactions are technically examples of oxidation reactions because oxygen is combined with the nitrogen. Nitrite and nitrate are charged molecules so are referred to as ions (specifically anions – molecules with a negative charge).

In the plant world nitrification, like fixation, involves specific micro-organisms.

Nitrosomonas europeae
Stan Watson,  Woods Hole Oceanographic Institute     http://genome.jgi-psf.org/niteu/niteu.home.html

In the first step ammonia is converted to nitrite by ammonia-oxidising archaea and also some ammonia-oxidising bacteria (mainly types of Nitrosomonas). Archaea look a lot like bacteria and until the 1970s were thought to be types of bacteria. Like bacteria, archaea are single-celled organisms and their genetic material is not contained within a nucleus. But more recently scientists have shown that genetically archaea are very different to bacteria so are now classified in their own group for the purposes of biological classification(1). Archaea are really interesting and I could go on a bit more about them but the details are not really relevant to my leaf question. It's thought that archaea are the most abundant ammonia-oxidising micro-organisms in many habitats(2), but I couldn't find a suitable image anywhere on the internet.
 

In the second step the main group of bacteria that are involved in converting nitrite to nitrate are called Nitrobacter.

 

Like in nitrogen fixation the micro-organisms involved in nitrification use specific enzymes to facilitate the conversions(3).

Unlike nitrogen fixation, in which the bacteria need energy (in the form of ATP) to convert atmospheric nitrogen to ammonia, nitrification actually releases energy that the micro-organisms use to make ATP (which then provides energy for other purposes). The term to describe this type of organism is a chemoautotroph – loosely translated meaning “obtaining own energy from a chemical reaction”.

The micro-organisms involved in both steps can be found in a wide range of environments, including, importantly for the purposes of my investigation, soil.

 

One of the important things about nitrate is that it is much more water soluble than ammonia, which means that it is easier for plants to take it up through their roots, and why it is most useful. Nitrite is also quite water soluble but is toxic to plants in large amounts so it is important that it is converted to nitrate.

Interestingly, nitrate can be formed directly when lightning strikes. The energy in a bolt of lightning enables atmospheric nitrogen to be directly combined with atmospheric oxygen. The resulting nitrate is soluble in moisture in the atmosphere and when it rains the nitrate is delivered to the soil, from where the plants take the nitrate up through their roots(4).
 



Diagram showing the stages in the conversion of atmospheric nitrogen to nitrate

 

So now we've found out how:

1. Atmospheric nitrogen is converted to ammonia

2. Ammonia is converted to nitrite and then to nitrate

 

Next I'm going to be looking at how the plants take nitrate up through their roots and what they then do with the nitrogen. This will involve a little break from the nitrogen cycle (don't worry we'll be coming back to it) and a diversion into cell biology.

 

 

References

(1) http://www.microbeworld.org/types-of-microbes/archaea accessed 17.8.13

(2) Bernhard, A. (2012) The Nitrogen Cycle: Processes, Players, and Human Impact. Nature Education Knowledge 3(10):25 Available http://www.nature.com/scitable/knowledge/library/the-nitrogen-cycle-processes-players-and-human-15644632 accessed 17.8.13

(3) http://nitrificationnetwork.org/Introduction.php accessed 17.8.13

(4) http://www.howstuffworks.com/nitrogen-fixation-info.htm accessed 17.8.13

Plant biology 1: getting hold of useful nitrogen

My mum had helpfully informed me that “nitrogen makes leaves”, when I asked her why she wasn't able to elucidate. She's a keen gardener so I thought this would qualify her to elucidate. I think she might also have an A-level in biology. I have an A-level in biology too. From 13 years ago – which is a scary thought, but probably not as scary as the pedigree of my mum's A-level in biology (if indeed she has one).

My very leafy squash plant - where do the leaves come from?

I do not recall any plant biochemistry from my A-level in biology. That may be because:

• I didn't learn any in the first place
• I have completely forgotten anything that I did learn, more likely – plant biochemistry not being high on my list of knowledge and skills for the last 13 years.

What I do recall is that nitrogen is needed for proteins – either structural or functional so this must come in somewhere.

But I wanted to find out more about what was going on with my leaves. I needed somewhere to start and from what I could recall from my A-level in biology (or maybe even chemistry) the nitrogen cycle might be a pretty good place. Things have moved on at great speeds since I did A-level biology, at that time I relied upon one owned text book, the library for any other text books and a very slow dial-up internet connection for a few more detailed bits of information if I had the patience to trawl around in a fairly haphazard way. Now I can sit on a bus connected to the internet, I will probably rely on the internet for most of my information and several qualifications after my A-level in biology I hope that I am much better at navigating the internet for the information I need.

2 nitrogen atoms make
an atmospheric
nitrogen molecule

Nitrogen (chemical symbol N) – a few facts:
Discovered in 1772 by Daniel Rutherford⁽¹⁾

Forms 78% of the Earth's atmosphere⁽¹⁾

Where it exists as the extremely stable (inert) N₂ molecule

The inert N₂ molecule doesn't do very much for plants. In order to fulfil its biological role nitrogen must be converted / integrated into other molecules and compounds. This can be done in a lab, but it's going on in nature all the time: this is part of the nitrogen cycle. When I learnt about the nitrogen cycle I remember it being rather more complicated than the water cycle and the carbon cycle. There are lots of arrows going all over the place with lots of long names to describe what is going on. I'm going to try to pick out the bits that are relevant to my plant question and look at these one at a time before making the cycle.

Nitrogen fixation: this is a misleading term for a start. It basically means any process by which the nitrogen atoms in the extremely stable N₂ molecules in the atmosphere are “converted” to other molecule(s) in which the nitrogen atom is more “free” (in my mind the opposite of fixed). Examples of these types of molecules are ammonia (where nitrogen is combined with hydrogen) and nitrous oxides (where nitrogen is combined with oxygen). One way to think of it I suppose is that nitrogen fixing takes nitrogen atoms from an unusable source (nitrogen in the air), and turns it (fixes it) into a more usable form.

In the plant world nitrogen is converted (“fixed”) to ammonia by micro-organisms (mostly bacteria). These bacteria are cunningly known as nitrogen fixing bacteria (they are also sometimes referred to as diazotrophs, which roughly translated means “nitrogen bond eating”); they have an enzyme (called nitrogenase) which facilitates this chemical reaction to take place. (An enzyme is basically a protein that makes processes happen more quickly by reducing the energy required for them to take place – like a biological version of the catalytic converter in your car.) To put this in perspective, to make ammonia industrially requires a temperature of 400-450ºC, a pressure of 200 atmospheres⁽²⁾ (the exact conditions vary depending on where you look but this gives you an idea) and an iron catalyst. (This is called the Haber-Bosch (not the same Bosch as the kitchen appliances) process and is a good example of the concept of chemical equilibria if you are interested.) So the bacteria really do do an impressive job with their enzymes.

A facility from the early days
of industrial ammonia production
http://www.deutsches-chemie-museum.de/index.php?id=57

Some nitrogen fixing bacteria
Jones D. H. Further Studies On The Growth Cycle Of Azotobacter.
 Journal Of Bacteriology, 1920, Vol. 5, No. 4 Р. 325-341

As well as the enzyme, the biological process of converting N₂ into ammonia requires energy and this comes in the form of an amazing compound called adenosine triphosphospate (ATP). Think of ATP as the biological world's petrol – basically it is involved in a reaction within cells that liberates energy for other purposes. And one of those purposes within the bacteria that we're talking about is nitrogen fixation.

There are a few different types of bacteria that can fix nitrogen⁽³⁾:

• Aquatic bacteria – cyanobacteria
• Free living soil bacteria – e.g. Azobacter
• Bacteria that interact with plants roots – e.g. Azospirrillum
• Bacteria that live in a mutually beneficial way with some plant roots – e.g. Rhizobium – these are known as symbiotic bacteria. These bacteria live in nodules in the plants' (typically legumes like beans and clover) roots and in this symbiotic relationship the plant provides the energy needed for the bacteria to fix the nitrogen, and the bacteria provide the plant with nitrogen in a form it can use for growth. In my garden the mangetout peas are an example of this type of plant. Once the crop is over I might try to take a closer look at their roots. The symbiotic relationship is incredible and I could go into lots of detail, but it's not really too relevant to where we started from.

Showing the nodules where nitrogen fixing
bacteria live symbiotically with certain plants
By Terraprima (Own work) [CC-BY-SA-3.0
(http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

In summary, there are loads of bacteria in the soil in my garden that are busy converting stable atmospheric N₂ into useful ammonia. When the plants can't get enough nitrogen through this process of biological nitrogen fixation is when fertiliser comes in. Some of the ammonia that is produced in the Haber-Bosch process goes on to be incorporated into fertiliser products that you can buy at garden centres, and this too can provide plants with nitrogen.

Diagram showing (I hope simply) biological nitrogen fixation

So, now we've found out about where the nitrogen comes from and how bacteria convert it into ammonia. Next time we'll try to find out about what happens next.

Just in this short bit of writing there's lots of tangents that I could have gone off on, and other information that I could have added but I've tried to resist. Feel free to ask questions and I'll try to answer or add comments (or your own facts).

References

(1) http://www.webelements.com/nitrogen/ accessed 26.7.2013

(2) http://www.chemguide.co.uk/physical/equilibria/haber.html accessed 26.7.2013

(3) Wagner SC. (2012) Biological nitrogen fixation. Nature Education Knowledge 3(10):15 http://www.nature.com/scitable/knowledge/library/biological-nitrogen-fixation-23570419 accessed 27.7.2013