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Biochemistry

Using nitrogen isotopes to start from the bottom…of the marine food web!

Article: K. Maki, N. Ohkouchi, Y. Chikaraishi, H. Fukuda, T. Miyajima, T. Nagata. (2014) “Influence of nitrogen substrates and substrate C:N ratios on the nitrogen isotopic composition of amino acids from the marine bacterium Vibrio harveyi.” Geochimica et Cosmochimica Acta, Vol. 140, pp. 521-530. DOI:10.1016/j.gca.2014.05.052

Background Information

Nitrogen is one of the most important elements for all living organisms. Biochemically speaking, nitrogen is one of the main building blocks of amino acids, which constitute proteins. Elemental nitrogen can be naturally found as two stable isotopes, nitrogen-14 and nitrogen-15. An isotope is an atom of the same element, but with different amounts of neutrons in the atom’s nucleus. What this means in biochemistry is that nitrogen-14 and nitrogen-15 have the same degree of “nutrition” but nitrogen-15 is a little bit harder for organisms to metabolize. The preference for organisms to metabolize nitrogen-14 over nitrogen-15 is called fractionation. Many marine creatures become enriched in nitrogen-15.

This fractionation of nitrogen, especially in amino acids, is a useful tool to figure out what trophic position a marine organism occupies (Figure 1). In general, nitrogen fractionation in an ocean critter’s amino acids becomes 6-7‰ more enriched than its prey. This not only helps to figure out where an organism lies on the food web, but can also help determine what they ate. In fish and larger mammals, we can look at the gut contents to figure out where the food they ate came from. However, this gets much harder to assess this at the bottom of the food web!

Figure 1: Using the stable isotope ratios of nitrogen (N) and carbon ( C). Credit: USGS, http://sofia.usgs.gov/publications/fs/2004-3138/

Figure 1: Using the stable isotope ratios of nitrogen (N) and carbon ( C). Credit: USGS, http://sofia.usgs.gov/publications/fs/2004-3138/

Bacteria are everywhere and can be difficult to place on the food web since they eat whatever they can get, often detritus, or dead organic matter composed of a complicated mix of amino acids, lipids, carbohydrates, and other decomposed molecules. There currently is little data avaible to assess the nitrogen isotopic ratios (δ15N ) for amino acids in microbes, and what is available varies greatly depending on different environmental and physiological factors.

In order to better understand and explore how nitrogen enrichment can affect the microbial level of the food web, Maki et al. set out to explore how the ratio of carbon to nitrogen (C:N) in the microbes’ food source affects the nitrogen fractionation of amino acids. They also compared the bacterial amino acid fractionation to marine phtyoplankton, which also occupy the bottom of the marine food web.

The Approach

The bacteria selected for this study was Vibrio harveyi since it is a surface water species found globally in the ocean (Figure 2). The bacteria was incubated (grown under closed conditions) in prepared artificial seawater with all of the minerals and vitamins required for bacterial growth (with the exception of nitrogen, which was the dependent variable).

vibrio

Figure 2: Vibrio harveyi, a cosmopolitan marine bacteria, is bioluminescent. Credit: http://genome.wustl.edu/genomes/detail/vibrio-harveyi/

That nitrogen was added as single amino acids so that the nitrogen fractionation could be easily measured as a reflection of metabolism. In the ocean, bacterial δ15N would be a sum of multiple metabolized amino acids, so this experimental set-up allows the researchers to look at nitrogen fractionation in reference to a specific food source.

The nitrogen food source added to the bacterial cultures were the amino acids glutamine (C:N=5) and alanine (C:N=3) as well as ammonia, which contains nitrogen but no carbon. Additional C:N ratios were created by adding glucose (sugar), which contains no nitrogen but is abundant in carbon. Bacterial incubations were carried out for 120 hours, but samples were taken every 24 hours.

The δ15N for the particulate organic carbon (this included everything in the culture that could be retained on a filter) was measured using an elemental analyzer coupled to an isotope ratio mass spectrometer. The δ15N for the specific amino acids were determined on a gas chromatograph coupled to an isotope ratio mass spectrometer. The concentration of amino acids were measured using high performance liquid chromatography.

The Findings

Fig. 3: The nitrogen-15 isotope ratio (δ15N) offset of individual amino acids relative to δ15N of the substrate initially added to the bacterial culture (a–c) or relative to mixture of glutamate and glutamine (Glx) δ15N (d–f) for bacteria grown on glutamate media (a, d), alanine media (b, e), and ammonium media (c, f). Horizontal bar with an asterisk (followed by the p level of the t-test) indicates that the offset relative to Glx δ15N values differ significantly between media with different C:N ratios. Amino acid abbreviations on the X-axis are as follows: Ala, alanine; Val, valine; Leu, leucine; Ile, isoleucine; Phe, phenylalanine; Glx, glutamate + glutamine; Met, methionine; Gly, glycine; Ser, serine. NA, data not available.

Fig. 3: The nitrogen-15 isotope ratio (δ15N) offset of individual amino acids relative to δ15N of the substrate initially added to the bacterial culture (a–c) or relative to mixture of glutamate and glutamine (Glx) δ15N (d–f) for bacteria grown on glutamate media (a, d), alanine media (b, e), and ammonium media (c, f). Horizontal bar with an asterisk (followed by the p level of the t-test) indicates that the offset relative to Glx δ15N values differ significantly between media with different C:N ratios. Amino acid abbreviations on the X-axis are as follows: Ala, alanine; Val, valine; Leu, leucine; Ile, isoleucine; Phe, phenylalanine; Glx, glutamate + glutamine; Met, methionine; Gly, glycine; Ser, serine. NA, data not available.

The δ15N of single amino acids in the marine bacteria Vibrio harveyi changed depending on the type of nitrogen (glutamine, alanine, or ammonia) used as a food source for the bacteria and the C:N ratio of the original media the bacteria was grown in.

The recovery of nitrogen (as ammonia) between the alanine and glutamate media used to grow the bacteria was significantly different depending on the C:N ratio. The glutamate media, for example, had a 25% recovery of nitrogen when the C:N ratio was low but only 1% was recovered when the C:N was high. This is important since it suggests that the organic matter (food) available to bacteria will change their metabolic capabilities, as seen by the δ15N fractionation.

In order to assess if the δ15N of amino acids in the grown Vibrio harveyi were fractionated, the researchers had to compare it to something. Maki et al. compared the bacterial δ15N relative to the original δ15N of the substrates (food source) added and normalized (put all the values on the same scale) it to the sum of the amino acids glutamate and glutamine (Glx).

The bacteria fed on the alanine substrate showed the greatest change in δ15N; the δ15N of the amino acids in the Vibrio harveyi became significantly more enriched compared to the Glx (Figure 3). This result also varied depending on the C:N of the original alanine food source. When the C:N of the alanine substrate was high, the bacteria had an average enrichment of δ15N at 3.1‰, but that fractionation increased to 10.0‰ when the C:N was low. This suggests that alanine δ15N analysis may be a useful indicator of the C:N present in the environments the bacteria are living in.

Maki et al. compared their results to 12 phytoplankton species from previously published data. While the Vibrio harveyi δ15N grown on ammonia was the same as the phytoplankton, the alanine and glutamine substrates created a more enriched δ15N in the bacteria compared to the phytoplankton! This could mean that looking at the δ15N of single amino acids could be helpful in distinguishing between phytoplankton and bacteria in food web studies.

Significance

This study showed that the δ15N of single amino acids in marine bacteria change depending on the C:N ratio and type of nitrogen used by the bacteria to grow. Additionally, the δ15N fractionation between the bacteria and phytoplankton (eukaryotic phytoplankton and cyanobacteria) were measurably different when grown under the same conditions. This ultimately suggests that the murky bottom of the food web can possibly be distinguished by using the δ15N of single amino acids.

Since these amino acid δ15N profiles were different between the bacteria and phytoplankton, Maki et al. propose that the metabolic pathways to uptake and use nitrogen may be different between the two organisms. This is an exciting biochemical idea that should be investigated in the further!

Finally the study gives us more insight into the transfer of amino acids, energy, and nitrogen up the different trophic levels in the ocean. For example, this fractionation could help determine differences between viral lysis and protist grazing.

Discussion

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  1. […] value may indicate changes in food web structure (For more detail on nitrogen isotopes, see this past oceanbites post).  Sixty-three samples of modern Pacific Cod tissue from the same area were also tested for Hg. […]

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