I think practically impossible, determine the concentration of CNP in seston and measure the biomass of seston and perform parallel sampling and estimation of biomass of phytoplankton. I wonder if somebody will give more optimistic answer:)

You can use flow cytometry to sort the cells, but you should determine the size range of the cells.

Hi!

Classical methods of bulk elemental analyses do not really allow to separate the phytoplankton from the seston (CN analyzers and colorimetric methods after digestion for P). But there is another method based on X-rays (X-ray microanalysis, see, e.g. Fagerbakke, et al 1996. Aquatic Microbial Ecology 10:15-27). This method allows one to measure the elemental content of individual cells, thus also providing one with an idea about the intra-population variation in stoichiometry!

Regards,

hi

you have to take individual cells nearly 30-100. and measure individual cell bio-volumes as per geometric shapes. then analysis determine C,N,P according processor. then we can calculate how much phytoplankton cell contains individually.

This is a really tough problem: you can try to separate the algal cells from the other particles in the water column by using a Percoll density gradient. http://202.38.193.234/spfx1/admin/picture/200752919841517.pdf

Percoll is an inert Si-material, and it is easy to make a density gradient in a centrifuge tube which is quite stable. When you load your (concentrated!) sample on it and centrifuge it in simple benchtop centrifuge (with swingout rotor) the material will seek its own density in the gradient and this might separate the cells from other particles. However, I'm not sure if you can recover enough material needed for the analysis and if you can load enough material on the Percoll gradient to start with: this will be trial and error. If you're water contains a lot of SPM (SESTON) and only a tiny fraction is algal material I'm pretty confident this will not work. The X-ray method suggested by Mehdi might be more practical in this case. Cell sorting is another option, but you need a high throughput in order to collect sufficient material for the analyses.

If you find a method, please circulate this around as many people without an X-ray microanalysis facility will be very pleased.

regards, Jacco

I think that the only way is to use flow cytometry to sort the cells, but you should determine the size range of the cells

I surpport the re-suspended method using a density gradient. Seperated algae cells through different size-membrane filter or gravitational water. If the algal cells are in the same range of several micrometer as seston, the re-suspend method is essential for algae cells in gravitational water. The algal cells can resuspended in the gravitational water, the other inorganic particles can settledown in the bottom. I suppose another method is in vivo fluorescence detect of algae cells, the inorganic seston cannot fluoresce.

You could try a very simple mesh filtration to separate phytoplankton from larger zooplanktpon, e.g. with a cutoff of 50µm or so? With different meshes, different size classes are possible. If you check the size fractions for Chlorophyll with a spectrometric/fluorometric method, you can at least estimate the contribution of phototrophs. For the low biomass i advise to filter several hundred mL up to a few liters of water onto precombusted (=clean) GF/F glass-fiber filters. A standard CN analyzer can then well resolve organic C and N. POP can be detected using the Lachat method, that works nice in microalgae as well: http://www.epa.gov/greatlakes/monitoring/sop/chapter_6/LG600.pdf

If a rough estimate can work, then you may get a known volume (known weight) of the water column (hopefully a large sample) contained in a suitable container to withstand high temperature and heat it in a muffle furnace to at least 500 C in order to determine what proportion of the sample (water column) is non-combustible and non-volatile (ash). Then you weight the remaining ash, which contains the amount of P and other elements. The amount combusted (volatilized) contained the C and N. Since you know the initial weight of the sample then you can estimate the proportion. This may give you a rough idea.

For more accurate estimation, you would need to send the sample to a lab where there is a TOC analyzer and a TN analyzer (these methods also use combustion).

Hope this is of some help.

If you measure chlorophyll a and filter increasing volumes of lake water, you can graph volume filtered (X) vs C, N, P, and chl a (Y). If you obtain similar slopes, the elemental chemistry is following the Chl a concentration. Positive ordinates suggest detrital or adsorbed C, N or P. For this to work, you need to start with the smallest possible volumes of filtered lake water that still allow detectable C, N, P concentrations; to minimze filter clogging and filter loading of adsorbed non-phytoplanktonic substances.

If you can remain onsite (on or near the lake) for 6-8 hours, another approach is to use a 'reverse passive filtration hydrostatic system', which will allow you to concentrate mixed layer or epilimnetic phytoplankton populations. To do this you obtain a short 30 cm by 4 or 6 inch diameter PVC tube. You buy an 8"5 x 11" polycarbonate 0.2 micron sheet (i.e. Nuclepore) and using the PVC tube diameter as a template, you cut a circle and glue it to the PVC tube using silicone caulk. You cap the other end leaving a small hole and a U shaped tube to prevent debris from falling into the tube while it fills up. The principle is to weigh the tube slightly so that it is slightly submerged relative to the lake surface. The higher outer hydrostatic pressure, relative to the air inside the tube, will cause water to flow through the filter until the tube is completely filled, which can take several hours. Leaving the submerged tube in the water (to prevent bursting of the membrane if you lift it out of the water), you must pump out the 0.2 filtered water, using a hand vacuum pump, to recover the material on the underside of the filter. The material is not tightly impacted and can be easily swept off the filter with a small artist's painI brush or tooth brush into a smaller vial to obtain very concentrated material. I have used this approach in oligotrophic lakes, by mounting six tubes in slightly larger diameter PVC tubes, attached to a floating raft at the center of a lake. The caps at the end of the tubes would act as a lock, preventing further submergence after 2 or so liters were filtered. i have to admit though, that is is really a 'field station approach', since it is very time consuming, and you need to watch your gear on the lake to prevent vandalisme or theft.

in a plexiglass tube (1m height, even more; 10cm in diameter), the water to be examined must be placed. The first 10-15 cm are left exposed to direct light, placed transversely to the tube.The rest of the tube is covered with paper of tin. The work-room should be dark, except for the small light. In modest volumes, the particulate tends to settle quickly, while the phytoplankton is towards the light source. In 2-3 hours, it is possible to draw off the top 10cm. it can be repeated with more tubes in series, or at different times, if it is possible to retain the material well. Certainly, in 2-3 hours the sample can undergo an evolution, but you can not have everything

This is a problem that has plagued phytoplankton scientists for a long time . The methods listed above can all work but they mostly require quite a lot of work for each data point. I recall that measuring ATP was once seen as the solution (as ATP was not found in detritus) and then using standard conversion factors estimate chla (e.g. Karl, D. M. and O. Holm-Hansen. 1978. Methodology and measurement of adenylate energy charge ratios in environmental samples. Marine Biology, 48, 185-197). Unless methods of measuring ATP have improved a lot over the years a simpler method was published by Karl Banse in Marine Biology 1977, Volume 41, pp 199-212 'Determining the carbon-to-chlorophyll ratio of natural phytoplankton' and has the advantage of being relatively quick and is not dependent upon too many assumptions (unlike the cell volume methods above). It also works for N and it is essentially the same answer given by Jean-christain. Working in the open ocean it is often true that phytoplankton are the vast majority of the particulate C and N so the problem can be much less severe and you could anticipate this latter method would be endorsed by a range of biological oceanographers.

Thanks, everyone!