John Rueter
September 4, 2004
During August and the beginning of September I did three pieces of work. First I took multiple samples for total organic carbon analysis. Second, I made measurements to compare the photosynthetic efficiency of cells from inside and outside the limno-corrals. Third, I continued to explore the conditions under which humic rich water will inhibit AFA photosynthesis.
Samples were collected on trips on July 27 and 28 and on August 23. These samples were kept on ice and then refrigerated. About 50 mL was filtered through Whatman GF/A filters using a syringe filtration system.40+ mL was used for TOC analysis and the rest was used for absorbance and fluorescence measurements.
Thirteen samples were analyzed for DOC using a Shimadzu total carbon analyzer. The samples were acidified prior to analysis. For a complete description of the sampling sites and the Lat-Long coordinates please see sampling-sites.html
# Sample site date comment TOC
mg/mLslope*
1 Moore Park Marina 2004.08.23 2 Lower Klamath Marsh channel 2004.08.23 3 Running Y Apple Tree 2004.08.23 4 Running Y Golf Course channel 2004.08.23 5 Shoal Water Bay 2004.08.23 6 ?Thompson Creek 2004.08.23 not sure if this creek leads into Thompson Creek 7 Wood River Marsh 2004.08.23 52.15
8 Wood River Marsh 2004.07.27
looks more muddy than brown 9 Canoe Trail 1 2004.07.27 from under the Wocus plants 24.40
10 Canoe Trail 2 2004.07.27 from open water in the trail 11 Running Y Golf Course 2004.07.27 12 Barley extract 1/1000 2004.07.27 13 Barley extract 1/100 2004.07.27 -0.0133
Running Y canoe dock 2004.09.01 afternoon Running Y canoe dock 2004.09.02 afternoon Running Y canoe dock 2004.09.02 evening * The "slope" is calculated as the decrease in the natural log of the absorbance against the wavelength between 250 and 500 nm.
These samples were examined with both spectrophotometer and spectrofluorometer analysis. I was trying to relate the total organic content and the little we know about each of these water sources with absorbance or fluorescence characteristics that have been shown to indicate particular processes.
The slope of the absorbance curve should indicate the photoreactivity. Lower slopes indicate that relatively less of the higher shorter wavelengths are being absorbed. I found a relationship between the total organic carbon and the slope of the decrease in absorbance (Figure 1a. "slope-vs-toc.gif"). This relationship implies that sources of water with higher organic content would probably contain a smaller proportion of UV absorbing compounds, and thus are likely to be less photoreactive. The highest point (28.23,-.109) is from Shoalwater Bay. The sample came from a was from the outflow of a large marsh area and was highly turbid with mud. The high turbidity could have protected the organics from degradation.
The UV absorbance at 250 nm (Figure 1c) and the fluorescence emission at 450 nm with excitation at 370 nm (Figure 1c) were both related to the dissolved organic carbon. These indicators could be used as rapid estimates for the amount of dissolved organic carbon. We still need a spectral or fluorescence signature that could be used to track or identify active humic water.
Figure 1 ("slope-vs-toc.gif"). The slope of the decrease in absorbance with wavelength plotted in relation to the total organic carbon concentrations.
Figure 1b ("abs250-vs-DOC.gif"). Absorbance of the filtered water at 250 nm.
Figure 1c ("fluor450-vs-DOC.gif"). Fluorescence for excitation at 350 nm and emission at 470 nm.
Photosynthesis response curves of light vs. ETR were run on samples collected from inside and outside the limno-corrals. Samples were taken in the morning and in the mid-afternoon just as the wind had built up. The afternoon sample was timed to determine if the AFA in ambient water was more mixed than those in the bags.
Figures 2a and 2b ("in-and-out-morning.gif" and "in-and-out-afternoon.gif"). ETR light curves measured by PAM fluorometery. ETR is related to other photosynthetic measurements such as oxygen production or 14C fixation. All but four points on these graphs represents the mean of two readings on two separate samples taken from either the lake or the limno-corral. There are four points at high light for the "IN" samples for the morning comparison where there are only duplicate readings of individual samples at 800, 1000, 1200, 1500, and 1600 uE m^-2 s^-1.
These differences in photosynthetic efficiency are minor and could be explained by a small (20%) difference in the cell count. The important result is that the cells in the limno-corrals were healthy and had high photosynthetic efficiencies. This indicates that there was no severe limitation by light or nutrients.
The potential inhibitory effect of humic rich water was tested by mixing filtered humic rich water with a healthy sample of AFA. In each run of this experiment, six conditions were run:
"E" 1/2 AFA source water + 1/2 filtered AFA source water
"F" 1/2 AFA source water + 1/2 filtered humic source water
"R" raw, undiluted, AFA source water
In each case cells were either left in the dark or exposed to natural sunlight. Cells that were left in the dark were denoted by a "-". After exposure, all the samples were sampled over a time course, looking for the level of inhibition and potential recovery of the Fv/Fm ratio.
Figure 3 ("inhibition-run1.gif"). Inhibition experiment run1, Sept1-04. Algae were collected from the limno-corral site. The brown water was collected from the canoe dock on the Running Y resort. The incubation took place from 4:30 PM to 5:04 PM. The PAR was not measured. The UV dose was 82.8 uE m^-2 s^-1 of UV (UVA and UVB). Each point in this graph is the mean of four readings on a single sample.
Figure 4 ("inhibition-run2.gif"). Inhibition experiment run2, Sept. 02, 2004. Algae were collected from the limno-corral site. The brown water was collected from the canoe dock on the Running Y resort. The samples were exposed to light for 21 minutes from 14:29 to 14:50.. The PAR dose averaged 1609 uE m^-2 s^-1. The UV dose was 106 uE m^-2 s^-1 of UV (UVA and UVB). The non-exposed points are the mean of four readings on a single sample at each time point. The light exposed points represent the mean of two separate samples with four readings on each sample.
Figure 5 ("inhibition-run3.gif"). Inhibition experiment run3, Sept. 02, 2004. Algae were collected from the boat ramp by the apple tree at the Running Y resort. The brown water was collected from the canoe dock on the Running Y resort. The samples were exposed to light for 10 minutes from 17:50 to 16:00.. The PAR dose averaged 610.8 uE m^-2 s^-1. The UV dose was 46.9 uE m^-2 s^-1 of UV (UVA and UVB). The non-exposed points are the mean of four readings on a single sample at each time point. The light exposed points represent the mean of two separate samples with four readings on each sample.
These inhibition experiments show definite inhibition of cells by light treatment, dilution and mixture with filtered brown water. Without further work, I can't be definitive about the statistical significance of these data however it is clear that the mixture of algae with humic rich water had a detrimental effect. In run 1 (Figure 3) the humic mixture "F" had a dramatic and unrecoverable inhibition. In run 2 (Figure 4) all exposed cells had much lower Fv/Fm ratios but the humic addition caused the most severe limitation. In run 3 (Figure 5) the humic addition had the lowest average Fv/Fm ratio and, importantly showed the least sign of recovery. In run 3 (and to a lesser extent in the other two runs) the addition of humics had a inhibitory effect even on the samples kept in the dark. This effect may be particularly important in the application of this to real mixing scenarios where the PAR and UV doses would be very small except for at the very top of the water column.
This work had three major results:
1. It would be difficult to identify reactive humic waters give the absorbance and fluorescence measurements that I am able to take. Maybe concentrated humics samples (from Mike Perdue) could be used in inhibition tests.
2. The cells in the limno-corrals seem to be as healthy (in terms of photosynthetic response) as the cells in the surrounding lake. The similarity between the corrals and the lake also would indicate that the cells in the bag were not light or nutrient limited. In order to really make the claim it is probably important to make sure that the water in the bag is not exchanging with the water in the lake. We could use a non-toxic conservative tracer in the bags.
3. The work over the last month helped clarify the value of the PAM fluorometer in these studies. It turned out that the dilution and light exposure regimes used were to "heavy handed". More work should be done that explores lower humic contributions (in the range up to 10% of the total rather than 50%) and lower light exposures. Dilutions and light exposures in these ranges are much more realistic for humic inputs during the spring.
