278
Exercise 20
9. The kinetic method assumes that the removal of the organic substratc by algae is negligible in
comparison to the active permease uptake system of bacteria. Do you believe that this
assumption is true for natural algal and bacterial communities at naturally occurring
substrate concentrations? Why?
10. Sometimes one organic substrate can inhibit competitively the uptake of another e.g.,
Burnison and Morita (1973)]. How could this potential problem be evaluated? What would
be the effect of such competitive inhibition?
11. Under acidic conditions, some substrates, such as acetic acid and glycollic acid, are volatile
and would be assayed as CO2 mineralized. What effects would this event have on the blank
(control) values? How would this complication affect the estimates of the various kinetic
parameters?
12. How could specific rates of decomposition of dissolved organic matter be determined?
[Cf., Cunningham and Wetzel (1989).J
Apparatus and Supplies
1. Reaction flasks (25-, 50-, or 100-ml) (e.g., K.882300, Kontes Glass Co., Vineland, NJ), with
serum stopper caps and suspended plastic cups (e.g., Kontes Center Well K-882320-0000).
Reaction flasks with a side-arm port (e.g., Kontes K-882360) facilitate addition of the killing
agent but are not essential.
2. Filter paper, e.g., Whatman No. 1.
3. Micropipets, e.g., Eppendorf of 10-, 50-, 100-lll capacities.
4. 2N H 2 S0 4 (50% v/v phosphoric acid can also be used as a killing agent).
5. Temperature control (bath or environmental chamber).
6. Rotary shaker.
7. Monoethanolamine. [Alternatively, f:i-phenethylamine can be used (Hobbie and Crawford,
1969a).J
8. Radioassay:
a. Standard scintillation vials.
b. Filter paper~cups~cocktail: Mix 15 g PPO (2,5-diphenyloxazole) and 1 g bis-MSB
[p-bis-(o-methylstyryl)-benzeneJ in I liter of scintillation grade toluene. Mix well three
parts of this solution to one part of spectral quality methanol. [Altcrnativc cocktails are
given in Hobbie and Crawford (1969a).J Caution: Some of these reagents are highly
toxic and flammable.
c. Bacteria on membrane filters: Place wet filters directly into Insta-gel (Packard Instrument
Co., Downers Grove, II) or similar scintillation solution.
d. Liquid scintillation counter.
9. Labeled organic substrates:
a. Uniformly carbon-14 labeled glucose, acetate, and/or glutamate or other amino acids,
e.g., glycine, of highest specific activity available (e.g., Amersham/Searle, Des Plaines, II).
b. Dilute the isotope to activities of the range of I !lCi/ml with sterile water of the greatest
purity available (deionized, organic-free, redistilled). Dispense into clean precombusted
(see Exercise 9) ampoules and seal and freeze immediately. All glassware and ampoules
must be cleaned scrupulously.
c. From the specific activity of the isotope, calculate the amount of substrate present in
!lg/ml in the final dilution from the specific activity (usually given as mCi/mmol).
d. Determine the radioactivity of the substrate(s) per ml by comparison to standards (see
Exercise 14).
10. Additions of substrate:
a. From Section 9, Part (d) above, determine the amount of "carrier" organic substrate that
is added with the labeled substrate. This amount is less than that needed for the serially
increasing substrate concentrations of the experiment.
b. The substrate concentration of the radioactively labeled ("hot") material must be
augmented with unlabeled ("cold") substrate of the same organic compound. The
Exercise 20
9. The kinetic method assumes that the removal of the organic substratc by algae is negligible in
comparison to the active permease uptake system of bacteria. Do you believe that this
assumption is true for natural algal and bacterial communities at naturally occurring
substrate concentrations? Why?
10. Sometimes one organic substrate can inhibit competitively the uptake of another e.g.,
Burnison and Morita (1973)]. How could this potential problem be evaluated? What would
be the effect of such competitive inhibition?
11. Under acidic conditions, some substrates, such as acetic acid and glycollic acid, are volatile
and would be assayed as CO2 mineralized. What effects would this event have on the blank
(control) values? How would this complication affect the estimates of the various kinetic
parameters?
12. How could specific rates of decomposition of dissolved organic matter be determined?
[Cf., Cunningham and Wetzel (1989).J
Apparatus and Supplies
1. Reaction flasks (25-, 50-, or 100-ml) (e.g., K.882300, Kontes Glass Co., Vineland, NJ), with
serum stopper caps and suspended plastic cups (e.g., Kontes Center Well K-882320-0000).
Reaction flasks with a side-arm port (e.g., Kontes K-882360) facilitate addition of the killing
agent but are not essential.
2. Filter paper, e.g., Whatman No. 1.
3. Micropipets, e.g., Eppendorf of 10-, 50-, 100-lll capacities.
4. 2N H 2 S0 4 (50% v/v phosphoric acid can also be used as a killing agent).
5. Temperature control (bath or environmental chamber).
6. Rotary shaker.
7. Monoethanolamine. [Alternatively, f:i-phenethylamine can be used (Hobbie and Crawford,
1969a).J
8. Radioassay:
a. Standard scintillation vials.
b. Filter paper~cups~cocktail: Mix 15 g PPO (2,5-diphenyloxazole) and 1 g bis-MSB
[p-bis-(o-methylstyryl)-benzeneJ in I liter of scintillation grade toluene. Mix well three
parts of this solution to one part of spectral quality methanol. [Altcrnativc cocktails are
given in Hobbie and Crawford (1969a).J Caution: Some of these reagents are highly
toxic and flammable.
c. Bacteria on membrane filters: Place wet filters directly into Insta-gel (Packard Instrument
Co., Downers Grove, II) or similar scintillation solution.
d. Liquid scintillation counter.
9. Labeled organic substrates:
a. Uniformly carbon-14 labeled glucose, acetate, and/or glutamate or other amino acids,
e.g., glycine, of highest specific activity available (e.g., Amersham/Searle, Des Plaines, II).
b. Dilute the isotope to activities of the range of I !lCi/ml with sterile water of the greatest
purity available (deionized, organic-free, redistilled). Dispense into clean precombusted
(see Exercise 9) ampoules and seal and freeze immediately. All glassware and ampoules
must be cleaned scrupulously.
c. From the specific activity of the isotope, calculate the amount of substrate present in
!lg/ml in the final dilution from the specific activity (usually given as mCi/mmol).
d. Determine the radioactivity of the substrate(s) per ml by comparison to standards (see
Exercise 14).
10. Additions of substrate:
a. From Section 9, Part (d) above, determine the amount of "carrier" organic substrate that
is added with the labeled substrate. This amount is less than that needed for the serially
increasing substrate concentrations of the experiment.
b. The substrate concentration of the radioactively labeled ("hot") material must be
augmented with unlabeled ("cold") substrate of the same organic compound. The