Experiment
For leaf litter leachate from each tree species, we prepared “control”
and “amended” treatments using 200 mL Nalgene incubation bottles. The
control treatment consisted of 150 mL of filtered leaf litter leachate,
and the amended treatment consisted of 147 mL of filtered leachate and 3
mL of basal COMBO medium, which contained macroelements (K, Ca, Mg, B),
trace elements (Fe, Mn, Cu, Zn, Co, Mo, Se, and V), and vitamins (B12,
biotin, and thiamin), but no inorganic N and P nutrients . We referred
these chemicals as micronutrients. In each leaf litter leachate, we set
up three replicates for each treatment.
We inoculated algal cells into each experimental bottle at an initial
incubation cell density of 2.2×104 cells
mL−1. The algae were incubated for 7 days in the
bottles, which were placed in an incubator at a temperature of 20 °C and
light intensity of 100 μE m−2 s−1.
The bottles were manually shaken every day to resuspend the cells. After
7 days, we preserved 20 mL of culture medium in Lugol’s solution for
estimating the cell number by microscopic enumeration. We also filtered
5 mL of culture medium on two combusted GF/F filters (Whatman), which
were dried for further analyses of the C, N, and P contents ofScenedesmus obliquus .