Experimental protocol 1: Assessment of soil trophic potential
Chemicals and Reagents
Algal medium Z8m (see below), deionized water
Material
Autoclave, centrifuges, microplate reader, pipettors, pipette tips, microtubes, centrifugation vials, paper filters, funnels, Erlenmeyer flasks, illuminated chamber, gas-tight incubation container, CO
2
supply, culture of Chlorella kessleri (strain
LARG/1)
Method protocol
1. Prepare soil extract a. resuspend 30 g of dry soil in 60 ml of deionized water for 20 minutes b. Centrifuge the suspension at 3044 × g for 30 min c. Filter the supernatant through a paper filter to remove floating woody particles d. Autoclave the extract at
121ºC for 25 minutes
2. Prepare fresh inoculum of Chlorella kessleri (strain LARG/1) a. Grow the algae for
4 days at 25 ºC under daylight illumination in 200 ml sterile liquid medium Z8m. b. Centrifuge t he culture at 180 × g for 5 min c. Resuspend the cells in 200 ml sterile deionized water d. Subject the algae to a 2-day period of mineral starvation under daylight illumination at 25 ºC.

e. Wash the cells once with sterile deionized water f. Dilute the suspension so as to reach the absorbance at 655 nm equal to 0.180 (corresponding to the concentration of 4 × 10 5 cells per ml).
This suspension is used as inoculum in the assay.
3. Establishment of the assay a. Distribute 100-
µl aliquots of the soil extracts to a 96-well flat-bottomed transparent serological plate. Use four analytical replicates (wells) per sample. b. Establish blanks, i.e. wells with 100µl aliquots of deionized water and serially diluted Z8m (dilutions 1:2, 1:9 and 1:29), with six analytical replicates each. c. Supply each well with 25 µl of algal inoculum d. Measure initial absorbance at 655 nm e. Incubate the samples for 7 days at 25 ºC, under the following conditions: i. illumination 3000 lux (cool-white fluorescent tubes) ii. 100% relative humidity iii. 5% CO
2
in the atmosphere f. Measure absorbance at 655 nm g. Subtract initial absorbance of the respective wells h. Calculate mean absorbance increments per sample, use this as a proxy of fertility of the soil (soil trophic potential)

Composition of the Z8m medium, the component amounts are given per 1 liter final concentration
K
2
HPO
4
31 mg
59 mg Ca(NO
3
)
2
.4H
2
O
MgSO
4
.7H
2
O 25 mg
21 mg Na
2
CO
3
NaNO
3
467 mg
FeNaEDTA
H
3
MnSO
Na
(NH
KBr
KJ
BO
2
3
4
WO
4
)
ZnSO
CdCl
CoCl
CuSO
NiCl
K
NaVO
Al
2
2
Cr
2
2
2
2
6
4
.5H
4
2
.2H
Mo
7
O
.2.5H
.6H
(SO
4
.6H
O
3
.7H
4
.5H
7
)
2
3
2
2
2
O
O
2
24
O
2
O
O
O
O
.4H
pH (KOH/HCl)
2
O
6.7 mg
3.1 mg
11.9 µg
12.5 µg
12 µg
2 µg
1.2 µg
17 µg
2.4 mg
3.5 µg
8.8 µg
12 µg
8.3 µg
28.7 µg
11.5 µg
7.0