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Appendix I: Composition of modified Z-medium (Zarrouk,1966)

Constituents Concentration (g/L)

NaHCO3 16.8

K2HPO4 0.5

NaNO3 2.5

K2SO4 1.0

NaCl 1.0

MgSO4.7H2O 0.2

CaCl2.2H2O 0.04

FeSO4.7H2O 0.01

Micronutrient solution (A5 solution) 1mL

pH 9.0

The A5 solution (Arnon, 1938) contained the following

constituents in g/L

H3 BO4 2.86

MnCl2 1.81

ZnSO4.7H2O 0.222

Na2MoO4.2H2O 0.0177

CuSO4.H2O 0.079

Appendix II: Preparation of Stacking gel and Separating gel

Stacking gel:

Seperating gel:

30% acrylamide/0.8% bis acrylamide solution 3.75 mL

4X Tris-Cl (pH 8.8) 3.75 mL

DDW 7.50 mL

10% APS 60 µL

TEMED 15 µL

30% acrylamide/0.8% bis acrylamide solution 3.75 mL

4X Tris-Cl (pH 8.8) 3.75 mL

DDW 7.50 mL

10% APS 60 µL

TEMED 15 µL

Appendix III: Composition of 16S rRNA gene amplification

Components/ sample Concentration

(stock)

Quantity/

reaction

PCR buffer with KCl

+ Mg Cl2 (15mM) 10 X 2.5l

MgCl2

dNTPs (mix)

25mM

100mM each

1l

0.2l

16S Primers (FD1 and RP2) 0.5 picomoles/μL each 5.0l

Taq polymerase

5 U/l 0.2l

Sterile water - 14.1l

Template DNA 25 ng/μL 2l

Total 25l

Appendix V: Composition of cpcB-IGS-cpcA gene amplification

Components/ sample Concentration

(stock)

Quantity/

reaction

PCR buffer with KCl

+ Mg Cl2 (15mM) 10 X 2.5l

MgCl2

dNTPs (mix)

25mM

100mM each

1l

0.2l

cpc_arF and cpc_arR 0.5 picomoles/μL each 5.0l

Taq polymerase

5 U/l 0.2l

Sterile water - 14.1l

Template DNA 25 ng/μL 2l

Total 25l

Appendix IV: Thermal cycler parameter for 16S rRNA gene amplification

Steps Temperature(oC)

Duration

(min) Cycles Activity

1. 94 5.00 1 Denaturation

2. 94 0.30

35

Denaturation

3. 64 0.75 Annealing

4. 72 2.00 Extension

5. 72 5.00 1 Final

Extension

6. 4 24 hours 1 Hold

Appendix VI: Thermal cycler parameter for cpcB-IGS-cpcA gene amplification

Steps Temperature(oC)

Duration

(min) Cycles Activity

1. 95 5.00 1 Denaturation

2. 95 0.25

35

Denaturation

3. 60 0.15 Annealing

4. 72 0.30 Extension

5. 72 5.00 1 Final

Extension

6. 4 24 hours 1 Hold