Supplementary material to
‘Understanding the Simultaneous Biodegradation of Thiocyanate and Salicylic
Acid by Paracoccus thiocyanatus and Pseudomonas putida ’
Combarros, R.G., Collado, S., Laca, A., Díaz, M*
Department of Chemical Engineering and Environmental Technology,
University of Oviedo, C/Julián Clavería s/n, E-33071, Oviedo, Spain
(10 Pages, 7 Figures, 1 Table)
Table of contents
1. Summary of biodegradation conditions and results of interaction effects between
P.putida-P. thiocyanatus - SA- SCN- system. (Table S1).
2. Evolution of salicylic acid or thiocyanate concentrations under different culture
conditions. (Figure S2).
3. Evolution of salicylic acid in presence of a pure culture of P. putida and growth
of P. thiocyanatus. (Figure S3).
4. Evolution of growth curves during the simultaneous biodegradation of both
contaminants by a co-culture and by pure culture of P. putida or P. thiocyanatus.
(Figure S4).
5. Specific growth rate of suspended biomass in a thiocyanate and salicylic acid
solution using a co-culture. (Figure S5).
6. Dot plots representing cFDA fluorescence versus PI fluorescence of co-culture of
Paracoccus thiocyanatus and Pseudomonas putida cells during shake-flask
fermentation. (Figure S6).
7. Dot plots representing side scatter light (SSC) versus forward scatter light (FSC)
signals of co-culture cells during shake-flask simultaneous biodegradation.
(Figure S7).
8. Evolution of salicylic acid or thiocyanate concentrations and growth curves
during the simultaneous biodegradation of both contaminants by a co-culture of
Paracoccus thiocyanatus and Pseudomonas putida. (Figure S8).
*Corresponding author’s e-mail: mariodiaz@uniovi.es
Phone: +34 985 10 34 39; Fax: +34 985 10 34 34
S 1: Summary of biodegradation conditions and results of interaction effects between P.putida-P. thiocyanatus - SA- SCN- system.
Row
Mineral
medium
Culture
conditions *
Bacteria
Substrate
1
PpMM
Pp conditions
P. putida
500 mg SA L-1
-1
ti (h)
Specific degradation rate
(mg substrate (h mg cells)-1)
μ (h-1)
0
2.5
0.2459
Conversion
(reaction time)
(%- h)
100% in 22
Volumetric degradation rate
(mg substrate (Lh)-1)
31
2
PpMM
Pp conditions
P. putida
500 mg SA L
500 mg SCN- L-1
0
-----
0.92
0
0.0007
73% in 140
0% in 140
6.25
0
3
PpMM
Pp conditions
P. putida
500 mg SCN- L-1
0
0
0
0% in 140
0
4
PtMM
Pt conditions
P. thiocyanatus
500 mg SCN- L-1
20
0.32
0.0314
100% in 48
20.7
-
-1
5
PtMM
Pt conditions
P. thiocyanatus
500 mg SCN L
500 mg SA L-1
48
-----
0.35
0
0.0093
100% in 144
0% in 144
6.7
0
6
PtMM
Pt conditions
P. thiocyanatus
500 mg SA L-1
0
0
0
0% in 144
0
-1
7
PpMM
CC conditions
P. putida
500 mg SA L
500 mg SCN- L-1
0
-----
0.99
0
0.2338
96% in 34
0% in 144
13.6
0
8
CCMM
CC conditions
P. putida
500 mg SA L-1
500 mg SCN- L-1
0
-----
0.86
0
0.3137
100% in 11
0% in 144
46.1
0
9
PtMM
CC conditions
P. thiocyanatus
500 mg SCN- L-1
500 mg SA L-1
60
-----
0.53
0
0.0097
100% in 135
0% in 144
6.67
0
10
CCMM
CC conditions
P. thiocyanatus
500 mg SCN- L-1
500 mg SA L-1
30
-----
0.36
0
0.0186
100% in 51
0% in 144
16.7
0
11
CCMM
CC conditions
P. putida
500 mg SA L-1
12
CCMM
CC conditions
13
CCMM
CC conditions
14
CCMM
CC conditions
15
CCMM
CC conditions
P. thiocyanatus
P. thiocyanatus
P. putida
P. thiocyanatus
P. putida
P. thiocyanatus
P. putida
0
0.89
0.23
100% in 12
39.3
-1
11
0.68
0.052
100% in 25
32.2
500 mg SCN- L-1
17
0.58
0.027
100% in 40
36.9
500 mg SA L-1
0
0.64
0.091
100% in 14
40
500 mg SCN- L-1
500 mg SA L-1
12
0
0.53
0.39
0.0186
0.3137
100% in 29
100% in 12
35.2
42.1
-
500 mg SCN L
* Pp conditions: 200 rpm, 30ºC and 100 mL of mineral medium in 250 mL Erlenmeyer flasks; Pt conditions: 250 rpm, 28ºC and 100 mL of mineral medium in 500 mL
Erlenmeyer flaks; CC conditions: 30°C, 250 rpm and 100 mL of mineral médium in 500 mL Erlenmeyer flasks
600
600
(a)
400
300
200
100
(b)
500
SCN- (mg/L)
SA (mg/L)
500
400
300
200
100
0
0
0
5
10
15
Time (h)
20
0
10
20
30
Time (h)
40
50
S 2: Evolution of salicylic acid or thiocyanate concentrations under different culture conditions. a)
Evolution of SA concentration by P. putida (), by P. putida in presence of P. thiocyanatus (■), by
P. putida in presence of SCN- () and by P. putida in presence of P. thiocyanatus and SCN- (). b)
Evolution of SCN- concentration by P. thiocyanatus (), by P. thiocyanatus in presence of P. putida
(■), by P. thiocyanatus in presence of SA () and by P. thiocyanatus in presence of P. putida and
SA (). In all cases: CCMM, initial SCN- or SA concentration: 500 mg L-1, 250 rpm, 30 ºC, 100mL
in 500 mL Erlenmeyer flasks.
600
4.5E+08
(b)
(a)
500
CFU/mL
SA (mg/L)
3.5E+08
400
300
200
2.5E+08
1.5E+08
100
5.0E+07
0
0
25
Time (h)
50
75
0
10
20
Time (h)
30
S 3: (a) Evolution of salicylic acid in presence of a pure culture of P. putida. Arrows denote the times
at which the bacteria was removed: 4.5 h (), 9.8 h (■) and 22 h (). These media were subsequently
inoculated with P thiocyanatus, following its bacterial growth as cfu/mL (b). In all cases: CCMM,
initial SA concentration: 500 mg L-1, 250 rpm, 30 ºC, 100mL in 500 mL Erlenmeyer flasks. The
arrows indicate the time when P. putida were removed from the medium and P. thiocyanatus were
inoculated in that medium.
0.12
C (g/L)
0.1
0.08
0.06
0.04
0.02
0
0
10
20
30 40
Time (h)
50
60
70
S 4: Evolution of growth curves during the simultaneous biodegradation of both contaminants by a
co-culture of Paracoccus thiocyanatus and Pseudomonas putida (■) and by pure culture of P. putida
() or P. thiocyanatus (). In all cases: CCMM, 30ºC, 250 rpm, inoculum size of 1·107 cfumL-1 of
both
(a)
0.02
0.01
(b)
0.03
μSCN- (h-1)
μSA(h-1)
0.03
0.02
0.01
0
0
0
1000
2000
3000
SA0 (mg L-1)
4000
0
1000
2000
3000
SCN0 (mg L-1)
4000
S 5: Specific growth rate of suspended biomass in a thiocyanate and salicylic acid solution using a
co-culture of P. putida and P. thiocyanatus (250-4000 mg L-1). a) P. putida specific growth rate b) P.
thiocyanatus ( Experimental data, …… substrate and toxic inhibition model, - - - Teisser model).
In all cases: 30ºC, 250 rpm, and inoculum size of 1·107 cfu mL-1 of each bacteria .
cFDA fluorescence
A
D
B
C
E
F
PI fluorescence
S 6: Dot plots representing cFDA fluorescence versus PI fluorescence of co-culture of Paracoccus
thiocyanatus and Pseudomonas putida cells during shake-flask fermentation. A, B and C plots
correspond to an initial SA and SCN- concentration of 250 mg L-1 at 0, 24 and 31 hours respectively.
D, E and F plots correspond to an initial SA and SCN- concentration of 2000 mg L-1 at 0, 48 and 144
hours respectively.
0h
48 h
72 h
168 h
192 h
S 7: Dot plots representing side scatter light (SSC) versus forward scatter light (FSC) signals of coculture cells during shake-flask simultaneous biodegradation to an initial SA and SCN- concentration
of 3000 mg L-1 at 0, 48, 72, 168 and 192 hours respectively
0.08
500
0.06
400
300
0.04
200
C co-culture (g/L)
SA or SCN- (mg/L)
600
0.02
100
0
0
0
10
20
30
Time (h)
40
S 8: Evolution of salicylic acid () or thiocyanate (■) concentrations and growth curves () during
the simultaneous biodegradation of both contaminants by a co-culture of Paracoccus thiocyanatus
and Pseudomonas putida. Previously to inoculation, the co-culture was acclimated with 4000 mg L-1
of both SCN- and SA during 120 hours. The culture conditions: CCMM, 30ºC, 250 rpm, initial SCNand SA concentrations of 500 mg L-1, and inoculum size of (1·107 cfu mL-1 of both bacteria).