Contents
General aim
Specific objectives
Advances
Phylogenetic identification
Parangueo Re-isolation of strains
Characterization of 18O-PAR strain
Los Azufres Electron donor and acceptor studies
Sulfide producing evaluation
Work to do
General aim
Evaluate the arsenic removal from acidic
effluents by precipitation with sulfide produced
in a sulfidogenic bioreactor inoculated with SRB
isolated from extremophile sites.
Specific objectives
Isolate SRB from Los Azufres and Parangueo.
Identify by molecular biology the isolated strains.
Characterize morphologically and physiologically strains of
interest.
Evaluate the optimal conditions for sulfide production.
Design a system for remediation of effluents contaminated
with arsenic.
Stablish the optimal operating conditions for arsenic removal.
Study the diversity changes of communities in the bioreactor
under different conditions.
•Identification of strains and
•Characterization studies
RESULTS
Identification of strains from Los
Azufres and Parangueo
28 samples:
12 from Spa “Los Azufres” obtained by roll-tube
16 from Parangueo Lake obtained by microplaques isolation
16S Tree from
Parangueo and Los
Azufres strains.
Constructed with MEGA 6.06.
using the statistical method
neighbor-joining and the
bootstrap method (500
bootstraps)
Desulfovibrionaceae Desulfovibrio gigas DSM 1382 (NR_121705)
Desulfonatronum cooperativum Z-7999 (NR_043143)
Desulfonatronum alkalitolerans HSRB (NR_108631)
PAR18O (PAR19O)
Desulfonatronum thiosulfatophilum (NR_116694)
Desulfonatronum thioautotrophicum ASO4-1 (NR_116693)
Desulfonatronum lacustre DSM 10312 (NR_041848)
PAR 13I (PAR 13F, PAR 12D, PAR 2B, PAR 18K)
PAR 4H11 (PAR 22N8, PAR 12B, PAR 14O)
Desulfonatronum thiodismutans MLF1 (NR_025163)
Desulfonatronaceae
Desulfococcus niacini DSM2650T (FR733668)
Desulfobacterium autotrophicum DSM 3382 (NR_041853)
Desulfobotulus sapovorans strain DSM 2055 (NR_117108.1)
22N
Desulfobotulus alkaliphilus ASO4-4 (NR_116806)
Desulfobacteraceae
Clostridium roseum strain ASU46 (KF372579.1)
Clostridium beijerinckii JCM 7990(AB678386)
AZB1 06 (AZB1 11, AZB109, AZB1 08)
Clostridium ghonii (JN048963)
Clostridium sordellii strain 2TT1 (HM163489)
AZB1 10
Clostridaceae
Desulfosporosinus orientis DSM 765 (NR_074131)
Desulfosporosinus auripigmenti strain OREX-4 (NR_025551)
Desulfosporosinus meridiei DSM 13257 (CP003629)
Desulfosporosinus youngiae JW/YJL-B18 (NR_115694)
AZB1 01 (AZLC 02, AZB1 04, AZLC 05, AZB1 07, AZB1 12)
Desulfosporosinus sp. enrichment culture clone H4 (JQ420043)
Desulfosporosinus acidiphilus SJ4 (NR_116868)
Desulfosporosinus acididurans (HG316991)
Peptococcaceae
Nitrospiraceae Thermodesulfovibrio yellowstonii (AB231858)
Desulfovibrionaceae Desulfovibrio gigas DSM 1382 (NR_121705)
Desulfonatronum cooperativum Z-7999 (NR_043143)
Desulfonatronum alkalitolerans HSRB (NR_108631)
PAR18O (PAR19O)
Desulfonatronum thiosulfatophilum (NR_116694)
Desulfonatronum thioautotrophicum ASO4-1 (NR_116693)
Desulfonatronum lacustre DSM 10312 (NR_041848)
PAR 13I (PAR 13F, PAR 12D, PAR 2B, PAR 18K)
PAR 4H11 (PAR 22N8, PAR 12B, PAR 14O)
Desulfonatronum thiodismutans MLF1 (NR_025163)
Desulfonatronaceae
Desulfococcus niacini DSM2650T (FR733668)
Desulfobacterium autotrophicum DSM 3382 (NR_041853)
Desulfobotulus sapovorans strain DSM 2055 (NR_117108.1)
22N
Desulfobotulus alkaliphilus ASO4-4 (NR_116806)
Desulfobacteraceae
Clostridium roseum strain ASU46 (KF372579.1)
Clostridium beijerinckii JCM 7990(AB678386)
AZB1 06 (AZB1 11, AZB109, AZB1 08)
Clostridium ghonii (JN048963)
Clostridium sordellii strain 2TT1 (HM163489)
AZB1 10
Clostridaceae
Desulfosporosinus orientis DSM 765 (NR_074131)
Desulfosporosinus auripigmenti strain OREX-4 (NR_025551)
Desulfosporosinus meridiei DSM 13257 (CP003629)
Desulfosporosinus youngiae JW/YJL-B18 (NR_115694)
AZB1 01 (AZLC 02, AZB1 04, AZLC 05, AZB1 07, AZB1 12)
Desulfosporosinus sp. enrichment culture clone H4 (JQ420043)
Desulfosporosinus acidiphilus SJ4 (NR_116868)
Desulfosporosinus acididurans (HG316991)
Peptococcaceae
Nitrospiraceae Thermodesulfovibrio yellowstonii (AB231858)
Desulfovibrionaceae Desulfovibrio gigas DSM 1382 (NR_121705)
Desulfonatronum cooperativum Z-7999 (NR_043143)
Desulfonatronum alkalitolerans HSRB (NR_108631)
PAR18O (PAR19O)
Desulfonatronum thiosulfatophilum (NR_116694)
Desulfonatronum thioautotrophicum ASO4-1 (NR_116693)
Desulfonatronum lacustre DSM 10312 (NR_041848)
PAR 13I (PAR 13F, PAR 12D, PAR 2B, PAR 18K)
PAR 4H11 (PAR 22N8, PAR 12B, PAR 14O)
Desulfonatronum thiodismutans MLF1 (NR_025163)
Desulfonatronaceae
Desulfococcus niacini DSM2650T (FR733668)
Desulfobacterium autotrophicum DSM 3382 (NR_041853)
Desulfobotulus sapovorans strain DSM 2055 (NR_117108.1)
22N
Desulfobotulus alkaliphilus ASO4-4 (NR_116806)
Desulfobacteraceae
Clostridium roseum strain ASU46 (KF372579.1)
Clostridium beijerinckii JCM 7990(AB678386)
AZB1 06 (AZB1 11, AZB109, AZB1 08)
Clostridium ghonii (JN048963)
Clostridium sordellii strain 2TT1 (HM163489)
AZB1 10
Clostridaceae
Desulfosporosinus orientis DSM 765 (NR_074131)
Desulfosporosinus auripigmenti strain OREX-4 (NR_025551)
Desulfosporosinus meridiei DSM 13257 (CP003629)
Desulfosporosinus youngiae JW/YJL-B18 (NR_115694)
AZB1 01 (AZLC 02, AZB1 04, AZLC 05, AZB1 07, AZB1 12)
Desulfosporosinus sp. enrichment culture clone H4 (JQ420043)
Desulfosporosinus acidiphilus SJ4 (NR_116868)
Desulfosporosinus acididurans (HG316991)
Peptococcaceae
Nitrospiraceae Thermodesulfovibrio yellowstonii (AB231858)
Desulfovibrionaceae Desulfovibrio gigas DSM 1382 (NR_121705)
Desulfonatronum cooperativum Z-7999 (NR_043143)
Desulfonatronum alkalitolerans HSRB (NR_108631)
PAR18O (PAR19O)
Desulfonatronum thiosulfatophilum (NR_116694)
Desulfonatronum thioautotrophicum ASO4-1 (NR_116693)
Desulfonatronum lacustre DSM 10312 (NR_041848)
PAR 13I (PAR 13F, PAR 12D, PAR 2B, PAR 18K)
PAR 4H11 (PAR 22N8, PAR 12B, PAR 14O)
Desulfonatronum thiodismutans MLF1 (NR_025163)
Desulfonatronaceae
Desulfococcus niacini DSM2650T (FR733668)
Desulfobacterium autotrophicum DSM 3382 (NR_041853)
Desulfobotulus sapovorans strain DSM 2055 (NR_117108.1)
22N
Desulfobotulus alkaliphilus ASO4-4 (NR_116806)
Desulfobacteraceae
Clostridium roseum strain ASU46 (KF372579.1)
Clostridium beijerinckii JCM 7990(AB678386)
AZB1 06 (AZB1 11, AZB109, AZB1 08)
Clostridium ghonii (JN048963)
Clostridium sordellii strain 2TT1 (HM163489)
AZB1 10
Clostridaceae
Desulfosporosinus orientis DSM 765 (NR_074131)
Desulfosporosinus auripigmenti strain OREX-4 (NR_025551)
Desulfosporosinus meridiei DSM 13257 (CP003629)
Desulfosporosinus youngiae JW/YJL-B18 (NR_115694)
AZB1 01 (AZLC 02, AZB1 04, AZLC 05, AZB1 07, AZB1 12)
Desulfosporosinus sp. enrichment culture clone H4 (JQ420043)
Desulfosporosinus acidiphilus SJ4 (NR_116868)
Desulfosporosinus acididurans (HG316991)
Peptococcaceae
Nitrospiraceae Thermodesulfovibrio yellowstonii (AB231858)
Desulfovibrionaceae Desulfovibrio gigas DSM 1382 (NR_121705)
Desulfonatronum cooperativum Z-7999 (NR_043143)
Desulfonatronum alkalitolerans HSRB (NR_108631)
PAR18O (PAR19O)
Desulfonatronum thiosulfatophilum (NR_116694)
Desulfonatronum thioautotrophicum ASO4-1 (NR_116693)
Desulfonatronum lacustre DSM 10312 (NR_041848)
PAR 13I (PAR 13F, PAR 12D, PAR 2B, PAR 18K)
PAR 4H11 (PAR 22N8, PAR 12B, PAR 14O)
Desulfonatronum thiodismutans MLF1 (NR_025163)
Desulfonatronaceae
Desulfococcus niacini DSM2650T (FR733668)
Desulfobacterium autotrophicum DSM 3382 (NR_041853)
Desulfobotulus sapovorans strain DSM 2055 (NR_117108.1)
22N
Desulfobotulus alkaliphilus ASO4-4 (NR_116806)
Desulfobacteraceae
Clostridium roseum strain ASU46 (KF372579.1)
Clostridium beijerinckii JCM 7990(AB678386)
AZB1 06 (AZB1 11, AZB109, AZB1 08)
Clostridium ghonii (JN048963)
Clostridium sordellii strain 2TT1 (HM163489)
AZB1 10
Clostridaceae
Desulfosporosinus orientis DSM 765 (NR_074131)
Desulfosporosinus auripigmenti strain OREX-4 (NR_025551)
Desulfosporosinus meridiei DSM 13257 (CP003629)
Desulfosporosinus youngiae JW/YJL-B18 (NR_115694)
AZB1 01 (AZLC 02, AZB1 04, AZLC 05, AZB1 07, AZB1 12)
Desulfosporosinus sp. enrichment culture clone H4 (JQ420043)
Desulfosporosinus acidiphilus SJ4 (NR_116868)
Desulfosporosinus acididurans (HG316991)
Peptococcaceae
Nitrospiraceae Thermodesulfovibrio yellowstonii (AB231858)
PARANGUEO LAKE
Mediums employed
Compuesto Concentración g/L (mM)
KH2PO4 0,2
NH4Cl 0,25
Na2SO4 4
KCl 0,5
CaCl2*2H2O 0,1
MgCl2*6H2O 1,2
Lactate (20)
Bis-tris-propano 3
NaCl 30
MPAR_1 MPAR_2 (based on 813 Desulfonatronum medium DSMZ)
Solución stock Concentración final
Vitaminas V7 100 µL/5mL
Adjust at pH 9 and after sterilization add:
Compuesto Concentración g/L (mM)
KH2PO4 0,2
MgCl2*6H2O 0,1
NH4Cl 0,25
Na2SO4 4
KCl 0,2
NaCl 15
Na2CO3 3,5
Yeast extract 0,2
Sol. Oligo de Widdel 1ml
Fe-Ni-Wo-Se 1000X 1 ml
Resazurine 0.1% 1 ml
Solución stock Concentración final
Lactato 0,5 M 20 mM
Vitaminas V7 100 µL/5mL
Na2S X 9H2O
(reductor)
0,4 g/L
Re-isolation of 18O, 19O, P1 y 22N
Serial dilution
Roll-tube (C4, Ethanol).
Caracterización fisiológica de la cepa 18O_PAR
0.000
0.005
0.010
0.015
0.020
0.025
0 10 20 30 40 50
µ (
h-1
)
Temperatura (°C)
0.000
0.005
0.010
0.015
0.020
0.025
0 50 100 150
µ (
h-1
)
g/L NaCl
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0 2 4 6 8 10 12
µ (
h-1
)
pH
Óptimos
• Temperatura: 35°C
• Salinidad :1-5 g/L
NaCl
• pH: 9
Dismutation assay: PAR18O
The inocula used for this trial was centrifuged and re-suspended with sulfate and lactate free medium.
Inoculation was made by duplicate in the following conditions:
Thiosulphate + Acetato: (+).
Thiosulphate + CO2 : (++).
Lactate + SO4 (control guarantee the good state of the cellules). (+++)
Lactate (to discard presence of sulphate): almost any growth.
Sulphate (to discard presence of lactate in inocula): (-)
Sulphate mM Thiosulphate mM Sulfide mM
Thiosulphate+Acetate 7,7 0,3 3,6
Tiosulfato+CO2 0,14 7,3 0,2
LOS AZUFRES
Mediums employed
Solución stock Concentración final
Lactate 0,5 M 5 mM (50µ/L/5mL)
Glycerol 0,25 M 5 mM (100µ/L/5mL)
Pyruvate 0,5 M 5 mM (50µ/L/5mL)
Acetate 0,25 M 5 mM (100µ/L/5mL)
Ethanol 1M 20 mM (100µ/L/5mL)
Peptone 125 g/L 10 g/L (400µ/L/5mL)
Vitamins V7 100 µL/5mL
MAZ-1
MAZ-2
Compuesto Concentración g/L (mM)
KH2PO4 0,25
MgSO4*7H2O (15)
(NH4)2SO4 1
Na2SO3 0,5
Na2S2O3*5H2O 0,5
Citrato de Fe 0,2
KCl 0,1
Ca(NO3)2*H20 0,05
L-cysteina HCl (reductor) 0,25
Yeast extract 0,2
Solución de oligo-elementos
de Widdel
1ml
Fe-Ni-Wo-Se 1000X 1 ml
Resazurina 0.1% 1 ml
Adjust at pH 5 after sterilization add:
Compuesto Concentración g/L (mM)
KH2PO4 0,1
NH4Cl 0,1
Na2SO4 2,13 (15 mM)
KCl 0,05
CaCl2 0,05
MgCl2*H2O 0,1
L-cysteine HCl 0,25
Yeast extract 0,2
Oligo de Widdel 1ml
Fe-Ni-Wo-Se 1000X 1 ml
Resazurine 0.1% 1 ml
Solución stock Concentración final
Lactato 0,5 M 10mM
Vitaminas V7 100 µL/5mL
Preliminary studies of electron aceptor
and carbon source
Electron aceptor
Cepa
Sulfite
SO3 (mM)
Sulfate
SO42- (mM)
Tiosulfate
S2O32- (mM)
T0 TF T0 TF T0 TF
AZB101 1,6 - 27,6 18,2 1,0 -
AZLC02 1,7 - 27,2 20,5 1,1 1,4
AZB103 1,6 - 27,2 17,9 1,1 -
AZB104 1,6 - 27,8 20,4 1,3 1,3
AZB105 1,6 - 27,6 19,4 1,2 1,3
AZLC07 1,7 1,6 27,5 20,0 1,4 1,6
AZB112 1,5 - 27,5 19,6 1,6 -
Since replicates at Marseille hadn’t grown well yet at 7 days, the TF is from
the cultures from Pau.
Carbon source and sulfide production
The HPLC results showed the next:
AZ02: consume piruvate and glycerol; producing
acetate, ethanol and butirato.
AZ05: consume glycerol, lactate and piruvate; a
producing butirate.
Sulfide production
Cepa mM H2S pH
AZ01 0,2 4,9
AZ02 1,4 5,7
AZ03 0,4 5,1
AZ04 0,1 4,9
AZ05 1,2 5,6
AZ07 0,1 4,9
AZ12 0,2 5
Inoculation in medium MAZ-2
In order to achieve a simplier medium, this medium
was used using lactate as carbon source and sulfate
as electron aceptor.
After 8 days, there wasn’t grown and sulfate
consumption at all.
Assay of carbon source
4 carbon sources (Lactate, formate, pyruvate and
H2/CO2) were tested, formate and pyruvate were
employed since their known to be used by
Desulfosporosinus sp.
All strains were tested by triplicate with each carbon
source.
•By day 3 only AZ05, AZ04 y AZ05 showed growth and only with pyruvate
•By day 7, AZ01 showed growth too and only with pyruvate.
•At the end of the experiment sulfide was measured, obtaining:
•AZ01 2,1mM H2S
•AZ02 0,7 mM H2S
•AZ04 0,4 mM H2S
•AZ05 0,4mM H2S
SULFIDE PRODUCTION
Parangueo
The medium was changed for MPAR-2 since MPAR-1 formed precipitates, which could interfere
in posterior studies, as well as to get a better growth.
This medium was suitable for all strains since they showed a good growth as well as sulfide
production as seen in next table:
Cepa H2S (mM) Cepa H2S (mM)
18O 2,4 18K 7,4
19O 3,6 12D 9,4
22N 10,1 12B 10,5
P1 1,0 2B 8,3
13F 11,0 9k-11 9,4
14O 8,0 17D 1,2
22N8 8,3 13I 11,5
4H11 9,9 11A-9 6,9
Desulfonatronum sp.
Desulfobotulus sp.
Substrate for sullfate reduction
Electron
donor HTR (h)
Sulfate
reduction
rate (SO42-
g/l*d
Pro (+), con (-)
Lactate 12-120 0.36-5.76
+Wide spectrum of SBR can grow
on lactate
+Generate large amount of
alkalinity
+Relieve the sulfide toxicity
-High cost
Ethanol 9.6-120 0.45-21
+Relative cheap reagent
+Easily converted by SRB
-Low biomass yield
-Incomplete oxidation to acetate
leading to high effluent COD
concentration.
0.00
0.05
0.10
0.15
0.20
0.25
0 50 100 150 200 250
DO
600n
m
Tiempo (h)
a)
Lactato Etanol
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 50 100 150 200
DO
600n
m
Tiempo (h)
b)
Lactato Etanol
0.00
0.05
0.10
0.15
0.20
0.25
0 50 100 150 200 250
DO
600n
m
Tiempo (h)
c)
Lactato Etanol
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0 50 100 150 200 250
DO
600n
m
Tiempo (h)
d)
Lactato Etanol
Strains
Lactate Ethanol
µ (h-1) Sulfide
(mM) µ (h-1)
Sulfide
(mM)
13F 0.019 11.0 0.011 9.40
12B 0.038 7.8 0.008 7.10
13I 0.024 8.6 0.007 10.1
4H11 0.036 9.8 0.013 9.2
13I-PAR
4H11 PAR
0
5
10
15
20
25
0 50 100 150 200 250 300
mM
Tiempo (h)
Etanol Acetato Sulfato
0.00
0.05
0.10
0.15
0.20
0
1
2
3
4
5
0 100 200 300
DO
60
0n
m
mM
Tiempo (h)
Sulfuro Biomasa
0.0
5.0
10.0
15.0
20.0
25.0
0 50 100 150 200 250
mM
Tiempo (h)
Sulfato Etanol Acetato
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
0 50 100 150 200 250
DO
60
0n
m
mM
Tiempo (h)
Sulfuro Biomasa
Kinetics of sulfide production
Cepa
DQO SO42- (mg/L)
DQO/SO4 Etanol oxidado/
SO42-reducido
Velocidad de
reducción de
SO42- (mg/L d) TO* TF** TO TF
13F 1576 2942 2086 1334 0.76 2.07 78.27
12B 1503 2760 2148 1368 0.70 1.93 78.27
13I 1451 662 2132 1949 0.68 1.56 18.32
4H11 1587 2559 2045 1324 0.78 2.16 72.39
Stoichiometry of sulfate reduction coupled with incomplete oxidation of ethanol
(acetate production).
Los Azufres
Cepa H2S (mM)
pH=7.5
H2S (mM)
pH=11
% sulfuro
acuoso
% sulfuro
gaseoso
C1501 5 7.5 67 33
C1502 5.6 7.1 79 21
DAM treatment
Fig. 1Basic scheme of DAM treatment
Factors affecting As precipitation.
Fig. 2 Dominance of oropimente amorphous
As2S3 (am) as function of pH sulfide content
Battaglia-Brunet et al., 2012.
Fig.3 Dependance of pH in metal sulfide
solubility.
(Lewis, 2010).
Work to do
Batch essays of sulfide production for optimal
conditions (pH and COD/SO42- rate).
Conceptual design of the bioprocess for both
conditions (acidic and alkaline).
Construction of sistem.
Gracias!!