Evaluation of Arsenic bio-remediation from industrial ......Desulfosporosinus auripigmenti strain...

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)




PAR18O (PAR19O)







Desulfonatronaceae




22N


Desulfobacteraceae



AZB1 06 (AZB1 11, AZB109, AZB1 08)



AZB1 10

Clostridaceae









Peptococcaceae





PAR18O (PAR19O)







Desulfonatronaceae




22N


Desulfobacteraceae



AZB1 06 (AZB1 11, AZB109, AZB1 08)



AZB1 10

Clostridaceae









Peptococcaceae





PAR18O (PAR19O)







Desulfonatronaceae




22N


Desulfobacteraceae



AZB1 06 (AZB1 11, AZB109, AZB1 08)



AZB1 10

Clostridaceae









Peptococcaceae





PAR18O (PAR19O)







Desulfonatronaceae




22N


Desulfobacteraceae



AZB1 06 (AZB1 11, AZB109, AZB1 08)



AZB1 10

Clostridaceae









Peptococcaceae


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:


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


Lactato 0,5 M 20 mM


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


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


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


Resazurina 0.1% 1 ml

Adjust at pH 5 after sterilization add:


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


Resazurine 0.1% 1 ml


Lactato 0,5 M 10mM


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!!

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Evaluation of Arsenic bio-remediation from industrial ......Desulfosporosinus auripigmenti strain...

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