Immobilization of sulfate reducing bacteria for the

removal of sulfate, metal toxicity and COD: Metal

removal mechanism and characterization

M Gopi kiran

Professor Kannan Pakshirajan and Professor Gopal Das

02/19/2021
 Introduction
 Heavy
 Heavy metals,
metals, sulfate,
sulfate, effects
effects of
of heavy
heavy metal
metal containing
containing wastewater
wastewater

 Brief
 Brief overview
overview of
of existing
existing technologies
technologies for
for metal
metal and
and sulfate
sulfate removal
removal and
and advantages
advantages of
of
biological
biological methods
methods and
and different
different mechanisms
mechanisms associated
associated with
with the
the biological
biological methods
methods

 Screening
 Screening of
of biomass
biomass from
from different
different sources
sources for
for aa metal
metal resistant
resistant sulfate
sulfate reducing
reducing bacteria
bacteria
(SRB)
(SRB) based
based on
on sulfate
sulfate and
and metal
metal removal
removal efficiencies
efficiencies and
and characterization
characterization of
of the
the
bioprecipitates
bioprecipitates
Sulfate and heavy metal removal from a multicomponent system
o To study the simultaneous effect of Cd, Cu, Ni, Fe, Pb and Zn on sulfate and metals on

their own removal by employing Plackett Burman design.

o Low and high level initial concentration levels of respective metals were chosen based on

the earlier studies with single metal solutions.

o Characterization of the precipitates.

o Preparation of SRB immobilized beads with different concentrations of sodium alginate

2 to obtain the optimum combination with chilled CaCl 2 02/19/2021

 Objectives
SRB immobilization for heavy metal and sulfate removal
To study the metal and sulfate removal with SRB immobilized beads and compare the results
with the results obtained from previous free biomass study
Characterization of the beads and precipitates.
Fabrication of a column reactor and device the performance of the metal and sulfate removal
using SRB immobilized beads
Kinetic studies of the removal of metals and evaluation of kinetic constants.

Continuous studies for metal and sulfate removal in rotating biological contactor
reactor (RBC)
o Immobilization of SRB on disks covered with bio-support material and start up of the reactor.
o To evaluate the performance of RBC by continuous experiments for metal and sulfate
removal with different concentrations basing on the results obtained from previous study
o Effect of different hydraulic retention time (HRT) for metal, sulfate and COD removal
o Effect of shock loading in terms of metal loading rate on the performance of the reactor.
o Characterization studies of metal removal by SRB in the anaerobic-RBC biofilm.
o Identification and characterization of SRB. 02/19/2021
3
 Methodology

Growth and maintenance of sulfate reducing bacteria in the mixed consortia with modified
Postgate medium

Screening of SRB immobilized beads with different concentrations of

sodium alginate (SA) for optimum composition of SA

Batch study for metal and sulfate removal with

SRB immobilized beads and compare the results

Precipitate Analysis

Fabrication of a column reactor for the metal and

sulfate removal using SRB immobilized beads

Precipitate Analysis
4 02/19/2021
 Initial stage of SRB immobilized
beads

Fig: SRB after being activated under growth

medium

Experimental conditions:
Temperature: 30 oC
Agitation: 100-120 rpm
pH: 7
COD/Sulfate: 0.67
Different metal concentrations : 10 and 50
Beads added to the serum bottles with metal mg/L
solution
5 02/19/2021
 60
10 mg/L f 10 m g/L
a 10 mg/L 60
50 mg/L
50 50 mg/L b 50 mg/L 50

Metal concentration (mg/L)

Metal concentration (mg/L)

50
Metal concentration (mg/L)

40 40
40
30 30
30

20 20
20
10
10
10
0
0 0 0 20 40 60 80 100 120
0 20 40 60 80 100 120 0 20 40 60 80 100 120 Time (h)
Time (h) Tim e (h)

More than 95% metal removal 10 mg/L and 50 mg/L, since they are lower More than 80% metal
in all the cases at 10 mg/L and upper limits for metal discharge removal at 50 mg/L

60
50 e 10m g/L
50m g/L f 10mg/L
f 10 mg/L 50 50mg/L
50 50 mg/L
Metal concentration (mg/L)

40

Metal Concentration (mg/L)

Metal concentration (mg/L)

40
40
30
30
30
20
20
20
10
10 10

0
0 0 20 40 60 80 100 120 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
Tim e (h)
Tim e (h) Tim e (h)

Fig. Metal removal efficiencies for a) Cd, b)Cu, c)Ni, d)Fe, e)Pb and f)Zn at 10 mg/L
and 50 mg/L, respectively
 Sulfate (10 m g/L) 1200
b
Sulfate and C OD concentration (m g/L)

1000
a Sulfate (50 m g/L) Sulfate (10 mg/L) c Sulfate (10 m g/L)

Sulfate and C OD concentration (m g/L)

Sulfate and C OD concentration (m g/L)

CO D (10 mg/L)
1000 Sulfate (50 m g/L)
Sulfate (50 mg/L)
CO D (50 mg/L) C OD (10 m g/L) 1000 C O D (10 m g/L)
800 C OD (50 m g/L) C O D (50 m g/L)
800
800
600
600
600

400 400
400

200 200
200

0 0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120
Time (h) Tim e (h) Time (h)

More than 80% removal in all More than 70 % removal at 50

the cases at 10 mg/L mg/L

1200
1200
d S ulfate (10 m g/L) Sulfate (10 m g/L)
Sulfate and C OD concentration (m g/L)

Sulfate and C O D concentration (m g/L)

1200 S ulfate (10 m g/L)
S ulfate (50 m g/L) Sulfate (50 m g/L)
e Sulfate (50 m g/L)
Su lfate and C O D con centration (m g/L)

C O D (10 m g/L) 1000 CO D (10 m g/L)

CO D (10 m g/L) CO D (50 m g/L)
1000 C O D (50 m g/L) CO D (50 m g/L)
1000
800
800
800
600
600 600

400
400 400

200
200 200

0
0 0 0 20 40 60 80 100 120
0 20 40 60 80 100 120 0 20 40 60 80 100 120
Tim e (h)
Tim e (h) Tim e (h)

Fig. Sulfate and COD removal efficiencies for a) Cd, b)Cu, c)Ni, d)Fe, e)Pb and f)Zn at 10 mg/L and
50 mg/L, respectively

7 (Min et al., 2008) 02/19/2021

Table: Metal, sulfate and COD removal with free suspended biomass

SN Sulfate COD removal % Metal removal %

removal%
Metal 10 50 10 50 10 50

ppm
Cd 98 92.3 88 83.2 99 78.6
Cu 98 95.5 92.8 85.6 99.76 99.44
Ni 94 91 85.6 78.5 95.6 82
Fe 98 98 88 80.8 96 83
Pb 98 92.7 88 78.5 95.5 81
Zn 98 91.9 85.6 80.8 95 77
Table: Metal, sulfate and COD removal with SRB
immobilized beads
SN Sulfate COD removal % Metal
removal% removal %
Metal 10 50 10 50 10 50

ppm
Cd 97.2
84.5 78.7 84.7 68.9 7 88.54
Cu 86 81.5 82 73.8 97.8 94.8
Ni 82.35 71 75 65.6 94.4 76.2
Fe 85.5 80 76 70 95.6 90.5
Pb 83.9 78.8 76.7 68.5 97.4 90
Zn 95.9
82 77.9 78.5 64.02 3 85.6

8 02/19/2021
 Fig: Beads after 24
h

9 02/19/2021
Fig: Beads after 120
 Cultivated in growth medium
Initial SRB immobilization
for SRB to get activated

SO42 SO42- SO42-

Mn+ SO42 Mn+
- SO42 Mn+
Mn+ SO42- - S2-
-
S2-
Mn+ S2-
SO42- SO42- SO42-
S2- S2- S2- S2- MS S2-
M n+
SO4 2-
M n+
Mn+
Mn+ MS
MS

Sulfate and metals in the media Sulfate reduction and sulfide layer formation Metal sulfide precipitation

10 02/19/2021
 Mn+ Possible Mn+
Metal precipitation mechanism
MS
MS MS

Fig. Possible metal precipitation mechanisms

11 (Min et al., 2008) 02/19/2021

 Cu
Cd

Ni Fe

Pb
Zn

12 02/19/2021
Fig: FESEM-EDX images of beads with cross Thanks to Narendra Naik and Srinu for FESEM-EDX
slots
Fig: FESEM-EDX images of beads

13 Fig: FESEM-EDX images of beads with cross 02/19/2021

section
 Cd Cu

Ni Fe

Zn
Pb

14 02/19/2021
Fig: FESEM-EDX images of beads
 Mn+ Possible Mn+
Metal precipitation mechanism
MS
MS MS

Confirmation of
Metal precipitation mechanism

Mn+

MS

15 (Min et al., 2008) Fig. Metal precipitation mechanism 02/19/2021

 Beads study
Reactor studies in different phases

Phase Activities Phase Activities

Phase I Single metal solution experiments Phase I Single metal solution batch experiments:
Thrice
Hydraulic retention time (HRT): 48 h Cd, Cu, Ni, Fe, Pb, Zn: 10 and 50 mg/L
Cd: 50 or 100 mg/L FESEM characterization, metal, sulfate and COD removal
Cu: 100 or 150 mg/L results
Ni: 50 or 100 mg/L
Phase II Column studies (packed bed Single metal solution
Fe: 50 or 100 mg/L
reactor) experiments
Pb: 50 or 100 mg/L
Dimensions: Hydraulic retention time
Zn: 50 or 100 mg/L
L: 12 inches (HRT): 24 h
Phase II Single metal solution experiments
Thrice Diameter: 1 inch Cd: 50 or 100 mg/L
Hydraulic retention time (HRT): 24 h
Hydraulic retention time Cu: 100 or 150 mg/L
Cd: 50 or 100 mg/L
(HRT): 48 h Ni: 50 or 100 mg/L
Cu: 100 or 150 mg/L
Cd: 50 or 100 mg/L
Ni: 50 or 100 mg/L
Cu: 100 or 150 mg/L Fe: 50 or 100 mg/L
Fe: 50 or 100 mg/L
Ni: 50 or 100 mg/L Pb: 50 or 100 mg/L
Pb: 50 or 100 mg/L
Fe: 50 or 100 mg/L Zn: 50 or 100 mg/L
Zn: 50 or 100 mg/L
Pb: 50 or 100 mg/L
Phase III Factorial design experiments
Zn: 50 or 100 mg/L
Phase IV Higher metal concentrations
Phase V

16 02/19/2021
 Conclusions
 The results in the study demonstrated the reduction of sulfate, COD and subsequent

precipitation of different metals using SRB immobilized beads. The study also showed very
high removal of different metals at both low and high level concentrations.
 However, at a high metal concentration combination, sulfate and COD reduction were

inhibited, thus resulting in a reduced removal of these metals by SRB.

 Any how , high metal removal was obtained at 50 mg/L of all the metals compared to the

results obtained from free biomass study

 FESEM-EDX analyses of the beads following metal removal further confirmed that the metal

precipitates were formed outside the bead surface and the heavy metal removal mechanism
was attributed to the capability of the SRB to reduce sulfate to sulfide, thereby resulting in
precipitation of the metals as sulfide salts.
 Overall, this study proved very good potential of SRB immobilized beads for metal precipitation

with a high tolerance towards different metals.

17 02/19/2021
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