Strategic development for the mitigation of heavy metal in the surface water around coal mining areas using native cyanobacterial strains

1. Strategic development for the mitigation of heavy
metals in the surface water around coal mining
areas using native cyanobacterial strains
By,
N. Arul Manikandan
Junior research fellow
Under the guidance of
Dr. K. Pakshirajan
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati

2. Contents
• Introduction
a) Heavy metal pollution
b) Robustness of cyanobacteria
• Results and discussion
a) Mechanism involved in N. muscorum
b) Kinetics and isotherm of metal uptake
c) Effect of heavy metals on lipid accumulation
• Conclusions
• References

3. Heavy metal pollution

4. Acid Mine drainage (AMD)
 Mines built as early as the 1800’s were developed in a manner which
utilized gravity drainage, to avoid excessive water accumulation in the
mines.
 As a result, water polluted by acid, iron, sulfur and aluminum drained
away from the mines and into streams
2FeS2(s) + 7O2(g) + 2H2O(l) 2Fe2+(aq) + 4SO4
2−(aq) + 4H+(aq)
Iron
pyrites
Iron Sulfuric acid
 The acid runoff further dissolves heavy metals such as copper, lead,
mercury into ground or surface water.

6. Methods to remove heavy metals
Coagulation
2
Extraction
3
Biosorption
4
Phytoremediation
5
Phycoremediation
6
Conventional chemical
methods
Novel biological methods
1
Precipitation

7. Robustness of cyanobacteria
 The oxygen atmosphere that we depend on was generated by numerous
cyanobacteria photosynthesizing during the Archaean and Proterozoic Era.
 Many species are filamentous, forming long, straight chains of cells or many
branching chains.
 There is growing interest in the field of application of cyanobacteria as
bioremedial agents to overcome the heavy metal-related environmental
problems:
a) They offer in situ remediation of contaminants without input of energy and
materials for their growth and biomass production.
b) They also require no organics for their growth, which is a major drawback with
other microorganisms, such as bacteria and fungi.

8. S. No Components Quantity/
L
1 citric acid 0.006 g
2 ferric citrate 0.006 g
3 EDTA (disodium
salt)
0.001 g
4 Na2CO3 0.02 g
5 MgSO4 · 7H2O 0.075 g
6 CaCl2 · 2H2O 0.036 g
7 K2HPO4 0.04 g
8 Trace minerals
BG-110 media for N. muscorum
cultivation
Materials and methods
Syiem et al. 2015
Hwang et al. 2014

9. Heavy metal removal
mechanism by cyanobacterium
N. muscorum

10. Four key aspects in heavy metal removal by
N. muscorum
1
23
4
Initial Passive
biosorption
Biosorption
following Ion-
exchange
Active intracellular
uptake
Metal
assimilation by
Redox reactions

11. Exopolysaccharides and
Proteins
Outer membrane
yielding to
sorption
Periplasmic
membrane to
transport metal ions
Export
Components involved in heavy metal removal by
cyanobacteria

12. FTIR image showing polysaccharide and protein
present in cell wall of N. muscorum
 Generally, the various functional groups such as hydroxyl, amino,
carboxyl, sulfhydryl etc., present on the cell surface confer negative
charge to the cell surface (Chojnacka et al. 2005).

13. Cu
Cu
Cu
Cu
Cu
Cu
Pb
Pb
Pb
PbPb
Cd
Cd
Cd
Zn
Cd
Cd
Zn
Zn
Zn
Zn
Zn
Zn
Redox
reactions
Metal removal by quick sorption and slow intracellular
uptake
 During the passive uptake, metal ions are adsorbed onto
the cell surface within a relatively short span of time.

14. Time (min.)
0 50 100 150 200 250
Cu(II)removal(mg/g)
0
2
4
6
8
10
Experimental observation of metal removal by
quick biosorption followed by slow
bioaccumulation
Biosorption of
heavy metals
Slow intracellular
uptake

15. Metal removal by Ion-exchange mechanism
Cu
Cu
Cu
Cu
Cu
Cu
Pb
Pb
Pb
PbPb
Cd
Cd
Cd
Zn
Cd
Cd
Zn
Zn
Zn
Zn
Zn
Zn
Redox
reactions
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
 Metals are likely to bind the adsorption sites on biomass by
displacing other cations linked through energetically weaker
bonds.
N
C

16. EDX image showing metal removal by N. muscorum
through Ion-exchange mechanism
Virgin biomass
Heavy metal treated biomass

17.  The metabolic activities in live species possibly help in
higher uptake of metal ions, and also, more binding sites are
available in live biomass as compared to dead biomass.
Comparison of biosorption and bioaccumulation
0
20
40
60
80
100
120
140
0 10 20 30 40 50 60 70
Cu(II)removal(%)
Time (h)
(a)
Run 1 Run 2 Run 3 Run 4
Run 5 Run 6 Run 7 Run 8
Run 9 Run 10 Run 11 Run 12

18. 18
S. No Cu
(mg/L)
Pb (mg/L) Cd
(mg/L)
Fe
(mg/L)
Zn
(mg/L)
Ca
(mg/L)
(%)Cu
Removal
(%)Pb
Removal
(%)Cd
Removal
1 10 15 10 5 5 10 56.72 99.19 61.22
2 10 20 10 1 10 10 62.04 54.60 74.91
3 5 20 10 1 10 5 93.64 99.05 78.89
4 10 20 5 5 10 5 89.55 99.50 79.02
5 5 15 10 5 10 5 96.46 99.30 83.68
6 10 20 5 5 5 5 95.07 99.07 73.01
7 10 15 5 1 10 10 92.90 52.40 66.96
8 5 15 5 1 5 5 95.89 99.18 88.52
9 10 15 10 1 5 5 53.23 90.00 52.34
10 5 20 10 5 5 10 94.69 87.93 86.00
11 5 15 5 5 10 10 96.88 99.29 86.60
12 5 20 5 1 5 10 89.19 82.50 76.06
Plackett- Burmann design
Results

19.  It has been suggested that different metals have preference
for binding with different ligands
Specific functional group present in biomass

20. Kinetic study
Single and multi metal system
A B
Heavy metal
removal
A B Heavy metal
removal
Study on effect of co-ions
A – Ligands present in the N. muscorum; B – Heavy metals present in the solution
Pseudo-second order
Pseudo-first order
when one of the reactants concentration is in excess (10 to 100
times) of the other reactant, then the reaction follows a first
order kinetics and such a reaction is called pseudo-first order
reaction.

21. Isotherm study
Cu
Cu
Cu
Cu
Cu
Cu
Pb
Pb
Pb
PbPb
Cd
Cd
Cd
Zn
Cd
Cd
Zn
Zn
Zn
Zn
Zn
Zn
Redox
reactions
Langmuir isotherm Freundlich isotherm Temkin isotherm
qm
(mg/g)
KL
(L/m
g)
R2 n
g/L
KF
(mg/g)
R2 kTm
(L/m
g)
bTm
(kJ/mol
)
R2
0.063 7.298 0.923 1.66 1.533 0.99 0.00
70
6.196 0.92
Biosorption Bioaccumulation

23. 6.8
7
7.2
7.4
7.6
7.8
8
8.2
8.4
8.6
8.8
0
1
2
3
4
5
6
0 50 100 150 200 250 300 350
pH
Biomassconcentration(g/L)
Cultivation time (hours)
Biomass concentration ( g /L )
pH
Reactor study
Photo bioreactor

24.  Among the different heavy metals examined for their bioremoval by
N. muscorum in this multicomponent study, Pb(II) was removed with a high
efficiency followed by Cu(II) and Cd(II).
 However, the time required for maximum metal removal was prolonged to
72 hrs due to the presence of co-ions.
 The metal removal by EDX analysis simply suggested ion-exchange as a
possible mechanism for binding of metal ions onto the biomass surface for
their uptake, and this is attributed to the presence of N-H and C=O functional
groups by FTIR analysis.
 The metal removal by N. muscorum followed the pseudo first-order kinetics
with very high estimated sorption capacity values for all these metals.
 Overall, this study proved a very good potential of the cyanobacterium
N. muscorum in the removal of heavy metals from a complex mixture
containing metals and other co-ions.
Conclusions

25. References
 Roy, A. S., Hazarika, J., Manikandan, N. A., Pakshirajan, K., & Syiem, M. B. (2015).
Heavy Metal Removal from Multicomponent System by the Cyanobacterium Nostoc
muscorum: Kinetics and Interaction Study. Applied biochemistry and
biotechnology, 175(8), 3863-3874.
 Manikandan, N. A., Pakshirajan, K., & Syiem, M. B. (2014). Cu(II) removal by
biosorption using chemically modified biomass of Nostoc muscorum–a cyanobacterium
isolated from a coal mining site. International Journal of Chemtech Research, 07(1), 80-
92.
 Hazarika, J., Pakshirajan, K., Sinharoy, A., & Syiem, M. B. (2014). Bioremoval of Cu (II),
Zn (II), Pb (II) and Cd (II) by Nostoc muscorum isolated from a coal mining site. Journal
of Applied Phycology, 1-10.
 Syiem, M. B., Goswami, S., Diengdoh, O. L., Pakshirajan, K., & Kiran, M. G. Zn
(II) and Cu (II) removal by Nostoc muscorum: a cyanobacterium isolated from a
coal mining pit in Chiehruphi, Meghalaya, India. Canadian Journal of
Microbiology.
 Hwang, J. H., Kim, H. C., Choi, J. A., Abou-Shanab, R. A. I., Dempsey, B. A., Regan, J.
M., ... & Jeon, B. H. (2014). Photoautotrophic hydrogen production by eukaryotic
microalgae under aerobic conditions. Nature communications, 5.

27. 27

28. 28

29. 29

30. 30

31. 31
The Critical Micelle Concentration（CMC）of Sophorolipids is 10～40mg/L, and γ-CMC is 30
～40mN/m, has very high efficiency as surfactants. This figure is 5 to 20 times better than
Sodium Dodecyl Sulfate(SDS), being considered due to its balky structure. However,
Sophorolipids generate only less foam, contributing to easy rinsing and lower skin stimulus.
High Degradability Sophorolipids has high degradability as same as Lauric Acid Sodium Salt,
far better Eco-Friendliness comparing to existing synthetic surfactants.
Properties

32. 32
Mulligan, C.N., Yong, R.N. and Gibbs, B.F.,
2001. Surfactant-enhanced remediation of
contaminated soil: a review. Engineering
Geology, 60(1), pp.371-380.
Chaprão, M.J., Ferreira, I.N., Correa, P.F.,
Rufino, R.D., Luna, J.M., Silva, E.J. and Sarubbo,
L.A., 2015. Application of bacterial and yeast
biosurfactants for enhanced removal and
biodegradation of motor oil from
contaminated sand. Electronic Journal of
Biotechnology, 18(6), pp.471-479.

33. 33
solubilization ratio (SR)
Emulsification activity and stability
Minimum surface tension, CMC and interfacial
tension determination

34. 34
The results suggested that the longer hydrophobic chain in SL
gives less CMC.
CMC of SLs ranges between 40 to 100 mg/l and
the value depends on the substrate used for its production.

35. 35
Emulsification

36. 36
n
Acidic sophorolipid Lactonic sophorolipid
n
O
OH
O
OH
OH
O
CH2OR2
CH2OR2
CH3
O
O CH
C = O
CH2
O
n
OH
O
OH
OH
O
CH2OR2
CH2OR2
CH3
O CH
C = O
CH2
O
O
OH
O
OH
OH
OH
O
CH2OR2
CH2OR2
CH3
O CH
COOH
CH2
O
OH
O
OH
OH
OH
O
CH2OR2
CH2OR2
CH3
O CH
CH2
SLs synthesis is associated with nitrogen
starvation.
Overall, it can be concluded that the physiological role of SLs synthesis is extracellular
carbon source storage, combined with dealing with a high-sugar niche and defending
it against other competing microorganisms (Van Bogaert et al., 2007).

37. 37
SLs and their derivatives have also shown promise as surfactants, emulsifiers, antimicrobials,
and a source of specialty chemicals such as sophorose and hydroxylated fatty acids ( Rau et
al., 2001 and Solaiman et al., 2007).

38. 38

39. Factors influencing the sophorolipids production
Operation
conditions
Physical
parameters
Medium
composition
1
23
4
Agitation
pHAeration
Temperature Nitrogen
source
21
Carbon
source
1
1
23
4
Fed-batch
Self cycling
fermentation
Resting cell
method
Batch
39

40. 40

41. But compared to chemical surfactants biosurfactants can be considered environmentally
safer, and besides this, they have several advantages over chemical or synthetic surfactants,
such as high ionic strength tolerance, high temperature tolerance, higher biodegradability and
lower toxicity, lower critical micelle concentration and higher surface activity (Bognolo, 1999).
41

42. 42
Classical commercial fermentation processes for the production of non-growth associated
products can be subdivided into three phases (Omstead et al., 1985) and SLs is no
exception: (1) the first stage is inoculum development; (2) the second phase is the stage
in which SLs are microbiologically synthesized and (3) the third phase is recovery of
SLs.
India has approximately 90 different vegetable
oil refineries located in different states of the
country.
Industrial wastewater treatment using sophorolipids

43. 43

44. Process Timeline Flow
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