cyanobacteria ppt

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 fellowUnder the guidance of

Dr. K. PakshirajanDepartment of Biosciences and Bioengineering

Indian Institute of Technology Guwahati

Contents

• Introductiona) Heavy metal pollutionb) Robustness of cyanobacteria• Results and discussiona) Mechanism involved in N. muscorumb) Kinetics and isotherm of metal uptakec) Effect of heavy metals on lipid accumulation• Conclusions• References

Heavy metal pollution

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) + 4SO42−(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.

Methods to remove heavy metals

Coa

gula

tion

2

Extra

ctio

n

3

Bio

sorp

tion

4

Phyt

orem

edia

tion

5

Phyc

orem

edia

tion

6

Conventional chemical methods

Novel biological methods

1

Prec

ipita

tion

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.

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

Heavy metal removal mechanism by cyanobacterium

N. muscorum

Four key aspects in heavy metal removal by N. muscorum

1

23

4Initial Passive

biosorption

Biosorption following Ion-

exchange

Active intracellular uptake

Metal assimilation by Redox reactions

Intracellular redox reaction

Exopolysaccharides and Proteins

Outer membrane yielding to

sorption

Periplasmic membrane to

transport metal ions

Export

Components involved in heavy metal removal by cyanobacteria

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).

Cu

Cu

Cu

Cu

Cu

Cu

Pb

Pb

Pb

PbPb

CdCd

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.

Time (min.)

0 50 100 150 200 250

Cu(

II) re

mov

al (m

g/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

Metal removal by Ion-exchange mechanism

Cu

Cu

Cu

Cu

Cu

Cu

Pb

Pb

Pb

PbPb

CdCd

Cd

Zn

Cd

Cd

Zn

Zn

Zn

Zn

Zn

Zn

Redox reactionsC

CC

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.

NC

EDX image showing metal removal by N. muscorum through Ion-exchange mechanism

Virgin biomass

Heavy metal treated biomass

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 10 20 30 40 50 60 700

20

40

60

80

100

120

140(a)

Run 1 Run 2 Run 3 Run 4Run 5 Run 6 Run 7 Run 8Run 9 Run 10 Run 11 Run 12

Time (h)

Cu(

II) r

emov

al (%

)

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 designResults

It has been suggested that different metals have preference for binding with different ligands

Specific functional group present in biomass

Kinetic studySingle and multi metal system

A BHeavy 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.

Isotherm study

Cu

Cu

Cu

Cu

Cu

Cu

Pb

Pb

Pb

PbPb

CdCd

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

0 50 100 150 200 250 300 3500

1

2

3

4

5

6

6.8

7

7.2

7.4

7.6

7.8

8

8.2

8.4

8.6

8.8

Biomass concentration ( g /L )

pH

Cultivation time (hours)

Bio

mas

s con

cent

ratio

n (g

/L)

pH

Reactor study

Photo bioreactor

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

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.

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

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

solubilization ratio (SR)

Emulsification activity and stability

Minimum surface tension, CMC and interfacial tension determination

34

The results suggested that the longer hydrophobic chain in SL gives less CMC.

CMC of SLs ranges between 40 to 100 mg/l andthe value depends on the substrate used for its production.

35

Emulsification

36

n

Acidic sophorolipid Lactonic sophorolipid

n

OOH

OOH

OH

O

CH2OR2

CH2OR2CH3

OO CH

C = O

CH2

O

n

OH

OOH

OH

O

CH2OR2

CH2OR2CH3

O CH

C = O

CH2

O

OOH

OOH

OH

OH

O

CH2OR2

CH2OR2CH3

O CH

COOH

CH2

OOH

OOH

OH

OH

O

CH2OR2

CH2OR2CH3

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

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).

http://www.sciencedirect.com/science/article/pii/S0963996909000192#bib18



39

Factors influencing the sophorolipids production

Operation conditions

Physical parameters

Medium composition

1

23

4Agitation

pHAeration

TemperatureN

itrog

en

sour

ce21

Car

bon

sour

ce

11

23

4

Fed-batchSelf cyclingfermentation

Resting cellmethod

Batch

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).



42

Classical commercial fermentation processes for the production of non-growth associatedproducts can be subdivided into three phases (Omstead et al., 1985) and SLs is noexception: (1) the first stage is inoculum development; (2) the second phase is the stagein which SLs are microbiologically synthesized and (3) the third phase is recovery ofSLs.

India has approximately 90 different vegetable oil refineries located in different states of the country.

Industrial wastewater treatment using sophorolipids

44

Process Timeline Flow

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cyanobacteria ppt

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