Extracellular polymeric substances (EPS) are metabolic byproducts of microorganisms. They are composed of lipids, carbohydrates and fats and have high molecular weight. They have many significant properties in soil aggregation, nutrient cycling, heavy metal adsorption, and antibiotics production. In this present slide i have presented the heavy metal removal capacity with mechanism. Go through the slides and let me know your valuable comments.

1. Credit
Seminar
SSAC-692

2. Bacterial Extra-cellular Polymeric Substances Mediated Heavy
Metal Removal from Soil
Dewali Roy
Roll No:11878
Ph.D. (II year)
Division of Soil Science and Agricultural Chemistry
ICAR-Indian Institute of Agriculture
Pusa, New Delhi

3. Introduction
Interactions between EPS and heavy metals
Factors influencing EPS activity
Applications of EPS in heavy metal remediation
Advantages and constraints of EPS
Conclusions
Path ahead
Seminar Outline

4. INTRODUCTION

5. What is bacterial extra-cellular polymeric substance(EPS)?
 EPS are a blend of high molecular weight microbial bio polymeric secretary by products by
several microrganism.
 These biopolymers mostly consist of proteins, polysaccharides, uronic acids, humic substances,
lipids and nucleic acids.
 EPS consists of quite viscous biofilm matrix. In general, EPS in a biofilm varies from 50% to 90%
of the total organic matter (Flemming and Wingender, 2001).
Classification basis EPS Remarks
Nature of EPS association with
cells
Slime Present in the supernatant after centrifugation of the
biomass. Mostly in soluble form or unattached to the
cells in the form of colloids.
Capsular The permanent part of the cell membrane and are bound
to pellets (bacterial cells).
Physicalechemical states and
composition of EPS
Soluble Secretion from the cells in dissolved form in the
surrounding environment. The main components are
macromolecules, colloids and slimes
Bound Attached to the cells. The components of bound EPS are
sheaths, capsular polymers, condensed gel, loosely
bound polymers and attached organic material.
More et al. (2017)

6. Components of the EPS Typical content in the EPS
matrix
Important properties of different
components
Polysaccharides 40 to 50% Adhesion, aggregation of bacterial cells,
retention of water, adsorption of organic and
inorganic compounds,nutrient source, and
protective barrier to cell.
Proteins 1 to 60% Adhesion, aggregation of bacterial cells,
retention of water, sorption of organic and
inorganic compounds, binding of enzymes,
electron donor or acceptor, and protective
barrier to cells.
Nucleic acids 1 to 10% Adhesion, aggregation of bacterial cells,
nutrient source, exchange of genetic
information, export of cell components, and
exchange of genetic information.
Lipids 1 to 10% Export of cell components.
Humic substances Adhesion, Electron donor or acceptor.
Composition and important properties of EPS
Flemming and Wingender. (2010); Tian (2008); Wingender et al. (1999)

7. EPS Type and monomeric
unit
Linkages Major producing
microorganisms
Application area
Xanthan Hetero
Glucose, mannose,
glucuronate
(1-4)-β-D-glucan Xanthomonas
campestris pv.
campestris
Food, cosmetics,
textile, feed industry
Levan Homo Fructose β-2-6 Halomonas
smyrnensis
Environment
Pharmaceutical,
medical
Dextran Homo Glucose α-1-6 Leuconostoc
mesenteroides
Food,
pharmaceutical
Pullulan Homo
Glucose
α-1-4 Aureobasidium
pullulans
Medical, food,
pharmaceutical,
Agricultural.
Cellulose Homo
Glucose
β-1-4 Gluconacetobacter
hansenii
Medical,,Pharmaceutic
Alginate Hetero
Guluronic acid,
β-1-4 Brown sea weed
Pseudomonas sp
Food, feed,Medicine
Curdlan Homo Glucose β-1-3 Agrobacterium sp Food, cosmetics,
medicine
Gellan Hetero
Glucose, rhamnose,
glucuronic acid
α-1-3 Sphingomonas
elodea
Construction
chemistry.
Some significant bacterial EPS
Bhavna et al. (2017)

8. Need of EPS
Priyadarshanee et al. (2020)

9. Methods Mechanism
Physical
Centrifugation EPS separates from cell surface and dissolve to solution under the centrifugal force.
Heating The molecular movement is enhanced that accelerates the EPS dissolution.
Sonication The EPS part of the biofilm matrix under the impulsive pressure
Chemical
Acidic treatment Improves the repulsive force and disrupts the interaction between EPS and cells,
causing the EPS to fall away from the cell surface.
Alkaline
treatment
NaOH causes the groups such as the Carboxylic groups to be ionized, resulting in a
strong repulsion between the EPS and the cells.
CER
(Cation exchange
resin)
CER removes the divalent cations, thus causing the EPS to fall apart.
Crown ether Combine divalent metals and disrupt the binding interaction between EPS and cells.
EDTA Removal of divalent cations using EDTA causes the EPS matrix to fall apart.
Enzymatic
Extraction
The carbohydrate and protein-hydrolysing enzymes disrupt the structure of sludge and
dissolve the EPS.
Ethanol
Extraction
Denatures the EPS and reduces the binding force between EPS and cells.
Different EPS Extraction Methods
Comte et al. (2007); Sesav et al. (2006); Wingender et al. (1999)

10. Sludge dewatering
Wastewater flocculation and settling
Landfill leachate treatment
Water treatment
Soil remediation and reclamation
Colour removal from the wastewater
Potential environmental pollution control applications of EPS

11. Fitness Factor
• Desiccation
Tolerance
• Protect nitrogenase
enzyme from oxygen
• Biofilm formation
Stress tolerance
• Cell and enzyme
immobilization
• Pesticide tolerance
• Waste water
treatment
• Metal adsorption/
removal
• Salt tolerance
Nutritional benefits
• Surface acting agent
• Carbon source
• Soil aggregation
• Agro ecological
restoration
Applications of EPS in Agriculture
More et al. (2017)

12. EPS and Heavy metal interactions
Barik et al. (2021)
Biosorption
Biovolatization
Bioleaching
Bioimmobilization

13. Biosorption
Bioleaching
Barik et al. (2021)
The capability of biological materials to
accumulate or bind heavy metals.
 Acts through metabolically facilitated or
physico-chemical pathways.
 Used in polluted water bodies and soil.
Metal cations are mobilized from almost
insoluble ores by complexation and biological
oxidation method.
EPS and Heavy metal interactions

14. Bioimmobilization
Biovolatization
Transformation of metals by microbes into their
volatile forms .
Contributes in the alteration of metal from
soluble state to gaseous state.
Metal can immobilized using microbial biomass
by biosorption to cell walls or by extracellular
substances.
Adsorption on exteriors, flocculation, cross
connectingofcells, nanocoating, entrapment,
covalent bonding to carriers and encapsulation.
Barik et al. (2021)
EPS and Heavy metal interactions

15. Factors influencing the heavy metal biosorption by EPS
Priyadarshanee et al. (2020)
pH
Temperature
Biosorbent dosage
Competing ions
Nature of biosorbent
Culture time

16. Bacillus licheniformis strain KX65783 isolated from earthworm (Metaphire posthuma).
Ethanol extraction method.
The metal sorption by EPS increased with increasing pH. EPS concentration of 25 mg/L metal solution, at pH 8,the
EPS removed 86 and 81% Cu(II) and Zn(II) respectively while 94.8% of Cu & Zn were removed at EPS
concentration of 100 mg /L.
Biswas et al. (2020)
Adsorption Efficiency of Cu and Zn at Varying pH and concentration by Bacillus sp.
Metal
removal
(%)
EPS concentration (mg/L)
pH

17. Effect of initial pH on metal adsorption by Rhodococcus sp.
EPS obtained from bacterial strain Rhodococcus opacus (89 UMCS )and Rhodococcus rhodochrous (202 DSM)
Stock solution Pb(NO3)2, Cd(NO3)24H2O, Co(NO3)24H2O, Ni(NO3)24H2O, K2Cr2O7
Heavy metal ions concentrations - 5 to 700 mg/L . The single adsorption system consisted of 5 mL of solution and 1.5 ±
0.03 mg of EPS.
A) Ni(II), B) Pb(II), C) Co(II), D) Cd(II) and E) Cr(VI)
Adsorbed
amount
mg/kg
pH
Dobrowolski et al.(2017)
open symbol= R opacus
Solid symbol= R rhodochrous

18. Effect of temperature on Metal adsorption
A) Ni(II), B) Pb(II), C) Co(II), D) Cd(II) and E) Cr(VI)
Dobrowolski et al. (2017)
Adsorbed
amount
mg/kg
Temperature °C

19. Effect of time on metal adsorption
A) Ni(II), B) Pb(II), C) Co(II), D) Cd(II) and E) Cr(VI)
Adsorbed
amount
mg/kg
Time (min)
Dobrowolski et al. ( 2017)
Open symbols=R.
Opacus
solid symbols=R.
Rhodochrous

20. EPS production by Bacillus sp. at varying concentrations of metals
The bacteria Bacillus sp. S3 used in this study was previously isolated from an antimony-mine area, China
The metal salts used CdCl2, K2Cr2O7, Cu(NO3)2·3H2O, and C8H4K2O12Sb2·3H2O(1000 mg/L)
The EPS content significantly increased as Cr(VI) and Cu(II) concentrations increase,and were peaked at 30 mg/L
(a) Cd(II), (b) Cr(VI), (c) Cu(II) and (d) Sb(III)
Zeng et al. (2019)

21. The concentrations of EPS indicators under different media
Gordon et al. (2016)
Samples of sandy soil (Cambic Arenosol, FAO classification) . pH- 5.95
Depth- surface horizon (0 to23 cm) of a permanent grassland area off the ‘Market Garden Experiment’at Rothamsted
Experimental Farm, UK.
biodiesel co-product

22. EPS
MEDIATED
HEAVY
METAL
REMOVAL

23. Prachim et al. 2009
Soil sample- from Mn mine spoil dump, Gumgaon India. (pH-7.8)
 Pot culture experiment.
Jensen’smedium was used for EPS production . Bacterial cells were incubated at 30 °C at 200 rpm for 120 h. -.
q =V(Ci - Cf)/1000W (V is the volume of solution in tube, W is the mass weight of adsorbent (whole cells or EPS)
(g), and Ci and Cf are the initial and final concentration of metal in solution mg /L respectively.
Azotobacter EPS on biosorption of Cd and Cr
0
5
10
15
20
25
Cd+ Cells Cd+EPS Cr+cells Cr+EPS
Treatments
Metal
biosorption
mg/kg

24. Prachim et al . (2009)
Treatment Details
GS= garden soil
Cr 1=Cr/Cd(5 ppm)
Cr 2=Cr/Cd (5 ppm) + free
cells
Cr 3=Cr/Cd (5 ppm) +
immobilized cells
Cr 4=Cr/Cd(10 ppm)
Cr 5=Cr /Cd(10 ppm) +
free cells
Cr 6=Cr/Cd (10 ppm) +
immobilized cells
Cr 7=Cr /Cd(15 ppm)
Cr 8=Cr/Cd(15 ppm) + free
cells
Cr 9=Cr /Cd(15 ppm) +
immobilized cells
Concentration of Heavy metal in wheat plant under different EPS treatments
0
0.2
0.4
0.6
0.8
1
1.2
1.4
GS Cr 1 Cr 2 Cr 3 Cr 4 cr 5 Cr 6 Cr 7 Cr 8 Cr 9
0
1
2
3
4
5
6
GS Cd 1 Cd 2 Cd 3 Cd 4 Cd 5 Cd 6 Cd 7 Cd 8 Cd 9
Metal
biosorption
mg/kg
Metal
biosorption
mg/kg

25. Zn adsorption onto the surface of P. aureofaciens
Drozdova et al. (2017)
The humic (surface, organic-rich, pH 5.6) and illuvial (mineral, organicpoor, pH 6.0) horizons of the podzol soil,
Northern Karelia, within the European boreal zone.
Bacterial strain used- P. aureofaciens CNMN PsB-03
Batch reactor technique.
EPS dosage- 0.1 gm/L
Humic horizons 1.5 times more adsorption capacity then illuvial horizon.
%
Zn
adsorbed

26. Adsorption of Cu(II) and Cd(II) with and without Escherichia coli
Nkoh et al. (2018)
•Oxisol derived from basalt collected from Haikou, Hainan Province.
•Alfisol derived from Loess from Nanjing, Jiangsu Province.
•EPS strain used- Escherichia coli (species No. 1.2389) centrifugation method.
•Alfisol-7.4% Cu & 17.1% Cd adsorption increased.
•Oxisol- 9.3% Cu % 8.9% Cd adsorption increased.

27. .
Enterobacter sp. was screened from local soil sample(Nandurbar, Maharashtra) for its ability to produce
EPS in yeast extract mannitol broth (YEMB) at 27 ºC at 120 rpm for 8-10 days.
Maximum absorption for MnCl2 (93.10%),CoCl2 (77.07%), ZnCl2 (90.03%) ,NiCl2 (70.24%), CuCl2
(82.02%) at 4.0 mg/L.
Sayyed et al. (2014)
Metal adsorption by Enterobactor EPS
0
0.5
1
1.5
2
2.5
3
3.5
4
MnCl2
ZnCl2
CoCl2
NiCl2
CuCl2
MnCl2
ZnCl2
CoCl2
NiCl2
CuCl2
MnCl2
ZnCl2
CoCl2
NiCl2
CuCl2
MnCl2
ZnCl2
CoCl2
NiCl2
CuCl2
MnCl2
ZnCl2
CoCl2
NiCl2
CuCl2
2 2.5 3 3.5 4
Absorbance
EPS concentration (mg/L)

28. % increase in germination of seeds
MnCl2 NiCl2 ZnSO4 ZnCl2 FeSO4 FeCl3 CuSO4 CuCl2 CoCl2 AgNO3 HgCl2
Wheat 40.0
(0.025)*
25.0
(0.28)NS
40.0
(0.025)*
40.0
(0.025)*
40.0
(0.025)*
40.0
(0.025)*
40.0
(0.025)*
40.0
(0.025)*
40.0
(0.025)*
25
(0.287) NS
25
(0.287) NS
Peanut 33.3
(0.158)NS
33.3
(0.158) NS
33.3
(0.158) NS
33.3
(0.158)NS
Nil
(01.0) NS
33.3
(0.158) NS
33.3
(0.158) NS
33.3
(0.158) NS
33.3
(0.158) NS
Nil
(01.0) NS
Nil
(01.0) NS
% increase in chlorophyll content of leaves
Wheat 22.58
(0.0056)*
18.51
(0.0808)
↓
(0.0704)
7.69
(0.196)
↓
(0.0742)
↓
(0.0704)
↓
(0.548)
08.33
(0.0096)*
↓
(0.0704)
↓
(0.0704)
↓
(0.120)
Peanut 21.42
(0.0096)*
04.34
(0.5733)NS
42.10
(0.175)NS
30.0
(0.334) NS
↓
(01.0) NS
08.30
(0.299)NS
21.42
(0.0096)*
↓
(0.196)NS
31.25
(0.071) NS
30.0
(0.299)NS
↓
(01.0) NS
% increase in CFU
Wheat 40.36
(0.00128) *
34.86
(0.0070)*
33.10
(0.0034) *
45.0
(0.00028)*
32.65
(0.0015)*
23.84
(0.0011)*
61.32
(0.0026)*
11.60
(0.0048) * 14.65
(0.0522)NS
02.94
(0.4772)NS
45.0
(0.00028)*
Peanut 71.34
(0.0037) *
43.01
(0.0028)*
56.77
(0.0164) *
31.03
(0.0025)*
44.80
(0.0073)*
31.08
(0.0011)*
28.36
(0.0229)*
49.90
(0.0141) * 40.69
(0.0042)*
65.54
(0.00060)*
08.92
(0.0306)*
*Values were taken to be statistically significant at P ≤ 0.05; NS Values were not statistically different at P ≤ 0.05.
Nil: No % increase or decrease as compared to control treatment in regarding parameter; ↓: % decrease as compared to control treatment
in regarding parameter
Influence of Enterobacter sp. EPS in heavy metal spiked soil
Sayyed et al. (2014)

29. Cd(II) adsorption by the Bacillus EPS, MMT and EPS-MMT composites
(a), (b) =Langmuir (c) and (d)=Freundlich isotherm
Yan et al. (2019)
Cd
adsorbed
mg/g
Strain used-Bacillus sp. NT10
Cd stock solution was prepared from Cd(NO3)2of analytical grade.
EPS- Montmorillonite composite with diff ratio of 5:50, 1:50 & 0.5:50 (w/w)

30. Do et al. (2020)
Species- Rahnella sp. LRP3
Soil sample (0 to 15 cm depth) of the mining area at
Panshi city, Jilin province, China. Total Cu content-
546 mg/kg
Formed Cu5(PO4)2(OH)4 via biomineralization.
EPS in the culture solution reduced 89.4 mg/kg of
DTPA-Cu content by 78.99% in soil in 10 days.
Cu immobilized in the precipitation process and
change of the EPS
cc
Cu removal by Rahnella EPS
Cu
removal
(%)
Cu
removal
(%)
Total
amt
of
EPS
(g/L)
Cu
Concentration
(mg/L)

31. Nazli et al. (2020)
•Rhizosphere soil samples were collected from heavy metal–contaminated fields near the industrial areas and
wastewater-irrigated fields around the cities of Bahawalpur,Pakistan.
•Three most efficient EPS- Cd-tolerant plant growth–promoting strains, i.e., FN13, FN14, and FN16, were selected
among 30 rhizobacterial strain (FN1-FN30) isolated.
PGP
characteristics
Rhizobacterial isolates
FN2 FN13 FN14 FN16 FN25 FN26 FN29 FN30
Zinc
solubilization
++ +++ +++ ++ - ++ - -
Phosphate
solubilization
++ ++ + - ++ + + ++
HCN production - ++ ++ +++ ++ ++ - ++
Ammonia
production
++ ++ ++ + ++ ++ - ++
Siderophore
production
++ +++ ++ +++ - ++ - ++
Catalase activity
- + + + + - - -
Root
colonization
(CFU g−1)
2.23 × 105 3.36 ×
106
2.48 ×
106
1.61 ×
106
1.43 ×
106
1.21 ×
106
4.12
× 105
1.07
× 105
HCN hydrogen cyanide
(+++) = (++) = (+) = Growth (-)No growth
Quantity of Cd removed by rhizobacterial
strains
Plant growth–promoting characteristics Cd-tolerant rhizobacterial EPS strains
260
270
280
290
300
310
320
330
FN13 FN14 FN16
Cd
removed
mg/kg

32. Species
Heavy
Metals
Initial
concentrations
(in ppm)
pH Temperature
(°C)
Contact time
( hour) Remediation
efficiency (in %)
Paenibacillus jamilae Pb 303.03 – 25 24 70
Azotobacter
chroococcum
Pb, Hg 33.5 of Pb 4.4 25 – 40.48
38.9 of Hg 4 25 47.87
Paenibacillus
Polymyxa
Cu, Pb 111.11 5 30 2 90
Ochrobactrum sp.
HG16
Hg 5 – 30 48 30
Serratia marcescens
HG19
Hg 2 – 30 48 90
Alteromonas
macleodii subsp.
Fijiensis
Pb 75 5 37 3 55
Alcaligenes feacalis
Burkholderia
cenocepalia
Cd, Pb
Cd
10 of Cd
10 of Pb
100 of Cd
7
–
30
30
72 of Cd
98 of Pb
48
70 of Cd
98 of Pb
60 of Cd
Bacillus cereus
KMS3-1
Cd, Cu,
Pb, Zn
5 7 27 48 –
Bacillus licheniformes
Cr 200 7.4 37 24 –
Bacillus sp. S3
Cd 20 5 28 7 –
Cr 150
Rasulov et al. (2013); Mokaddem et al. (2014); Wang et al.(2019): Krishnamoorthy et al. (2020)
EPS producing various bacterial species dealing with heavy metal pollution

33. Easily bioavailable
Non living biosorbent
No pathogenicity issues
Higher stability to environmental stress
Higher surface to volume ratio
Advantages of EPS
Zhang et al. (2021)
Constraints of EPS application
 No universal extraction method available so far to determine accurately the quantitative
extraction of the EPS from the different microbial suspensions or aggregates
 Lacking of more research based studies in polluted agricultural fields.
 The culture requirement (growth medium, temperature, pH ) varies with species.
 Lack of flexibility in wide range of soil type and land use.

34. Conclusions
• Bioremediation is gradually substituting the conventional methods of heavy metal remediation because of
being cost effective, eco friendly and more efficient.
• Extra cellular polymeric substance (EPS) are the bio polymeric secretary microbial by-products which can
adsorb or bind with the heavy metals through several mechanisms (biosorption,
bioleaching,biovolatilization,bioimmobilization) and reduce their contamination.
• Several factors such as temperature, pH, concentration of metals, growth media controls the EPS activity
and alters their efficiency.
• Bacterial spp like Pseudomonas, Bacillus, Azotobacter, E.coli, Rahnella have biosorption capacity towards
Zn Cd, Cr,Cu respectively. Whereas Enterobacter shows multi metal resistance towards Ni, Co, Mn, Cu &
Zn.
• Rhizobacterial EPS producing strains also have PGPR (plant growth promoting) characteristics that
enhance overall growth of plant with other beneficial functions.
• EPS can be a potential bioremediation tool with several other scopes in future if the challenges related to its
extraction , isolation and application can be addressed properly.

35. Path ahead
• More research should be conducted to identify
and develop the most easy EPS extraction
method that can be feasible and time saving.
• More EPS based study on farmers field to
evaluate their removal efficiency under
different land use and soil type.
• Developing EPS formulation which can be
easily available in market.
• More studies on their specific selectivity and
tolerance towards particular heavy metals to
improve their application aspect.

36. Thank You