Heavy metal absorption studies

1. Rishav Prakash
(1MV15BT026)
VIIIth Semester
Guides
R Manjunath
Ishwar Chandra
Sir M Visvesvaraya Institute of
Technology, Bangalore

2. Trace amounts of heavy metals are
required by living organism
including copper, iron, zinc but
however excessive levels of these
metals can be toxic to the organism
due to their toxicity and
accumulation behavior(Franke S, Grass G,
Rensing C, Nies DH (2003) )

3. Sources of Heavy Metals
Pacyna JM, Pacyna EG (2001) An assessment of global and regional emissions of trace metals to the atmosphere from anthropogenic sources worldwide. Environmental Reviews 9: 269-
298.
Czekalski N, Gascón Díez E, Bürgmann H (2014) Waste-water as a point source of antibiotic-resistance genes in the sediment of a freshwater lake. ISME J 8: 1381-1390. 8. Duruibe J,
Ogwuegbu M, Egwurugwu J (2007) Heavy metal pollution and human biotoxic effects. International Journal of Physical Sciences 2: 112-118.
Sources of heavy
metal
Mining Agriculture and
Farming Solid Waste
Automobile spear
parts
Emission of fossil fuels gases
Batteries and E-waste

4. METAL REMOVAL
TECHNOLOGIES
Reverse
osmosis
Precipitation
Ion
Exchang
e
Adsorption
Biosorption

5. Basic concepts and
terminology
Biosorption passive sequestration by non-metabolizing non-living
biomass
Bioaccumulation Metabolically mediated transport and deposition of
chemical species in living cells
Adsorption Involves the interface accumulation or
concentration of substances at a surface or interface
Sorption +
Absorption Molecules or atoms of one phase
interpenetrate among other of another phase to form a
«solution»
Davis TA, Volesky B, Mucci A. Water Research. 2003

6. Properties of heavy metals
• They occur near the bottom of the periodic table
• Have high densities
• Toxic in nature
• Nondegradable
Note: Arsenic is not actually a metal but is a semimetal
i.e. its properties are intermediate between those of
metals and nonmetals.

7. • It is particularly the cell wall structure of certain algae, fungi and bacteria
which was found responsible for this phenomenon (Volesky, 1986)
• Bioaccumulation is nothing but a biosorption but it happens in a living
cells
Biosorption is a property of certain types of inactive, dead,
microbial biomass to bind and concentrate heavy metals.

8. Mechanism Involved in Biosorption

10. Non-living biomass Adsorption
amines
thioethers
carboxylates
hydroxyls
thiols
phosphates
amides
Living biomass Biological
Processes
Reduction, oxidation, methylation…
+ Adsorption
Transport through the membrane
Cellular membrane binding
Biosorption processes

11. Biosorption of zinc ion: a deep comprehension ;Vishal Mishra (2013)

12. Ca,K, Na,Mg
Zn,Cr,Pb,Cd,

13. Transport across cell membrane
 transport across microbial cell membranes may be mediated by the
same mechanism used to convey metabolically important ions such as
potassium, magnesium and sodium.
Basically biosorption by living organisms comprises of two steps.
1. Metabolism independent binding where the metals are bound to
the cell walls and
2. Metabolism dependent intracellular uptake, whereby metal ions
are transported across the cell membrane. ( Costa, et.al., 1990,
Gadd et.al., 1988, Ghourdon et.al., 1990, Huang et.al., 1990.,
Nourbaksh et.al., 1994)

15. Physical adsorption
• In this category, physical adsorption takes place with the help
of van der Waals' forces.
– Kuyucak and Volesky 1988, hypothesized that uranium,
cadmium, zinc, copper and cobalt biosorption by dead
biomasses of algae, fungi and yeasts takes place through
electrostatic interactions between the metal ions in
solutions and cell walls of microbial cells.
• Electrostatic interactions have been demonstrated to be
responsible for copper biosorption by bacterium Zoogloea
ramigera and alga Chiarella vulgaris (Aksu et al. 1992), for
chromium biosorption by fungi Ganoderma
lucidum and Aspergillus niger .

16. Ion Exchange
Cell walls of microorganisms contain polysaccharides
and bivalent metal ions exchange with the counter
ions of the polysaccharides.
o Kuyucak and Volesky 1988: the alginates of
marine algae occur as salts of K+, Na+, Ca2+, and
Mg2+. These ions can exchange with counter ions
such as CO2+, Cu2+, Cd2+ and Zn2+ resulting in the
biosorptive uptake of heavy metals
o Muraleedharan and Venkobachr, 1990: The
biosorption of copper by fungi Ganoderma
lucidium and Aspergillus niger was also up taken
by ion exchange mechanism.

17. Complexation
 removal can take place by complex formation on the cell surface
after the interaction between the metal and the active groups.
Metal +Active group---- complex
 Aksu et al. 1992 hypothesized that biosorption of copper by
C. vulgaris and Z. ramigera takes place through both adsorption and
formation of coordination bonds between metals and amino and
carboxyl groups of cell wall polysaccharides.
Microorganisms + metals -----chelate  metallo-organic
(citric, oxalic, gluonic, fumaric, lactic and malic acids)
 Metals may be biosorbed or complexed by carboxyl groups found in
microbial polysaccharides and other polymers.

18. Precipitation
 Precipitation may be either dependent
cellular metabolism or independent of it. In the former
case, the metal removal from solution is often associated
with active defense system of the microorganisms.
 They react in the presence of a toxic metal producing
compounds, which favour the precipitation process.
 In the case of precipitation not dependent on the cellular
metabolism, it may be a consequence of the chemical
interaction between the metal and the cell surface.

19. it affects the solution chemistry of
the metals, the activity of the
functional groups in the biomass
and the competition of metallic
ions (Friis and Myers-Keith, 1986,
Galun
Temperature seems not to influence the
biosorption performances in the range of 20-
35 0C (Aksu et al. 1992)
Biomass concentration in solution seems to influence the specific
uptake: for lower values of biomass concentrations there is an
increase in the specific uptake (Fourest and Roux, 1992; Gadd et al.
1988). Gadd et al. 1988 suggested that an increase in biomass
concentration leads to interference between the binding sites.
Fourest and Roux, 1992 invalidated this hypothesis attributing the
responsibility of the specific uptake decrease to metal concentration
shortage in solution.

20. Bacteria as Biosorbents
• Bacterial biosorption is mainly used for the removal of metals
ions and dyes.
Leitão AL (2009) Potential of Penicillium species in the bioremediation field. Int J Environ Res Public Health 6: 1393-1417.
. Li X, Li A, Long M, Tian X (2015) Equilibrium and kinetic studies of copper biosorption by dead Ceriporia lacerata biomass isolated from the litter of an invasive plant in China. J Environ
Health Sci Eng 13: 37.
Cho DH, Kim EY, Hung YT (2010) Heavy metal removal by microbial biosorbents Environmental Bioengineering Springer 11: 375-402.
Lone MI, He ZL, Stoffella PJ, Yang XE (2008) Phytoremediation of heavy metal polluted soils and water: progresses and perspectives. J Zhejiang Univ Sci B 9: 210-220.
. Green-Ruiz C, Rodriguez-Tirado V, Gomez-Gil B (2008) Cadmium and zinc removal from aqueous solutions by Bacillus jeotgali: pH, salinity and temperature effects. Bioresour Technol 99:
3864-3870.

21. Algae as Biosorbents
• Algae are efficient and cheap biosorbents
– algae absorb about 15.3% - 84.6% which is higher as
compared to other microbial biosorbents.
• Brown algae
– known to have high absorption capacity. Biosorption of
metal ions occurs on the cell surface by means of ion
exchange method.
– absorbed metals like Cd, Ni, Pb through chemical
groups on their surface such as carboxyl,
Sulfonate, amino, as well as sulfhydryl.

23. Fungi as Biosorbents of Heavy Metals
• The use of fungi as bio sorbents material has been shown to be efficient
material is also one of the cost-effective and eco-friendly methods with
serves as an alternative to chemically bound treatment process.
• The capability of the many type of fungi to produce extracellular enzymes
for the assimilation of complex carbohydrates for former hydrolysis makes
capable the degradation of various degrees of pollutants.
• They also have the benefit of being relatively uncomplicated to grow in
fermenters, therefore being appropriate for large scale production.
• Another benefit is the easy separation of fungal biomass by filtration
because of its filamentous structure. In comparison to yeasts, filamentous
fungi are less sensitive to variations in nutrients, aeration, pH,
temperature and have a lower nucleic content in the biomass

25. Biosorption of heavy metals by
obligate halophilic fungi
Highlights
 First report of using obligate halophilic fungi for biosorption of
heavy metals.
 Cadmium, copper, ferrous, manganese, lead and zinc were
effectively removed from their medium by obligate halophilic
fungi.
 A. flavus and S. halophilus showed best performance for the
biosorption of heavy metals.
 Over all, Fe and Zn were most removed by obligate halophilic
fungi.
 This study provides a cost effective solution of removing
heavy metals
Javaid Hussain , Chemosphere 199 (2018) 218-222

26. Abstract
The presence of heavy metals in the environment poses a serious threat to human
health. Remediation of this problem using microorganisms has been widely
researched to find a sustainable solution.
 Obligate halophilic fungi comprising Aspergillus flavus, Aspergillus gracilis,
Aspergillus penicillioides (sp. 1), Aspergillus penicillioides (sp. 2), Aspergillus
restrictus and Sterigmatomyces halophilus were used for the biosorption of
cadmium, copper, ferrous, manganese, lead and zinc.
 The metals were supplemented as salts in potato dextrose broth for the growth of
obligate halophilic fungi and incubated for 14 days. The supernatant and biomass
were obtained by the acid digestion method.
 The biosorption was screened by atomic absorption spectroscopy. All tested fungi
showed moderate to high adsorption of heavy metals, amongst which A. flavus
and S. halophilus showed the best average adsorption of all heavy metals studied,
with an average of 86 and 83%, respectively.
 On average, Fe and Zn are best removed from the liquid media of obligate
halophilic fungi, with an average of 85 and 84%, respectively.

27. Materials and methods
Aspergillus restrictus
Aspergillus pencillodes
Aspergillus gracilis
A.flavus
(5%Nacl)
Potato broth Agar
Or
Sterigmatomyces
halophilus

28. Innoculated in a series of Erlenmeyer flask
50 ml of PDB
Initial concentration of heavy metal -1000 ppm
Heavy metal used - 𝐶𝑑𝐶𝑙2. 𝐻2 𝑂, 𝐶𝑢𝐶𝑙2. 2𝐻2 𝑂
𝐹𝑒(𝑁𝐻4)2 𝑆𝑂4. 6𝐻2 𝑂, 𝑀𝑛𝐶𝑙2,
𝑃𝑏(𝑁𝑂3)2, 𝑍𝑛(𝑁𝑂3)2
 Flask was incubated for 14 days at lab
 Control medium having no fungal presence
 Fungal were ontained from media, using
Whatman Filter Paper
 Dried at oven 80© for 12 hrs(jack etal,2005)

29. Acid Digestion Protocol
0.15 mg of dried sample placed in
100 ml Erlenmeyer flask
20 ml of 65% HN𝑂3
+
3 ml Deionized water
24 hrs, heated on hot plate
@95-105©
Further heating adding
35% HCl
The sample digested by refluxes
After cooling filtered through ash
less filter paper.

30. Species %removal
A.flavus 85
Aspergillus
restrictus
60
Aspergillus
gracilis
70
Aspergillus
pencillodes
55,60
Sterigmatomy
ces halophilus
89

31. Species %removal
A.flavus 82
Aspergillus
restrictus
73
Aspergillus
gracilis
55
Aspergillus
pencillodes
33,40
Sterigmatomyce
s halophilus
85

32. Species %removal
A.flavus 95
Aspergillus
restrictus
65
Aspergillus
gracilis
80
Aspergillus
pencillodes
85,86
Sterigmatomy
ces halophilus
70

33. Species %removal
A.flavus 90
Aspergillus
restrictus
70
Aspergillus
gracilis
75
Aspergillus
pencillodes
70,68
Sterigmatomyces
halophilus
85

34. Species %removal
A.flavus 65
Aspergillus
restrictus
85
Aspergillus
gracilis
90
Aspergillus
pencillodes
80,79
Sterigmatomyces
halophilus
87

35. Species %removal
A.flavus 87
Aspergillus
restrictus
43
Aspergillus
gracilis
82
Aspergillus
pencillodes
75,74
Sterigmatomyces
halophilus
50

36. Result
Heavy Metal Species showing maximum or absorption
Cadmium Aspergillus flavus, Sterigmatomyces halophilus
Copper A.flavus , A.restrictus ,S.halophilus
Iron A.restrictus (least)
Manganese A.flavus , S.halophilus
Lead A.restrictus, S.halophilus
Zinc A.grailis, S.halophilus(90%)

37. Inference
 It was observed that out of six species three species shows
excellent absorption property for heavy metal such as zinc
lead , cadmium
 Species such as Aspergillus flavus , A.restrictus,
Sterigmatomyces halophilus
 In that also Sterigmatomyces halophilus showed absorbance
in every heavy metal
 In case of Zinc Sterigmatomyces halophilus absorbed 90% of
heavy metal.

38. Mechanism of Pb2+ removal from aqueous solution
using a nonliving moss biomass
Highlights
 The moss plant Barbula lambarenensis (RBL) will be useful for
metal adsorption from aqueous solution.
 " The maximum adsorption capacity (Qo) for Pb adsorption is
62.50 mg/g at 298 K and 90.91 mg/g at 323 K.
 " The pH for optimum adsorption is 5.0 while maximum
adsorption was attained in 30 min.
 " The adsorption data obeyed the pseudo-second-order
model.
 " The free energy changes (DGo) are positive and the reaction
is exothermic.
Bamidele I. Olu-Owolabi, Paul N. Diagboya, William C. Ebaddan ,Chemical Engineering
Journal (2012), 270–275

39. Protocol
• Sampling, pre-treatment and characterization of biosorbent
material
 The B. Lambarenensis was obtained in August 2011 in
Agbor(611060 0E; 615090 0N), Delta State, Nigeria; washed
severally withtap water and deionized water to remove impurities,
then air-dried,ground, sieved through a 0.5 mm mesh size sieve,
and the sieved particles were used for the study. This was called the
raw BL

40. • 1000 mg/L of standardized Pb2+ stock solution was prepared from
the analytical grade chloride salt. Working solutions of required
concentrations were prepared from this stock as required.
• Replicate batch experiments were used to determine metal sorption
capacity of the biomass in 60 mL polyethylene bottles by contacting
approximately 0.1 g of the RBL biomass with 20 mL of metal solution,
except where otherwise stated, for determining effect of pH, time,
temperature and sorbate concentration.
• The sorbent–sorbate mixtures were shaken on a mechanical shaker
duringthe course of the adsorption experiment and concentrations of
metal in the filtrate solutions were determined using the Buck
Scientific 205 Atomic Absorption Spectrometer (AAS) with air-
acetyleneflame on absorbance mode.

41. FT-IR test spectra of unloaded RBL biomass
showed several absorption peaks between the
scanning frequency range of4500–500 cm1

42. The water content in the RBL biomass and the numerous free hydroxyl groups in
the polysaccharide structure of this moss plant wall may explain the presence of the
band.
The sharp absorption just below 3000 𝑐𝑚−1
is indicative of CAH stretch likely from
alkanes while the band at around 2350 𝑐𝑚−1
is suggestive of cumulative double bonds
stretch of
The band around 1700 𝑐𝑚−1
is characteristic of the stretch of carbonyl double bond
either from free or esterified carboxyl groups. However, closer band frequencies at 1655
𝑐𝑚−1
have been attributed to amide-I of protein secondary structures.
The absorption peak at about 1424 𝑐𝑚−1 suggests aromatic methyl group/methyl ketone
and carboxylate vibrations or likely CAH deformations of alkanes. The strong band around
1090 cm1 is due likely to the bond and stretching, which are characteristic
for polysaccharides.
Peaks below 1000 𝑐𝑚−1
have been attributed to such groups as aromatic bending
vibrations (874 𝑐𝑚−1), thioesters (672 𝑐𝑚−1), but majorly plane deformations.
The broad band positioned around 3400 𝑐𝑚−1
is indicative of the stretching band of the
carbonyl double bond from some carboxylic acids and their salts, a stretching vibration of
free hydroxyl functional groups of aromatic and aliphatic origins, and possibly NH stretch
of amides

43. Effects of pH

44.  The uptake of Pb2+ ions is pH dependent. At the low pH value of 3.0, RBL
adsorbed lower amount (56.4 mg/g or 57.8%) of Pb2+ onto its surface active
adsorption sites. With increase in solution pH, the quantity adsorbed increased
steadily until pH 5.0 (70.6 mg/g or 71.6%) and 6.0 (72.4 mg/g or 73.3%) where
the quantities of Pb2+ adsorbed made a plateau on the graph before
precipitation of Pb2+ ions set in.
 In summary, steady increment in adsorption was observed from pH 3.0 to 5.0,
indicating that more Pb2+ ions were adsorbed on RBL surfaces as pH increased.
 Then adsorption reached a plateau between pH 5.0 and 6.0. The optimum pH
for adsorption of Pb2+ was recorded at pH 5.0.
 Above this pH, there was an apparent increase in adsorption of Pb2+.
 This has been attributed mainly to solvation and hydrolysis of Pb2+ ion products
leading to precipitation of Pb2+ from solution.
 In aqueous solutions of pH less than 5, Pb2+ ions exist as either Pb2+ or Pb(OH)+
or both. However, the formation of Pb2+ hydrolysis products begins to occur at
pH values between 5 and 6, and this brings about precipitation.
 Due to this reason all the experiments were carried out at pH 5.

45. Effect of time on Pb2+ adsorption

46. Effect of varying biosorbent dose on Pb2+ adsorption.

47. Effect of initial adsorbate concentration on Pb2+ adsorption.

49.  The ability of B. lambarenensis biosorbent to remove Pb2+ from
aqueous solution was investigated in equilibrium, kinetics and
thermodynamics studies.
 The results obtained show that the biomass of B. lambarenensis has
an optimum pH for Pb2+ adsorption at 5.0, nearly attains maximum
adsorption within 1 h at 298 K, and obeys the pseudo-second order
kinetics with an exothermic reaction. Pb2+ adsorption increased with
increase in temperature.
 The Langmuir isotherm described the equilibrium data better than
other isotherms indicating adsorption is monolayer with monolayer
adsorption capacity of 62.50 mg/g at 298 K and 90.91 mg/gat 323 K.
 The FT-IR analysis showed that possible functional groups responsible
for metal adsorption are carboxyl, carbonyl, amides, hydroxyl and
possibly other smaller groups that cannot be identified using only FT-
IR spectra.

50. The satisfaction and euphoria that accompany the successful completion of any
seminar should be, but incomplete without the mention of the people who made
it possible.
We sincere thank to our guide Mr. R Manjunath and Mr.Ishwar Chandra
(Assistant Professor, Sir M. Visvesvaraya Institute of Technology, Bangalore)
for his constant guidance, support and encouragement in conceptualizing,
developing and executing the presentation.
I also thank Dr. Mrinalini menon for her constant support and encouragement.
We express our gratitude to Dr. H. G. Nagendra (HOD, Sir M. Visvesvaraya
Institute of Technology, Bangalore) for giving us the opportunity to present such
an enriching topic.
Our heartfelt thanks to the entire faculty of Sir M. Visvesvaraya Institute of
Technology for enabling us with the necessary knowledge, skills and capabilities
to present this topic successfully.
ACKNOWLEDGEMENT

52. Extra Slides
• Potato dextrose broth and agar were prepared
using 250 g of potato boiled in 100 ml of
distilled water for 30 min and the filtrate is
mixed with 2 g of dextrose and for agar plates
1.5 g of agar was added with this mixture.
• Extreme or obligate halophiles require high
osmotic pressure (up to 30% salt)
Facultative halophiles tolerate high osmotic
pressure.