8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 1/35
BIOREMEDIATION OF COBALT AND NICKEL IN ACIDIC
MINES USING SULPHATE REDUCING BACTERIA AND
PAENIBACILLUS POLYMYXA Document By: Bharadwaj
Visit my website
www.Engineeringpapers.blogspot.com
More Papers and Presentations available on above site
ABSTRACT:
Acid Mine Drainage (AMD) or Acid Rock Drainage (ARD) has been one of the most important problems
both the active and abandoned mining industries are being affected with. It is characterized by its high
[Type the abstract of the document here. The abstract is typically a short summary of the contents of
the document. Type the abstract of the document here. The abstract is typically a short summary of the
contents of the document.]
[Type the abstract of the document here. The abstract is typically a short summary of the contents of
the document. Type the abstract of the document here. The abstract is typically a short summary of the
contents of the document.]
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 2/35
acidity, high concentration of metals like Cu,Cd, etc and high concentration of sulphates.
Inorder to mitigate a part of the problem caused by Co and Ni ions in AMD, a bioremediation process
using SRB (Sulphate Reducing Bacteria) namely Desulfovibrio desulfuricans (DD) & Desulfotomaculum
nigrificans (DN) and Paenibacillus polymyxa has been considered in the present studies. Bioremediation
means Destroying hazardous contaminants or transforming them into less harmful forms by the use of
Microorganisms(mainly Bacteria).
From research it was found that Desulfovibrio desulfuricans and Desulfotomaculum nigrificans are
capable of 99% removal of Cobalt sulfate and 96% removal of Nickel sulfate when taken 100ppm of each
separately or together as precipitates of their sulfides. The rates of removal of metal ions of Cobalt and
Nickel were found decreasing over the period of conversion of sulfates to sulfides and their
precipitation. In the studies with biosorption of Cobalt and Nickel ions by Paenibacillus polymyxa wasfound upto 25% in a period of 7 days. The rate of biosorption of Cobalt and Nickel ions by Paenibacillus
polymyxa was also found decreasing. 10 % of 10ppm Nickel was adsorbed by Desulfotomaculum
nigrificans and the rate was found to be constant after 1 day.
KEYWORDS
Acid mine drainage, bioremediation, biosorption, Sulfate reducing bacteria, Paenibacillus polymyxa,
adsorption, precipitation of sulphides, reducing the toxicity of metal ions.
INTRODUCTION
It is known that contaminated land is a potential threat to human health and has adverse effect on our
environment. This has led the mankind to take up remedial measures.
Remediation
Remediation is the removal of pollution or contaminants from land (including sediments in waterways)
for the general protection of the environment.
Bioremediation
"Remediate" means to solve a problem, and "bio-remediate" means to use biological organisms to solve
an environmental problem such as contaminated soil or groundwater.
Need for Bioremediation:
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 3/35
The quality of life on Earth is linked inextricably to the overall quality of the environment. Contaminated
lands generally result from past industrial activities(oil drilling, mining) when awareness of the health
and environmental effects connected with the production, use, and disposal of hazardous substances
were less well recognized than today. The problem is worldwide, and the estimated number of
contaminated sites is significant. It is now widely recognized that contaminated land is a potential threat
to human health, and its continual discovery over recent years has led to international efforts to remedy
many of these sites, either as a response to the risk of adverse health or environmental effects caused
by contamination or to enable the site to be redeveloped for use.
The conventional techniques used for remediation have been to dig up contaminated soil and remove it
to a landfill, or to cap and contain the contaminated areas of a site. The methods have some drawbacks.
The first method simply moves the contamination elsewhere and may create significant risks in the
excavation, handling, and transport of hazardous material. Additionally, it is very difficult and
increasingly expensive to find new landfill sites for the final disposal of the material. The cap and contain
method is only an interim solution since the contamination remains on site, requiring monitoring and
maintenance of the isolation barriers long into the future, with all the associated costs and potential
liability.
A better approach than these traditional methods is to completely destroy the pollutants if possible, or
at least to transform them to innocuous substances. Some technologies that have been used are high-
temperature incineration and various types of chemical decomposition (e.g., base-catalyzed de-
chlorination, UV oxidation). They can be very effective at reducing levels of a range of contaminants, but
have several drawbacks, principally their technological complexity, the cost for small-scale application,
and the lack of public acceptance, especially for incineration that may increase the exposure to
contaminants for both the workers at the site and nearby residents.
Bioremediation is an option that offers the possibility to destroy or render harmless various
contaminants using natural biological activity. As such, it uses relatively low-cost, low-technology
techniques, which generally have a high public acceptance and can often be carried out on site. It will
not always be suitable, however, as the range of contaminants on which it is effective is limited, the
time scales involved are relatively long, and the residual contaminant levels achievable may not always
be appropriate. Although the methodologies employed are not technically complex, considerable
experience and expertise may be required to design and implement a successful bioremediation
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 4/35
program, due to the need to thoroughly assess a site for suitability and to optimize conditions to achieve
a satisfactory result.
Because bioremediation seems to be a good alternative to conventional clean-up technologies research
in this field is rapidly increasing. It has been used at a number of sites worldwide with varying degrees of
success. Techniques are improving as greater knowledge and experience are gained, and there is no
doubt that bioremediation has great potential for dealing with certain types of site contamination.
Some tests make an exhaustive examination of the literature of bioremediation of organic and inorganic
pollutants, and another test takes a look at pertinent field application case histories.
Advantages of bioremediation
• Bioremediation is a natural process and is therefore perceived by the public as an acceptablewaste treatment process for contaminated material such as soil. Microbes able to degrade the
contaminant increase in numbers when the contaminant is present; when the contaminant is
degraded, the biodegradative population declines. The residues for the treatment are usually
harmless products and include carbon dioxide, water, and cell biomass.
• Theoretically, bioremediation is useful for the complete destruction of a wide variety of
contaminants. Many compounds that are legally considered to be hazardous can be
transformed to harmless products. This eliminates the chance of future liability associated with
treatment and disposal of contaminated material.
• Instead of transferring contaminants from one environmental medium to another, for
example, from land to water or air, the complete destruction of target pollutants is possible.
• Bioremediation can often be carried out on site, often without causing a major disruption of
normal activities. This also eliminates the need to transport quantities of waste off site and the
potential threats to human health and the environment that can arise during transportation.
• Bioremediation can prove less expensive than other technologies that are used for clean-up of
hazardous waste.
Acid Mine Drainage (AMD)
It is characterized by:
High acidity
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 5/35
High concentration of metals like Cu, Fe, Zn, Co, Ni, As, Pb, Cd, etc
High Sulfate Concentration
SRB for treatment of acid mine drainage
SRB
Sulfate-reducing bacteria comprise several groups of bacteria that use sulfate as an oxidizing agent,
reducing it to sulfide. Most sulfate-reducing bacteria can also use other oxidized sulfur compounds such
as sulfite and thiosulfate, or elemental sulfur. This type of metabolism is called dissimilatory, since sulfur
is not incorporated - assimilated - into any organic compounds. Sulfate-reducing bacteria have been
considered as a possible way to deal with acid mine waters that are produced by other bacteria.
Sulfate-reducing bacteria (SRB) form one group of sulfate reducing prokaryotes. Desulfovibrio
desulfuricans and Desulfotomaculum nigrificans are often used to immobilize dissolved heavy metals as
metallic sulfides.
Chemical Mechanisms of Treatment
SRB are involved in several of the in situ and ex situ treatment technologies and are often used in
conjunction with other technologies. The general purpose of using SRB in AMD treatment is to produce
sulfides for metal sulfide precipitation, while generating alkalinity. Biologically Sulfate Reduction may be
divided into two categories:
Assimilatory
Dissimilatory
Assimilatory:
In general, sulfate is converted to a protein containing Sulfur by:
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 6/35
Sulfate must be 'activated' initially by PAP before the reaction can proceed. Assimilatory sulfate
reduction occurs anaerobically as well as aerobically.
Dissimilatory:
In soils that become deficient in oxygen, usually the result of flooding, the sulfide level will
increase to relatively high concentrations.The basic reaction:
The formation of sulfide by sulfate reduction in nature is enhanced in warm, wet, or water logged
soils with a pH of above 6.0. Sulfide accumulation may be particularly pronounced in sulfate-
rich saline areas in which plant excretions (release of carbon compounds) serve as the carbon
source in addition to the hydrogen. Thus, like denitrification, an oxidizable carbon source serves
as the electron donor, while the sulfate serves as the electron acceptor.
The metabolic dissimilatory process is similar to the assimiliatory sulfate reduction in that the
sulfate must be first activated by a molecule called ADP (adenosine-5-phosphate).
Or if tetrathionate is reduced
The chemical basis involves microbially-mediated sulfate reduction coupled with organic matter
(represented by CH2O) oxidation. A typical overall conversion equation is (neglecting the small amount
of organic material required to produce biomass):
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 7/35
Eight electrons are transferred from the energy source acetic acid to the electron acceptor sulphate in
order to produce sulphide. The reaction equation shows that in the same process also alkalinity is
produced. This leads to an increase in the pH of the water, often to a near neutral value.
Typically, a certain amount of metals is present together with the sulfate. These metals will react with
the dissolved sulfide to form highly insoluble metals sulfides.
These bacterial precipitated metal sulphides can be recovered and recycled.
Cadmium, Copper, Iron, Lead, Mercury, Cobalt, Nickel, and Zinc are some of the metals that will
precipitate as metal sulfides. In addition, Arsenic, Antimony, and Molybdenum form more complex
sulfide minerals. Metals such as Manganese, Iron, Nickel, Copper, Zinc, Cadmium, Mercury, Cobalt,
Nickel and Lead may also be removed to some extent by co-precipitation with other metal sulfides.
Furthermore, SRB species have been found, that can reduce certain metals to a more insoluble form,
such as reduction of uranium (VI) to uranium (IV). Sulfate reduction also consumes acidity, raising the
pH. Increasing the pH facilitates the above precipitation reactions and creates suitable conditions for
precipitation of metal hydroxides.
The reduction product of reaction (1), hydrogen sulphide, is a volatile gas. The form in which sulphide
occurs depends on the pH:
HS-
and S2-
which occur at neutral and high pH respectively are both water soluble. H2S is the
predominant form at low pH (<6).
Sulphide is distributed over the gas phase (g) and the liquid phase (l) according to:
α is a dimensionless distribution coefficient. The unionised H2S concentration also depends on the
temperature. Sulphide is highly reactive, corrosive and toxic to microorganisms. The toxicity increases at
low pH while only the un-ionised hydrogen sulphide form is able to permeate through the cell
membrane. H2S affects the intracellular pH of the microorganism and impedes its metabolism.
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 8/35
There are several different bioremediation techniques. They can lead to the results striven for.
Indigenous populations of microbial bacteria can be stimulated through the addition of nutrients
or other materials. Exogenous microbial populations can be introduced in the contaminated
environment. The addition of extra bacteria is known as bio augmentation. If necessary,
genetically altered bacteria can also be used.
Biosorption of heavy metals by Paenibacillus polymyxa
Paenibacillus polymyxa is a Gram-positive neutrophilic, periflagellated heterotroph, occurring
indigenously associated with several mineral deposits. It secretes exopolysaccharides, proteins
and several organic acids such as acetic, formic and oxalic acids. They also generate levan
forming the capsule of the organism from sucrose by the activity of a specific enzyme known as
levan sucrase. The extracellular polysaccharide (ECP) aids in biological uptake of metal ions
necessary for metabolism and growth. Heavy metals are known to bind to the cell walls through
the ECP.
MATERIALS AND METHODS
Microorganisms
The pure bacterial strains of Desulfovibrio desulfuricans, Desulfotomaculum nigrificans and
Paenibacillus polymyxa were obtained from the national collection of industrial microorganisms,
National Chemical Laboratory(NCL), Pune, India, were used for the studies.
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 9/35
Growth media were prepared in conical flasks by adding all the components and adjusting the
pH corresponding to the medium. The flasks were covered with nonabsorbent cotton plugs andthen with aluminium foil. Then they were autoclaved, and cooled.
Desulfovibrio desulfuricans & Desulfotomaculum nigrificans were inoculated in air tight 125
ml bottles containing sterilized Postgate’s medium & Barr’s medium separately.
Postgate’s medium composition:
Components g/L
Tryptone 10.0
Sodium sulphite 1.0
Sodium sulphate 1.0
Ferric citrate 0.5
Distilled water 1000ml
pH 7-7.5
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 10/35
Barr’s medium composition:
Components g / L
K2HPO4 0.5
NH4Cl 1.0
CaSO4 2.0
MgSO4 2.0
Sodium lactate 7.0
Ferrous ammonium
Sulphate
0.5
Distilled water 1000ml
pH 7.5
The two bacteria, Desulfovibrio desulfuricans & Desulfotomaculum nigrificans inoculated in theabove two media, i.e., the 4 bottles were kept in an incubator maintained at 35º.
Paenibacillus polymyxa was inoculated in Nutrient Broth medium taken in a conical flask byadding the pure culture of bacteria to the medium. Inoculation of the bacteria has to be done in
the UV-chamber inorder to avoid contamination. Then placed in an orbital shaker rotating at 205rpm maintained at 37 º for one week and the cell count readings were taken.
Nutrient Broth’s medium composition:
Components g / L
Peptone 10
Yeast extract powder 10
Nacl 5
Distilled water 1000ml
PH 7-7.5
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 11/35
Cell Count:
Periodically cell count was calculated using Petroff Hausser Counter on a Leitz phase contrast
microscope. A drop of microbial culture was placed over the slide at the marked region and had
put under the microscope.
The counter consists of ruling covering a square millimeter. The center square is ruled into 25groups, each consisting of 16 squares. All the 25 groups are separated with a triple ruling
whereas each of the single squares of 16 squares is singly ruled.
The height of the ruling is 0.02 mm.
The area of each square is 1/400 mm2. The bacterial cells were counted in the center square.
Depth of small square = 1/50 mm
Area of small square= 1/400 mm
2
Volume of small square= 1/50 * 1/400 mm3 = 1/50 * 1/400 *10-3
Number of cells per milliliter = Average number of cells counted per small square / Volume
in cm3
= Average number of cells counted per small square x 24 x 103
Analytical Methods
Determination of sulfate concentration:
Turbidimetric method has been used to measure the concentrations of sulphate ion using a UV-
Visible Spectrophotometer. The absorbance of the sample was measured at a wavelength of 420
nm.
A blank solution was prepared in a 100ml standard round bottomed flask using 5ml of conditioning reagent and filled upto the mark with distilled water.
Samples for the estimation are made in 100ml standard flasks by adding 5ml of conditioning
reagent, 1ml of bacterial cells from the growing cultures of SRB and filled upto the mark with
distilled water.
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 12/35
Conditioning Reagent composition:
Components ml
Glycerol 50
Concentrated HCl 30
Distilled water 300
Isopropyl Alcohol 100
Distilled water 1000
NaCl 75gm
Then the blank and samples were added with more or sufficient amount of BaCl 2 and thoroughlystirred on a magnetic stirrer. Then after five minutes the undisturbed blank and samples were
taken in cleaned cuvettes and placed in the Spectrophotometer for the estimation of sulphate
concentration.
Determination of pH and ESCE
Glass pH-electrode combined with the reference Ag/AgCl electrode and Standard Calomel
Electrode were used to measure pH and redox potential (Eh) respectively.
Calibration of pH meter:
The electrode is first washed with alcohol and dried. It is dipped in pH 7 solution and the knob is
adjusted till the instrument reads 7.00. Then it is removed from the buffer, washed and dried.Later it is dipped in pH 4 buffer solution and the slope screw is turned to get the instrument read
4.00.
For the bioremoval studies of Cobalt and Nickel, 1000ppm stock solutions of each were
prepared.
Preparation of Standard Stock Solutions
Standard stock solutions containing 1000ppm of Co2+
and 1000ppm of Ni2+
were prepared bydissolving 0.4789gm of CoSO4 and 0.4478gm of NiSO4 respectively with distilled water in
100ml standard flasks.
Precipitation studies of metal sulfates as sulfides by SRB
100 ppm of Co2+
, 100ppm of Ni2+
solutions separately and 100 ppm of Co2+
& 100ppm of Ni2+
together in a solution were made from the standard stock solutions of 1000ppm by diluting with
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 13/35
Postgate’s medium. Then these solutions were taken in 125ml air tight bottles and inoculated by
adding 1ml of pure bacterial culture of Desulfovibrio desulfuricans & Desulfotomaculum
nigrificans. These bottles were maintained at 35º in an incubator and the metal ion
concentrations were determined periodically by Atomic Absorption Spectrophotometer (AAS).
Bioremoval of Cobalt and Nickel ions using Paenibacillus polymyxa
100 ppm of Co2+, 100ppm of Ni2+ solutions separately and 100 ppm of Co2+ & 100ppm of Ni2+
together in a solution were made from the standard stock solutions of 1000ppm by diluting with
Nutrient Broth medium. Then pure bacterial strains were added to the previously prepared
solutions in conical flasks and placed in an orbital shaker rotating at 205rpm and maintained at35º.
Scanning Electron Microscope (SEM) photograph of Paenibacillus polymyxa & SRB adhesionto pyrite ore were obtained.
Procedure for taking Scanning Electron Microscope (SEM) photograph:
1. A drop of the bacterial sample is placed on a Cover slip and it is allowed to air dry. If there
is some surface on which bacteria has been deposited, that also has to be air dried.
2. The samples are chemically fixed for a period of 24-96hrs using a final
concentration of 2.5% (W/V) gluteraldehyde.
3. The samples are rinsed in distilled water 3 times to remove traces of gluteraldehyde.
4. Then the samples are dehydrated in graded series of ethanol 30, 50, 75, 85, 95, 100%,
three minutes in each.
5. Finally air dried under vacuum and kept in a desiccator until used.
6. The films are later on, coated with Gold palladium and loaded for SEM.
Preparation of bacterial cell pellets:
The grown cultures of bacteria were centrifuged in a Beckman Coulter centrifuge with JH-10
rotor rotating at 15000rpm at a temperature of 4ºC for about 30 minutes.
Adsorption Studies on SRB
10ml of 1000ppm stock solutions of Cobalt and Nickel were taken separately as well as togetherin 100 ml standard flasks and made upto the mark with 10 -3 M KNO3 solution to prepare
solutions containing 100ppm of Cobalt & Nickel separately and together respectively.
For the adsorption studies by the bacterial cells pellets of Desulfovibrio desulfuricans &
Desulfotomaculum nigrificans were obtained and were mixed with the above prepared 10-3
MKNO3 solutions with 100ppm of Cobalt & 100ppm of Nickel separately and as well as together
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 14/35
in 125 ml air tight containers and incubated at 35º. The metal ion concentrations were
determined periodically by Atomic Absorption Spectrophotometer (AAS).
Adsorption studies on Paenibacillus polymyxa
Cell pellets of Paenibacillus polymyxa were mixed with the above prepared 10
-3
M KNO3 solutions with 100ppm of Cobalt & 100ppm of Nickel separately and as well as together inconical flasks and placed in the orbital shaker for 7 days and Co & Ni ion concentrations were
determined periodically by Atomic Absorption Spectrophotometer (AAS) with Zeeman furnace
of Thermo Electron Corporation.
RESULTS AND DISCUSSION
Growth curves of SRB:
The decrease of sulphate concentration and Eh, the increase of pH values, the formation of black
precipitates and the sensorial detection of H2S smell were observed.
The following figure 1 depicts the Growth Curve of Desulfovibrio desulfuricans in Postgate’s
medium. It clearly shows that the cell number increased from 107
to 5x108
and the sulphate
concentration got reduced from 1.6mg/L to 0.5mg/L in 7 days . As the number of cells increased,the conversion of Sulphate to sulphide increased thereby resulting in the reduction of Sulphate
Concentration.
Fig (I). Growth curve of Desulfovibrio desulfuricans in Postgate’s medium
0 1 2 3 4 5 6 7 810
7
108
109
C e l l s / m l
Time(Days)
Cell Count
0.0
0.4
0.8
1.2
1.6
2.0
SO4
-2
Concentration
S u l p h a
t e i o n c o n c e n t r a t i o n ( m g / L )
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 15/35
Similarly the figure 2 depicts the Growth Curve of Desulfotomaculum nigrificans in Postgate’s
medium where the cell number increase from 0.8x107
to 2.8x108
and the Sulphate Concentration
reduction from 1.6mg/L to 0.7mg/L were observed in 7 days .
0 1 2 3 4 5 6 7 8
107
108
C e l l s / m l
Time(Days)
Cell Count
0.5
1.0
1.5
2.0
SO4
-2Concentration
S u l p h a t e I o n C o n c e n t r a t i o n ( m g / L )
Fig (II). Growth curve of Desulfotomaculum nigrificans in Postgate’s medium
0 1 2 3 4 5 6 7 8
107
108
109
1010
C
e l l s / m l
Time(Days)
Cell Count
-180
-160
-140
-120
-100
-80
-60
-40
-20
E
S C E
( m V )
ESCE
Fig (III). Growth curve of Desulfovibrio desulfuricans in Barr’s medium
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 16/35
The above figure 3 and the below figure 4 depict the growth curves of Desulfovibrio
desulfuricans and Desulfotomaculum nigrificans respectively in Barr’s medium where the
increase is cell number is accompanied with the decrease in the ESCE can be clearly observed.
Growth curve of Paenibacillus polymyxa
0 25 50 75 100 125 150 175 200 225 250
5.0x108
1.0x109
1.5x109
2.0x109
2.5x109
3.0x109
C e l l s / m l
Time(hrs)
Cell Count
2
3
4
5
6
7
8
p H
pH
0 1 2 3 4 5 6 7 8
107
108
109
C e l l s / m l
Time(Days)
Growth Curve
-160
-140
-120
-100
-80
-60
-40
-20
E S C E
ESCE
Fig (IV). Growth curve of Desulfotomaculum nigrificans in Barr’s medium
Fig (V). Growth curve of Paenibacillus polymyxa in Nutrient broth medium
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 17/35
From figure 5, clearly it can be seen that the cell number increased from 1x108
to 2.5x109
in a
period of 125 hours and the pH decreased from 7.0 to 6.2 during the cell growth. All the phases
lag, log, and death phases involved in the growth of bacteria can be observed.
Fig (VII) (a) SEM image of Paenibacillus polymyxa
Fig (VI). Microscopic image of fully grown culture of Paenibacillus polymyxa
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 18/35
The decrease in aqueous metal concentrations is the result of the following processes:
1. Biosorption to cell sufaces
2. Release of Extra Cellular Polymeric substances
3. Precipitation of Sulphides
4. Intra cellular penetration and accumulation
Fig (VII.b). SEM image of Desulfovibrio desulfuricans adhering to pyrite surface
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 19/35
Bioremoval of Cobalt and Nickel as Sulfide precipitates:
0 1 2 3 4 5 6
0
20
40
60
80
100
C o b a l t r e t a i n e d a s C o S O 4
( % )
Time(days)
% CoSO4
retaining
(a)
Fig (VIII.b) % Precipitation of CoS by Desulfovibrio desulfuricans
during their growth in Postgate’s medium.
0 1 2 3 4 5 6
0
20
40
60
80
100
P r e c i p i t a t i o n o
f C o b a l t a s s u l f i d e ( % )
Time(days)
% Conversion to CoS
(b)
Fig (VIII.a) %CoSO4 Retained by Desulfovibrio desulfuricans
during their growth in Postgate’s medium.
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 20/35
Figure 8 (a) & (b) depicts the percentage of CoSO4 remaining in the solution and the percentage
of CoSO4 converted to CoS precipitated respectively when Desulfovibrio desulfuricans are
grown in Postgate’s medium. It can be observed that 100% precipitation of CoSO4 as CoS was
achieved in a period of 6 days.
The rate of conversion of CoSO4 to CoS in the absence of Nickel by Desulfovibrio desulfuricans
was about 1.0268mg/L/hr initially & later decreased to 0.02954mg/L/hr.
0 1 2 3 4 5 6
0
20
40
60
80
100
% NiSO4
retaining
N i c k e l r e t a i n e d a s N i S O
4 ( % )
Time(Days)
(a)
0 1 2 3 4 5 6
0
20
40
60
80
100
N i c k e l p r e c i p i t a t i o n a s s u l f i d e ( % )
Time(days)
% Conversion to NiS(b)
Fig (IX.a) % of NiSO4 Retained by Desulfovibrio desulfuricans during their growth in Postgate’s
medium
Fig (IX.b) % Precipitation of NiS by Desulfovibrio desulfuricans during their growth in Postgate’s
medium
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 21/35
Figure 9 (a) & (b) depict the percentage of Nickel remaining in the medium and % of Nickel
precipitated as sulfide as a function of time during the growth of Desulfovibrio desulfuricans in
Postgate’s medium containing 100 ppm of Nickel ions. It can be observed from the above figures
that Nickel concentration decreased from 100ppm to 3.125ppm in 4 days, that is 96.875% of the
Nickel was adsorbed by the bacterial cells. Further the Nickel concentration got reduced to
2.556ppm in 6 days, that is 97.444% remediation was achieved in a period of 6 days.
The rate of conversion of NiSO4 to NiS was about 1.015mg/L/hr initially and later got reduced
to 0.0533mg/L/hr
0 1 2 3 4 5 6
0
20
40
60
80
100
C o b a l t r e
t a i n e d a s C o S O 4
( % )
Time(days)
% CoSO4
retaining
(a)
Fig (X.a) % CoSO4 Retained by Desulfotomaculum nigrificans during their growth in Postgate’s
medium
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 22/35
From figures 10 (a) & (b) we can observe that the 100% conversion of cobalt sulfate to CobaltSulfide precipitate was achieved by the growing culture of Desulfotomaculum nigrificans in a
period of 6 days.
The rate of conversion was about 1.015mg/L/hr for the initial period and later it was decreased to
0.0533mg/L/hr.
Fig (X.b) % Precipitation of CoS by Desulfotomaculum nigrificans during their growth in Postgate’s medium
0 1 2 3 4 5 6
0
20
40
60
80
100
(b)
C o b a l t s u l f a t e p r e c i p i t a t e d a s s u l f i d e ( % )
Time(days)
% Conversion to CoS
0 1 2 3 4 5 6
0
20
40
60
80
100 % NiSO
4retaining
N i c k e l r e t a i n e d a s N i S O
4 ( % )
Time(Days)
(a)
Fig (XI.a) % NiSO4 Retained by Desulfotomaculum nigrificans during their growth in Postgate’s medium
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 23/35
.
From figures 11 (a) & (b) we can observe that during the growth of Desulfotomaculum
nigrificans in Postgate’s medium, 92.084% of the Nickel was precipitated in a period of 3 days
and total 96.574% removal as precipitate was achieved in 6 days.
The rate of conversion to sulfide was about 0.9592mg/L/hr initially & later was found to
decrease to 0.04677mg/L/hr.
0 1 2 3 4 5 6 7 8
0
20
40
60
80
100
% CoSO4
retaining
% NiSO retaining
M e t a l r e
t a i n i n g a s s u l f a t e ( % )
Time(Days)
(a)
0 1 2 3 4 5 6
0
20
40
60
80
100
N i c k e l p r e c i p i t a t i o n
a s s u l f i d e ( % )
Time(Days)
% Conversion to NiS
(b)
Fig (XI.b) % Precipitation of NiS by Desulfotomaculum nigrificans during their growth in Postgate’smedium
Fig (XII.a) % NiSO4 and CoSO4 Retained by Desulfovibrio desulfuricans during their growth in
Postgate’s medium
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 24/35
Figure 12 (a) and (b) depict the percentage precipitation of Cobalt and Nickel as sulphides with
time. Here the Cobalt sulfide precipitation is little faster than that of Nickel when both the metals
were present in the growing culture of Desulfovibrio desulfuricans.
Here the rate of precipitation of CoS was found to be 1.01527mg/L/hr initially & the decreased
rate 0.02639mg/L/hr was observed for the later period and that of NiS was 0.9489mg/L/hr in the
starting and later it was about 0.0723mg/L/hr.
Fig (XII.b) % Precipitation of NiS and CoS by Desulfovibrio desulfuricans during their growth in
Postgate’s medium
Fig (XIII.a) % NiSO4 and CoSO4 Retained by Desulfotomaculum nigrificans during their growth in Postgate’s medium
0 1 2 3 4 5 6 7 8
0
20
40
60
80
100 CoSO
4retaining
NiSO4
retaining
M e t a l s u l f a t e r e t a i n e d ( % )
Time(Days)
(a)
0 1 2 3 4 5 6 7 8
0
20
40
60
80
100 CoSO
4retaining
NiSO4
retaining
M e t a l s
u l f a t e r e t a i n e d ( % )
Time(Days)
(a)
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 25/35
Figures 13 (a) & (b) depict the precipitation of Cobalt and Nickel as sulphides where the Cobalt
sulfide precipitation is almost same as that of Nickel when both the metals together were added
to the growing culture of Desulfotomaculum nigrificans.
Initially the rate of CoS precipitation was 0.5907mg/L/hr & later it decreased to about
0.3371mg/L/hr and the rate of NiS precipitation for the initial period was 0.5892mg/L/hr &
decreased to 0.4730mg/L/hr for the later period.
Bioremoval of Cobalt and Nickel by Paenibacillus polymyxa
0 1 2 3 4 5 6 7 80
20
40
60
80
100
C o b a l t r e t a i n e d ( % )
Time(days)
% Cobalt retaining
(a)
Fig (XIII.b) % Precipitation of NiS and CoS by Desulfotomaculum nigrificans during their growth in Postgate’s medium
Fig (XIV.a) % Cobalt Retained by Paenibacillus polymyxa in Nutrient Broth medium
0 1 2 3 4 5 6 7 8
0
20
40
60
80
100
% Conversion to CoS
% Conversion to NiS
M e t a l p r e c i p i t a t e
d a s s u l f i d e ( % )
Time(Days)
(b)
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 26/35
From previous figures 14 (a) & (b) it can be observed that 62.542 % of Cobalt ions were taken in
by the cells of Paenibacillus polymyxa over a period of 7 days.
Initially the rate of uptake of Cobalt ions was about 0.49204mg/L/hr and lastly it got reduced to
0.1153mg/L/hr.
0 1 2 3 4 5 6 7 8
0
20
40
60
80
100
(b)
C o b a l t b i o s o r b
e d ( % )
Time(days)
% Biosorption of Cobalt
0 1 2 3 4 5 6 7 80
20
40
60
80
100
N i c
k e l r e t a i n e d ( % )
Time(days)
% Ni retained
(a)
Fig (XIV.b) % Biosorption of Cobalt by Paenibacillus polymyxa in Nutrient Broth medium
Fig (XV.a) % Nickel Retained by Paenibacillus polymyxa in Nutrient Broth medium
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 27/35
From figures 15 (a) & (b) it can be seen that 74.651 % of Nickel ions were taken in by the cells
of Paenibacillus polymyxa over a period of 7 days.
Initially the rate of uptake of Cobalt ions was about 0.4595mg/L/hr and later it reduced to
0.0289mg/L/hr.
Fig (XVI.a) % Nickel & Cobalt Retained by Paenibacillus polymyxa in Nutrient Broth medium
0 1 2 3 4 5 6 7 8
0
20
40
60
80
100
(b)
N i c k e l b i o s o r b e d ( % )
Time(days)
Ni Biosorption
Fig (15.b) % Biosorption of Nickel by Paenibacillus polymyxa in Nutrient Broth medium
0 1 2 3 4 5 6 7 8
0
20
40
60
80
100
% Nickel retaining
% Cobalt retaining
M e t a l r e t a i n
i n g i n s o l u t i o n ( % )
Time(Days)
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 28/35
From figures 16 (a) & (b) it can be observed that Nickel intake was slightly greater than that of
Cobalt by the cells of Paenibacillus polymyxa.
Rate of intake of Cobalt ions was 0.4465mg/L/hr initially and later it decreased to 0.0289mg/L/hr
and that of Nickel was 0.5123mg/L/hr and finally reduced to 0.02852mg/L/hr.
Adsorption studies on Paenibacillus polymyxa
0 5 10 15 20 250
20
40
60
80
100
% of Cobalt retained
C o b a l t
R e t a i n e d ( % )
Time(hrs)
(a)
0 1 2 3 4 5 6 7 8
0
20
40
60
80
100
% Nickel Biosorption % Cobalt Biosorption
M e t a l b i o s o r b
e d ( % )
Time(Days)
Fig (XVI.b) % Biosorption of Nickel & Cobalt by Paenibacillus polymyxa in Nutrient Broth medium
Fig (XVII.a) % Cobalt Retained by Paenibacillus polymyxa
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 29/35
Figures 17 (a) & (b) show the biosorption of Cobalt by the cells in an interaction period of 1hr,2hr, 4hr, 6hr, 8hr and 24 hrs with the growing culture of Paenibacillus polymyxa in Nutrient
Broth medium. It can be observed that 42.258% of the Cobalt was adsorbed in 8hrs and later
desorption had taken place. After 24hrs the adsorbed amount of Cobalt was 77.325%.
0 5 10 15 20 25
60
70
80
90
100
(a)
% of Nickel retaining
N i c k e l R e t a i n e d ( % )
Time(hrs)
0 5 10 15 20 250
20
40
60
80
100
% of Cobalt adsorption
C o b a l t a d s o r
b e d ( % )
Time(hrs)
(b)
Fig (XVII.b) % of Cobalt adsorbed by Paenibacillus polymyxa
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 30/35
Figures 18 (a) & (b) depict the amount of Nickel that was adsorbed in 1hr, 2hr, 4hr, 6hr, 8hr and
24 hrs interaction periods with Paenibacillus polymyxa. It can be observed that 24.081% of the
Nickel was adsorbed in 8hrs and 37.591% in 24 hrs.
0 5 10 15 20 25
0
20
40
60
80
100
Nickel biosorption
N i c k e l b i o s o r b e d ( % )
Time(hrs)
(b)
0 5 10 15 20 25
0
10
20
30
40
M e t a l a d s o r b e d ( % )
Time(hrs)
Cobalt biosorption Nickel biosorption
Fig (XVIII.a) % Nickel Retained (b) % of Nickel adsorbed by Paenibacillus polymyxa
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 31/35
Figures 19 (a) & (b) are plotted for adsorption studies of Cobalt of initial concentration 100ppm
and Nickel of 100ppm initial concentration when taken together in the growing culture of
Paenibacillus polymyxa in Nutrient Broth medium. It can be observed that 24.868% of Cobalt
and 21.676% of Nickel were adsorbed in a time span of 8 hrs. In 24 hrs Cobalt adsorption was
found to be 34.243% and that of Nickel was found to be 28.12%.
Adsorption studies on Desulfotomaculum nigrificans
0 5 10 15 20 250
20
40
60
80
100
M e t a l r e t a i n e d ( % )
Time(hrs)
% Cobalt retained
% Nickel retained
Fig (XIX) a) % Cobalt and Nickel Retained (b) % of Cobalt and Nickel adsorbed by Paenibacillus
polymyxa
0 1 2 3 48.0
8.4
8.8
9.2
9.6
10.0
A m o u n t o f N i c k e l r e t a i n i n g ( p p m )
Time(Days)
Adsorption studies of Nickel
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 32/35
It can be observed from the figures 20 (a) & (b) that about 10 % of Nickel was adsorbed by the
cells of Desulfotomaculum nigrificans when 10 ppm of Nickel was taken for adsorption studies.
CONCLUSIONS
The following conclusions are based on the above work:
1. SRB namely Desulfovibrio desulfuricans and Desulfotomaculum nigrificans are capable
of 99% removal of Cobalt sulfate and 96% removal of Nickel sulfate when taken
separately or together as precipitates of their sulfides.
2. The rates of removal of metal ions of Cobalt and Nickel were found decreasing over the
period of conversion of sulfates to sulfides and their precipitation.
3. Biosorption of Cobalt and Nickel ions by Paenibacillus polymyxa was upto 25% in a
period of 7 days.
4. The rate of biosorption of Cobalt and Nickel ions by Paenibacillus polymyxa was also
found decreasing.
5. 10 % of 10ppm Nickel was adsorbed by Desulfotomaculum nigrificans and the rate was
found to be constant after 1 day.
Fig (XX) a) % Nickel Retained (b) % of Nickel adsorbed by Desulfotomaculum nigrificans
0 1 2 3 4
0
20
40
60
80
100
N i c k e l A d s o r b e d (
%
)
Time(Days)
% of Nickel adsorbed
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 33/35
6. These results thereby show that this process helps in decreasing in the toxic metal ion
concentration in the mined lands.
7. Modelling a suitable method maintaining suitable environmental conditions would help
to remediate sites on a large scale based on research work.
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 34/35
REFERENCES
1. Evvie Chokalingam, S. Subramanian, 2005. Studies on removal of metal ions andsulphate reduction using rice husk and Desulfotomaculum nigrificans with reference
to remediation of acid mine drainage. Chemosphere 62: 699-708
2. Rajesh Kumar Sani, Brent M. Peyton, Laura T. Brown, 2001. Copper inducedinhibition of growth of Desulfovibrio desulfuricans G 20: Assessment of its toxicity
and correlation with those of Zinc and Lead. Applied and Environmental
Microbiology, p. 4765-4772.
3. Ralf Cord-Ruwisch and Friedrich Widdel, 1986. Corroding Iron as a Hydrogen
source for sulfate reduction in growing cultures of SRB. Appl. Microbiol. Biotechnol.
25: 169-174.
4. Look W. Hulshoff Pol, Piet N. L. Lens, Alfons J. M. Stams & Gatze Lettinga 1998.
Anaerobic treatment of Sulphate rich waste waters. Biodegradation 9: 213-224.
5. Graciela Gonzalez-Gil, Robbert Kleerebenzem & Gatze Lettinga, 1999. Effects of
Nickel and Cobalt on kinetics of methanol conversion by methanogenic sludge as
assessed by online CH4 monitoring. Applied and Environmental Microbiology, p
1789-1793.
6. M. Sai Ram, L. Singh, M.V. S. Suryanarayana and S. I. Alam, 2000. Effect of Iron,Nickel and Cobalt on bacterial activity and dynamics during anaerobic oxidation of
organic matter. Water, Air and Soil Pollution 117: 305-312.
7. Namita Deo and K. A. Natarajan, 1998. Studies on interaction of Paenibacillus
polymyxa with iron ore minerals in relation to beneficiation. International journal of
Mineral Processing, Vol 55,p: 42-60.
8. C. Garcia, D. A. Moreno, A. Ballester, M. L. Blazquez & F. Gonzalez, 2001.
Bioremediation of an industrial acid mine water by metal tolerant sulfate reducing
bacteria. Minerals Engineering, Vol. 14, No. 9, pp. 997-1008.
9. Asa Kolmert and D Barrie Johnson, 2001. Remediation of acidic waste waters using
immobilized, acidophilic sulfate reducing bacteria. Chem. Technol. Biotechnol. 76:
836-843.
10. R. D. Norris, R. E. Hinchee, R. Brown, P. L. McCarty, L. Semprini, J. T. Wilson, D.
H. Kampbell,
11. M. Reinhard, E. J. Bouwer, P. C. Borden, T. M. Vogel, J. M. Thomas, C. H. Ward.
Handbook of Bioremediation. Lewis, Boca Raton, FL (1993).
8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
http://slidepdf.com/reader/full/bio-remediation-of-cobalt-and-nickel-in-acidic-mines-using-sulphate-reducing 35/35
12. J. G. Mueller, C. E. Cerniglia, P. H. Pritchard. Bioremediation of Environments
Contaminated by Polycyclic Aromatic Hydrocarbons. In Bioremediation: Principles
and Applications, pp.125 – 194, Cambridge University Press, Cambridge (1996).
13. A. S. Allard and A. H. Neilson. Int. Biodeterioration Biodegradation 39, 253 – 285
(1997).
Document By: Bharadwaj
Visit my website
www.Engineeringpapers.blogspot.com
More Papers and Presentations available on above site