bio remediation of cobalt and nickel in acidic mines using sulphate reducing bacteria and...

8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa

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BIOREMEDIATION OF COBALT AND NICKEL IN ACIDIC

MINES USING SULPHATE REDUCING BACTERIA AND

PAENIBACILLUS POLYMYXA Document By: Bharadwaj

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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

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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:



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



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



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:

http://en.wikipedia.org/wiki/Bacterium

http://en.wikipedia.org/wiki/Sulfate

http://en.wikipedia.org/wiki/Sulfide

http://en.wikipedia.org/wiki/Sulfite

http://en.wikipedia.org/wiki/Thiosulfate

http://en.wikipedia.org/wiki/Thiosulfate

http://en.wikipedia.org/wiki/Sulfite

http://en.wikipedia.org/wiki/Sulfide

http://en.wikipedia.org/wiki/Sulfate

http://en.wikipedia.org/wiki/Bacterium



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):



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.

http://wiki.biomine.skelleftea.se/wiki/index.php/Acetic_acid%20/%20Acetic%20acid

http://wiki.biomine.skelleftea.se/wiki/index.php/PH%20/%20PH

http://wiki.biomine.skelleftea.se/wiki/index.php/Hydrogen_sulphide%20/%20Hydrogen%20sulphide


http://wiki.biomine.skelleftea.se/wiki/index.php/Temperature%20/%20Temperature

http://wiki.biomine.skelleftea.se/wiki/index.php/Sulphide%20/%20Sulphide

http://wiki.biomine.skelleftea.se/wiki/index.php/Sulphide%20/%20Sulphide

http://wiki.biomine.skelleftea.se/wiki/index.php/Temperature%20/%20Temperature


http://wiki.biomine.skelleftea.se/wiki/index.php/Hydrogen_sulphide%20/%20Hydrogen%20sulphide


http://wiki.biomine.skelleftea.se/wiki/index.php/Acetic_acid%20/%20Acetic%20acid



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.



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



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


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


PH 7-7.5



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.



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



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



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 )



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



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



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



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



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.



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



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



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



.

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



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)



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)



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



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)



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



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



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



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



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



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.



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.

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1789-1793.

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organic matter. Water, Air and Soil Pollution 117: 305-312.

7. Namita Deo and K. A. Natarajan, 1998. Studies on interaction of Paenibacillus

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bacteria. Minerals Engineering, Vol. 14, No. 9, pp. 997-1008.

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836-843.

10. R. D. Norris, R. E. Hinchee, R. Brown, P. L. McCarty, L. Semprini, J. T. Wilson, D.

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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).



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and Applications, pp.125 – 194, Cambridge University Press, Cambridge (1996).

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