BIOREMEDIATION OF COBALT AND NICKEL IN ACIDIC MINES USING SULPHATE REDUCING BACTERIA AND PAENIBACILLUS POLYMYXADocument 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 ofthe 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 ofthe document. Type the abstract of the document here. The abstract is typically a short summary of the contents of the document.]
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Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
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8/3/2019 Bio Remediation of Cobalt and Nickel in Acidic Mines Using Sulphate Reducing Bacteria and Paenibacillus Polymyxa
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.
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
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
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
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
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
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
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
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
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
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