BIOREMEDIATION OF COBALT AND NICKEL IN ACIDIC MINES USING SULPHATE REDUCING BACTERIA AND PAENIBACILLUS POLYMYXA

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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 was found 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 hightemperature incineration and various types of chemical decomposition (e.g., base-catalyzed dechlorination, 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 acceptable waste 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:

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 sulfaterich 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:

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.

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 and then with aluminium foil. Then they were autoclaved, and cooled. Desulfovibrio desulfuricans & Desulfotomaculum nigrificans were inoculated in air tight ml bottles containing sterilized Postgates medium & Barrs medium separately. Postgates medium composition: 125

Components Tryptone Sodium sulphite Sodium sulphate Ferric citrate Distilled water pH

g/L 10.0 1.0 1.0 0.5 1000ml 7-7.5

Barrs medium composition:

Components K2HPO4 NH4Cl CaSO4 MgSO4 Sodium lactate Ferrous ammonium Sulphate Distilled water pH

g/L 0.5 1.0 2.0 2.0 7.0 0.5 1000ml 7.5

The two bacteria, Desulfovibrio desulfuricans & Desulfotomaculum nigrificans inoculated in the above two media, i.e., the 4 bottles were kept in an incubator maintained at 35. Paenibacillus polymyxa was inoculated in Nutrient Broth medium taken in a conical flask by adding 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 205 rpm maintained at 37 for one week and the cell count readings were taken. Nutrient Broths medium composition:

Components Peptone Yeast extract powder Nacl Distilled water PH

g/L 10 10 5 1000ml 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 25 groups, 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 mm2 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 UVVisible 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 Glycerol Concentrated HCl Distilled water Isopropyl Alcohol Distilled water NaCl

ml 50 30 300 100 1000 75gm

Then the blank and samples were added with more or sufficient amount of BaCl2 and thoroughly stirred 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 by dissolving 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

Postgates 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 35 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 at 35. Scanning Electron Microscope (SEM) photograph of Paenibacillus polymyxa & SRB adhesion to 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 4C for about 30 minutes. Adsorption Studies on SRB 10ml of 1000ppm stock solutions of Cobalt and Nickel were taken separately as well as together in 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 10-3M KNO3 solutions with 100ppm of Cobalt & 100ppm of Nickel separately and as well as together

in 125 ml air tight containers and incubated at 35. 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 in conical 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 Postgates 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.

10

2.0

1.6

1.2

10

0.8

0.4

10

0.0 0 1 2 3 4 5 6 7 8

Time(Days)

Fig (I). Growth curve of Desulfovibrio desulfuricans in Postgates medium

Sulphate ion concentration(mg/L)

SO4 Concentration Cell Count

-2

Cells/ml

2.0

Cell Count

10

1.5

1.0

10

0.5 0 1 2 3 4 5 6 7 8

Time(Days)

Fig (II). Growth curve of Desulfotomaculum nigrificans in Postgates medium

Similarly the figure 2 depicts the Growth Curve of Desulfotomaculum nigrificans in Postgates 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 .

10

10

-20

Cell Count ESCE

10
9

-40 -60 -80 -100

10

-120 -140 -160 -180 0 1 2 3 4 5 6 7 8

10

Time(Days)

Fig (III). Growth curve of Desulfovibrio desulfuricans in Barrs medium

ESCE(mV)

Cells/ml

Sulphate Ion Concentration(mg/L)

SO4 Concentration

-2

Cells/ml

The above figure 3 and the below figure 4 depict the growth curves of Desulfovibrio desulfuricans and Desulfotomaculum nigrificans respectively in Barrs medium where the increase is cell number is accompanied with the decrease in the ESCE can be clearly observed.

-20 -40 -60 -80

10

Growth Curve ESCE

Cells/ml

10

-100 -120 -140

10

-160 0 1 2 3 4 5 6 7 8

Time(Days)

Fig (IV). Growth curve of Desulfotomaculum nigrificans in Barrs medium

Growth curve of Paenibacillus polymyxa

3.0x10

2.5x10

Cell Count pH

2.0x10

Cells/ml

1.0x10

5.0x10

25

50

75

100

125

150

175

200

225

2 250

Time(hrs)

Fig (V). Growth curve of Paenibacillus polymyxa in Nutrient broth medium

pH

1.5x10

E SCE

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 (VI). Microscopic image of fully grown culture of Paenibacillus polymyxa

Fig (VII) (a) SEM image of Paenibacillus polymyxa

Fig (VII.b). SEM image of Desulfovibrio desulfuricans adhering to pyrite surface

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

Bioremoval of Cobalt and Nickel as Sulfide precipitates:

100

Cobalt retained as CoSO4(%)

80 60 40 20 0 0 1 2

% CoSO4 retaining

(a)

Time(days)

Fig (VIII.a) %CoSO4 Retained by Desulfovibrio desulfuricans during their growth in Postgates medium.

100
Precipitation of Cobalt as sulfide(%)

80 60 40 20
(b) % Conversion to CoS

0 0 1 2 3 Time(days)
Fig (VIII.b) % Precipitation of CoS by Desulfovibrio desulfuricans during their growth in Postgates 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 Postgates 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.

100
Nickel retained as NiSO4(%)

% NiSO4 retaining 80 60 40 20 0 0 (a) 1 2 3 Time(Days) 4 5 6

Fig (IX.a) % of NiSO4 Retained by Desulfovibrio desulfuricans during their growth in Postgates medium

Nickel precipitation as sulfide (%)

100 80 60 40 20 0 0 1 2 3 Time(days) 4 5 6 % Conversion to NiS (b)

Fig (IX.b) % Precipitation of NiS by Desulfovibrio desulfuricans during their growth in Postgates 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 Postgates 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

100

Cobalt retained as CoSO4(%)

80 60 40 20 0 0 1 2

% CoSO4 retaining

(a)

3 Time(days)

Fig (X.a) % CoSO4 Retained by Desulfotomaculum nigrificans during their growth in Postgates medium

Cobalt sulfate precipitated as sulfide(%)

100 80 60 40 20 0 0 1 2 3 Time(days) 4 5 6
(b)

% Conversion to CoS

Fig (X.b) % Precipitation of CoS by Desulfotomaculum nigrificans during their growth in Postgates medium

From figures 10 (a) & (b) we can observe that the 100% conversion of cobalt sulfate to Cobalt Sulfide 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.

100
Nickel retained as NiSO4(%)

% NiSO4 retaining 80
(a)

60 40 20 0 0 1 2 3 Time(Days) 4 5 6

Fig (XI.a) % NiSO4 Retained by Desulfotomaculum nigrificans during their growth in Postgates medium

Nickel precipitation as sulfide(%)

100 80 60 40 20 0 0 1 2 3 4 5 6 % Conversion to NiS

(b)

Time(Days)
Fig (XI.b) % Precipitation of NiS by Desulfotomaculum nigrificans during their growth in Postgates medium

From figures 11 (a) & (b) we can observe that during the growth of Desulfotomaculum nigrificans in Postgates 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.

100
Metal retaining as sulfate(%)

80 60

% CoSO4 retaining % NiSO retaining

(a)

40 20 0 0 1 2 3 4 5 6 7 8

Time(Days)

Fig (XII.a) % NiSO4 and CoSO4 Retained by Desulfovibrio desulfuricans during their growth in Postgates medium

100
Metal sulfate retained(%)

80 60 40 20 0 0 1 2 3 4

CoSO4 retaining NiSO4 retaining

(a)

Time(Days)
Fig (XII.b) % Precipitation of NiS and CoS by Desulfovibrio desulfuricans during their growth in Postgates 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.
100
Metal sulfate retained(%)

80 60 40 20 0 0 1 2 3 4

CoSO4 retaining NiSO4 retaining

(a)

Time(Days)
Fig (XIII.a) % NiSO4 and CoSO4 Retained by Desulfotomaculum nigrificans during their growth in Postgates medium

Metal precipitated as sulfide(%)

100 80 60 40 20 0 0 1 2 3 4 5 6 7 8
(b)

% Conversion to CoS % Conversion to NiS

Time(Days)
Fig (XIII.b) % Precipitation of NiS and CoS by Desulfotomaculum nigrificans during their growth in Postgates medium

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

100 80

Cobalt retained (%)

60
% Cobalt retaining

40
(a)

20 0 0 1 2 3 4 5 6 7 8
Time(days)

Fig (XIV.a) % Cobalt Retained by Paenibacillus polymyxa in Nutrient Broth medium

100 80
% Biosorption of Cobalt

Cobalt biosorbed (%)

60
(b)

40 20 0 0 1 2 3 4 5 6 7 8

Time(days)
Fig (XIV.b) % Biosorption of Cobalt by Paenibacillus polymyxa in Nutrient Broth medium

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.

100 80

Nickel retained(%)

60 40
(a)

% Ni retained

20 0 0 1 2 3 4 5 6 7 8 Time(days)

Fig (XV.a) % Nickel Retained by Paenibacillus polymyxa in Nutrient Broth medium

100 80
Nickel biosorbed(%)
(b) Ni Biosorption

60 40 20 0 0 1 2 3 4 5 6 7 8

Time(days)
Fig (15.b) % Biosorption of Nickel 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.

100
Metal retaining in solution(%)

80 60 40 20 0 0 1 2 3 4 5 6 7 8 % Nickel retaining % Cobalt retaining

Time(Days)
Fig (XVI.a) % Nickel & Cobalt Retained by Paenibacillus polymyxa in Nutrient Broth medium

100 80 60 40 20 0 0 1 2 3 4 5 6 7 8 % Nickel Biosorption % Cobalt Biosorption

Metal biosorbed(%)

Time(Days)
Fig (XVI.b) % Biosorption of Nickel & Cobalt by Paenibacillus polymyxa in Nutrient Broth medium

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

100
% of Cobalt retained

Cobalt Retained(%)

80 60 40 20 0
0 5 10 15 20 25 Time(hrs)
Fig (XVII.a) % Cobalt Retained by Paenibacillus polymyxa
(a)

100
% of Cobalt adsorption

Cobalt adsorbed(%)

80 60 40
(b)

20 0
0 5 10 15 20 25 Time(hrs)
Fig (XVII.b) % of Cobalt adsorbed 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%.

100 % of Nickel retaining

Nickel Retained(%)

90 80 70 60 0 5 10 15 20 25
(a)

Time(hrs)

100

Nickel biosorbed(%)

80 60
(b)

Nickel biosorption

40 20 0 0 5 10 15 20 25

Time(hrs)
Fig (XVIII.a) % Nickel Retained (b) % of Nickel 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.
40

30

Metal adsorbed(%)

20 Cobalt biosorption Nickel biosorption

10

0 0 5 10 15 20 25

Time(hrs)

100 80 60 40 20 0 0 5 10 15 20 25 Time(hrs)
Fig (XIX) a) % Cobalt and Nickel Retained (b) % of Cobalt and Nickel adsorbed by Paenibacillus polymyxa

Metal retained(%)

% Cobalt retained % Nickel retained

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

10.0
Amount of Nickel retaining(ppm)

9.6 9.2 8.8 8.4 8.0 0 1

Adsorption studies of Nickel

2
Time(Days)

100 80
Nickel Adsorbed( % )

% of Nickel adsorbed

60 40 20 0 0 1 2
Time(Days)

Fig (XX) a) % Nickel Retained (b) % of Nickel adsorbed by Desulfotomaculum nigrificans

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.

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.

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12. J. G. Mueller, C. E. Cerniglia, P. H. Pritchard. Bioremediation of Environments Contaminated by Polycyclic Aromatic Hydrocarbons. In Bioremediation: Principles and Applications, pp.125194, Cambridge University Press, Cambridge (1996). 13. A. S. Allard and A. H. Neilson. Int. Biodeterioration Biodegradation 39, 253285 (1997).

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