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Biological removal of some flotation collector reagents from aqueous solutions and mineral surfaces
Minerals Engineering, Vol. 11, No. 8, pp. 717-738, 1998
Pergamon 0892-6875(98)00058-2
BIOLOGICAL REMOVAL FROM AQUEOUS
© 1998 Elsevier Science Lid All fights reserved 0892-6875/98/$ - see front matter
OF SOME FLOTATION COLLECTOR REAGENTS SOLUTIONS AND MINERAL SURFACES
N. DEO and K.A. NATARAJAN Department of Metallurgy, Indian Institute of Science, Bangalore - 560 012, India Email: kan @metalrg.iisc.ernet.in
(Received 31 January 1998; accepted 20 May 1998)
ABSTRACT
The utility of BaciUus polymyxa, a heterotrophic bacterium indigenous to ore deposits in the degradation of several flotation collectors has been demonstrated. Dodecyl amine, diamine, isopropyl xanthate and sodium oleate could be efficiently removed from alkaline solutions through bioremediation in an environmentally acceptable fashion. The bacterial cells were also found to be capable of stripping adsorbed collector reagents from various mineral surfaces. Mechanisms of such a biodegradation process are illustrated through results obtained from zeta potential, adsorption, bacterial growth and surface analysis studies. © 1998 Elsevier Science Ltd. All rights reserved. Keywords Flotation collectors; bacteria; biotechnology; environmental; mineral processing
INTRODUC~ON Froth flotation is extensively used the world over for the beneficiation of sulfide and oxide ores. Alkyl xanthates are used as collector reagents in sulfide ore flotation, while amines and fatty acids find application in iron ore flotation and the beneficiation of calcite, fluorite and quartzite. Although these collector reagents react preferentially with the concerned minerals in the treated ore pulp, excess and unreacted concentrations of these organic surfactants end up in the mill process effluents. Actual concentrations of various organic flotation collectors remaining in the effluent and process waters of mineral process operations are not readily available. It has been known that even smaller concentrations of these reagents in water streams are toxic to water life, besides their deleterious influence on end stream processes during recycling. Bioremediation has long been recognized as a cheap, flexible and environmentally benign technique in waste water treatment. Organic flotation reagents are also amenable to biological degradation. However, no systematic study on the potential of biological processes to detoxify flotation plant effluents has so far been reported. In this paper, the utility of a biological process for the efficient removal of some typical organic flotation collectors from aqueous solutions is illustrated. Two types of amines, namely, Deodecylammonium acetate (a primary amine) and a diamine, DA-16 which are extensively used in iron ore flotation were taken as examples. Further, sodium isopropyl xanthate, a universal sulfide collector reagent and sodium oleate a fatty
717
718
N. Deo and K. A. Natarajan
acid collector also were chosen. Also, the utility of a biological process to strip residual adsorbed collector reagents from mineral surfaces is demonstrated. A soil bacterium, namely, Bacillus polymyxa, which occurs indigenously, associated with several ore deposits was used in this work. M A T E R I A L S AND M E T H O D S
Bacterial strain and growth conditions A pure strain of Bacillus polymyxa NCIM 2539 obtained from national collection of industrial microoranisms, National Chemical Laboratory, Pune, India. was used in all the studies. A 10% v/v of an active inoculum was added to Bromfield medium [1] and incubated at 30°C on a rotary shaker at 240 rpm. The bacterial growth was studied by microscopic counting using a Petroff-Hausser counter under a phase contrast microscope and also by colony counting after plating, pH changes during bacterial growth was monitored and enough cell mass (>109 cells/ml) was generated by continuous growth for 8 hours. The culture was filtered thorough whatman No. 1 filter paper to remove precipitates and centrifuged at 15000 rpm for 15 minutes. The cell pellet was washed several times and resuspended in deionised double distilled water (pH 7 and specific conductivity less than 1.5 la mhos/cm).
Biodegradation of flotation collector reagents Biodegradation of sodium oleate, sodium isopropyl xanthate, isododecyl oxypropyl aminoprypyl amine (DA16) and dodecyl ammonium acetate (DAA) was studied under different conditions and concentrations. All the above collector reagents were of analytical grade and prepared in double distilled deionised water at the desired pH. The various conditions for biodegradation studies were: (a) (b) (c) (d)
During bacterial growth, In presence of metabolite alone In presence of cells only, and In presence of fully-grown culture (cells + metabolite + medium)
(a)
To 100 ml of each previously autoclaved Bromfield medium (without sucrose), different concentrations (50, 100, 150 ppm) of xanthate, oleate, DA-16 and DAA were added and the pH was adjusted to 9.0 and 10% of active inoculum was added to each flask. Two of each, uninoculated control flasks were maintained separately.
(b)
To 100 ml of each cell-free culture filtrate (the culture which reached stationary phase), different concentrations (50, 100, 150 ppm) of xanthate, oleate, DA-16 and DAA were added.
(c)
To 100 ml of each cell suspension (6 x 109 cells/ml in 10-3 M KNO 3, solution) different concentrations (50, 100, 150 ppm) of xanthate, oleate, DA-16 and DAA were added and the pH was adjusted to 9.0. Two of each cell free control flasks were also maintained separately.
(d)
To 100 ml of each active culture (the culture at mid-exponential phase) different concentrations (50, 100, 150 ppm) of xanthate, oleate, DA-16 and DAA were added.
All flasks were incubated at 30"C on a rotary shaker at 240 rpm for different periods of time. After each interval of time, the pH was checked and the residual concentrations of oleate [2], xanthate [3] DA16, and DAA [4] were measured in the cell-free supernatant by acid-titration and by U.V-visible spectrophotometry. The stages during biodegradation of the various flotation reagents, namely, oleate, xanthate, DA-16, and DAAwere also monitored through UV-visible and FTIR spectra. For this purpose, an initial blank spectrum for the different reagents was obtained. The progress of degradation and disappearance from the solutions of the various collector reagents in the presence of cells, metabolite, and cells + metabolites, under the four
Biologicalremovalof flotationcollectorreagents
719
different conditions mentioned above was then monitored through the disappearance, shifting of the initial absorption peaks and also the appearance of new peaks. Growth of Bacillus polymyxa in presence of oleate, xanthate, DA-16 and DAA To 100 ml of each previously autoclaved inorganic medium (without sucrose in 500 ml Erlenmeyer flasks) 100 ppm of xanthate, oleate, DA-16 and DAA were added. Two of each control flasks without the above collector reagents were also taken. The pH of all the flasks was maintained at 9.0. To all these above flasks, 10% of the fully grown culture along with the cells were added. After inoculation, the flasks were incubated on a rotary shaker at 240 rpm at 30°C. The bacterial growth was monitored by microscopic counting using a Petroff-Hausser counter and pH changes during growth were also measured at regular intervals of time. During growth, the protein and polysaccharides produced by the bacteria were also measured frequently from the cell-free supernatant by Bradford method [5] and phenol sulphuric acid method [6] after dialysis. Adsorption of collector reagents on bacterial cells Adsorption of collector reagents onto cell surfaces was estimated at different collector concentrations. A 10.3 M KNO 3 solution was used as the base electrolyte for the purpose. 0.5 g of each wet biomass were interacted with different concentrations (50, 100, 150, 200, 300, 400 ppm) of xanthate, oleate, DA-16, and DAA at pH 9.0 for different intervals of time. After each interval of time, the residual concentrations of collectors were estimated through analysis of the supernatant solution after centrifugation. Electrokinetic studies on bacterial cells after interaction with collector reagents The Zeta potentials of bacterial cells as a function of pH were determined using Zeta meter-3.0. Cell suspensions (109 cells/ml) were interacted with different concentrations of oleate, xanthate, DA-16 and DAA at pH 9.0 for different periods of time. After such interaction, the cell suspension was centrifuged at 15000 rpm for 15 minutes. The cell pellet was washed several times with 10.3 M KNO 3 and then resuspended in 10.3 M KNO 3 solution and adjusted to the required pH for electrokinetic measurements. In this manner, the surface chemical changes on bacterial cell surfaces before and after interaction with various organic reagents could be established as also the probable mechanisms of interaction. Flotation of bacteria after interaction with collectors 4 g of wet biomass was interacted with 1000 ppm of various collector reagents for 5 minutes at pH 9.0. After 5 minutes interaction, the cell suspension was centrifuged and the cell pellets were washed several times with 10.3 M KNO3. Then the cells were suspended in 10.3 M KNO3 solution and the flotation was carried out for five minutes under a nitrogen flow rate of 40 ml/minute using a Leaf and Knoll cell [7]. Biological stripping of adsorbed collectors from mineral surfaces The respective mineral samples, namely, hematite, calcite and pyrite were initially reacted with amine, oleate and xanthate collectors, respectively and subsequently subjected to biotreatment with cells of Bacillus polymyxa. The removal of adsorbed organic reagents from the mineral surfaces was monitored through FTIR spectroscopy.
RESULTS AND DISCUSSION Zeta potential and adsorption studies Surface affinity of the various flotation collectors towards bacterial ceils was assessed initially throgh electrokinetic and adsorption studies. Both the amine reagents, namely, Dodecylammonium acetate (DAA) and Isododecyl oxypropyl aminopropyl amine (DA-16) exhibited enhanced surface adsorption onto cells of
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N. Deo and K. A. Natarajan
Bacillus polymyxa as shown in Figure 1. For example, the isoelectric point (IEP) of bacterial cells shifted from the initial value at about pH 2 to about pH 2.8 and 3.3, respectively, after one hour interaction with 50 ppm of DAA and DA-16. Similarly, after similar interaction with 100 ppm concentrations of the above amines, the IEP of the cells shifted to pH 3.0 and 4.3, respectively (not shown in the Figure). The IEP shifts were thus found to be a function of amine concentration and indicate chemical interaction between the amine and cell surface components. Also, electrostatic attraction between negatively charged cell surfaces and cationic aminium ions would facilitate biosorption. On the otherhand, interaction with 50-100 ppm of the anionic collectors such as sodium isopropyl xanthate and sodium oleate did not result in any significant surface chemical changes on the bacterial cells as attested by measured zeta potentials. Electrostatic repulsion between negatively charged bacterial cells and anionic collector reagents retard mutual interaction. 20 50 ppm for
1 hr each
0 A
Cells before ht~octlon Atte¢ kltero~tion with
• I"1 •
[
I
I
i
,,
.
')
"
,,
I
DAA IPX Na- Oote
,t
I
DA-16
1
L
I
vE z
~-20
-40
-60
Fig. 1
Zeta potentials as a function of pH for cells of Bacillus polymyxa before and after interaction with various collector reagents.
Adsorption behaviour of various collector reagents onto bacterial cell surfaces was then established through adsorption isotherms at a solution pH of 9.00 as illustrated in Figure 2. Enhanced affinity of the two amine collectors towards bacterial cell surfaces could be readily seen in comparison with the two anionic collectors. For example, the maximum adsorption density for DAA and DA-16 corresponded to about 45 and 36 mg/g, respectively, compared to only about 2.5 mg/g for xanthate and oleate. The isotherms follow a Langmuir model which describes the sorption reaction as m--
MbC l+ bC
or by the linearised form,
C
1
C
m
bM
M
where
m = mass of solute sorbed per unit weight of sorbent, M = mass of solute sorbed per unit weight of sorbent at saturation b = constant accounting for energy of interaction C = measured equilibrium concentration
Biological removal of flotation collector reagents
721
From a plot of C/m vs C, the Langmuir model constants were derived as described in Table 1.
60
~40
"1o .Q
O~
~d •
2o E <~
I
"~ 40
0
J
A
Sodium oleate Xanthate
• O
OAA DA-16
A
I & i i -r I 80 120 150 Residual concentration , mg /L
~-T 200
I 240
Fig.2 Adsorption isotherms for various collector reagents.
TABLE 1 Langmuir adsorption model constants obtained from isotherms Organic reagent DAA D A - 16 Xanthate Oleate
M, mg/g 50.70 38.50 2.90 2.90
b 0.20 0.17 0.12 0.04
Another significant observation was that such amine adsorption rendered the bacterial cell surfaces more hydrophobic as attested to by increased flotability of amine-interacted bacterial cells. For example, 96% and 82% of the cells after 5 minute interaction with DA-16 and DAA could be readily floated and recovered from the aqueous solution at pH 9.0. No such flotation response was observed with xanthate and oleate treated cells.
Bacterial growth in the presence of collector reagents Although only the two amine collectors among the four reagents tested in this work exhibited significant biosorption tendency, all the organic surfactants however promoted bacterial growth in the solution as illustrated in Figures 3a and b. In the absence of any added carbon source such as sucrose, Bacillus polymyxa could grow effectively utilising the carbon present in the amines, sodium oleate and isopropyl xanthate and the nitrogen present in the amines. In such bacterial growth studies, the Bromfiled medium contained all other constituents except sucrose, which is the essential cabon source for normal growth of Bacillus polymyxa in the absence of other organic reagents. Substantial increase in cell population and pH decrease due to organic acid production by bacterial metabolism could be observed. The cell data have been plotted using the Logistic equation, thus enabling direct comparison of rate constants.
o dt
where
-" ~lmax
- Xma x
1
X = cell density (cells/ml)
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N. Deo and K. A. Natarajan
Xm= = maximum cell density lam~ = growth rate (h -1) X(t~o) = X o
x ] i xo t
Xm~x- X
LXm~x - X o Um"xt
Assuming various values of Xm~ from the experimental data, l a ~ could be estimated from the slope of the linear plot of
lfXm:X] vst The various values of Xm~ and ~tm~x thUS estimated are given in Table 2. From lamx, from Xm~,
DA-16 > oleate > xanthate > DAA oleate > DA-16 > DAA = xanthate
10.0
1011 Oleate
E
1010
8.0
ro
Xanthate
c "1o
7.0 "16 . 0 °"
10 9 Xanthate
E tO
9.0
5.0
lo8 j
Oleate
4.0
E3 0
3.0 107 ~ 0
10
20
30
40
2.0 50
Time (hr) Fig.3a
Logistic curves for growth of Bacillus polymyxa in the Bromfield medium (no sucrose) in the presence of collector reagents--Isopropyl xanthate and sodium oleate.
Biological removalof flotation collectorreagents
723
T A B L E 2 M a x i m u m cell number and growth rate constants Organic reagent Oleate Xanthate D A - 16 DAA
Xm~x(cells/ml) 6.7 x 10'" 6.2 x 10~ 2.65 x 10" 6.4 x 10~
It=,= (h-') 0.2609 0.2188 0.2643 0.1783
~ I:,,.
]1o.o
~/
'°'°F\~
19.o
.--/
[
i
t 'o
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° 1°1
,0,ISo 0
10
t,.0
20
30
40
50
Time (hr)
Fig.3b
Logistic curves for growth of Bacillus polymyxa in the Bromfield medium (no sucrose) in the presence of collector reagents--DA-16 and DAA.
Amounts of proteins and polysaccharides generated due to bacterial utilization of the various flotation collectors in a sucrose-free Bromfield medium are illustrated in Figure 4 a and b. It becomes readily apparent that the bacteria could effectively utilize all the organic surfactants and generate polysaccharides '
'
i=
-i
Imthc~'~u
Fig.4a
-~o
TW,*E(h)
Protein and polysaccharide generation by Bacillus polymyxa in the presence of Isopropyl xanthate and sodium oleate.
724
N. Deo and K. A. Natarajan
240
DAA DA16
• •
0 []
21:10
E z
o
E 120 u
8O
4Ol-
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ot~-mr " L
//
--14o
i 12
I
1
24
36
I Io
48
INTERACTION TiME 3 h
Fig.4b
Protein and polysaccharide generation by Bacillus polymyxa in the presence of DA-16 and DAA.
and proteins as the major metabolic products besides organic acids. Protein production (amino acids) was found to be the highest in the presence of amines, while that of polysaccharides in the presence of xanthate and oleate. This could be expected since the amines containing nitrogen and amino groups are degraded by the cells to generate amino acid containing proteins. It has also been subsequently observed that the amino acids, polysaccharides and fatty acids are further utilised by the bacteria for metabolism and growth leading to complete dissociation of all the collector reagents. The above observation was substantiated by continuous analysis of the solution with time over a period of 4 days.
Biodegradation of collector reagents in aqueous solutions The above results indicate that the bacterium, Bacillus polymyxa can be effectively used to degrade and remove various organic collector reagents from aqueous solutions. Biodegradation of the various collector reagents as a function of time at three initial concentrations under conditions of growth of Bacillus polymyxa was then studied and the results are presented in Figures 5a,b,c and d. In these tests, the initial cell concentrations was low at about 106 cells/ml which gradually increased with time upto 101° cells/ml after full growth in the presence of the collector reagents. Under such conditions, the initial rate of biodegradation was slower, especially at higher concentrations of the collector reagents. As the biomass increased, the rate of removal of the collector reagents also increased as a function of time. Also, the pH decreased from an initial value of 9 to about 5 during the period due to organic acid generated by bacterial metabolism. The following observations are note worthy:
a)
The overall rate of biodegradation of the various collector reagents was found to be influenced by their initial concentrations.
Biological removal of flotation collector reagents
b) c) d)
725
There was an initial lag period for the biodegradation at higher collector concentrations. The amines are biodegraded at a faster rate than oleate and xanthate. With respect to the rate of biodegradation for a given concentration and duration (for example, 30 ppm and 4 hours) the following order holds good. DA16 > DAA > Na- Oleate > Xanthate
r
O
30 ppm
A
100 ppm
O
150 ppm
8C z
40
0
Fig.5a
O
24 36 INTERACTION TIME (h)
12
48
Biodegradation of isopropyl xanthate flotation collector reagent in the presence of Bacillus polymyxa .
.
.
.
O 30 ppm Zl 100 ppm 0 150 ppm E z
Q
i
12
Fig.5b
24
36 48 INTERACTION TIME (h)
GO
72
Biodegradation of sodium oleate flotation collector reagent in the presence of Bacillus polymyxa.
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N. Deo and K. A. Natarajan
•
30 ppm
I'1 100 ppm
0 150 ppm 12q
! E
0
Fig.5c
-
4 8 INTERACTION TIME (h)
12
Biodegradation of DA-16 flotation collector reagent in the presence of Bacillus polymyxa.
12] 30 pprn A 100 ppm 0
120
150 ppm
2
Z
Q
~ 8c U
40
2
Fig.5d
4
6 8 10 INTERACTION TIME (h)
12
Biodegradation of DAA flotation collector reagent in the presence of Bacillus polymyxa.
Biodegradation of the collector reagents was further evaluated under other experimental conditions as well such as
a) b) c)
in the presence of harvested cells alone (101° cells/ml) Cell-free metabolites and using a fully-grown culture containing bacterial cells (101° cells/ml), medium and metabolites.
Biological removalof flotation collectorreagents
727
Typical results under the above three conditions are illustrated in Table 3. 150 ppm of DAA and DA-16 at an initial solution pH of 9 could be completely removed within about 2 hours in the presence of a fully grown culture, whereas it took between 6-8 hours for their complete degradation in the presence of cells alone. Bacterial metabolites alone could remove only about 45% of both the amines in 12 hours. In the case of sodium oleate, 75-80% could be removed in 12 hours in the presence of either cells alone or fully-grown bacterial culture. Bacterial metabolites alone, on the otherhand, could remove only about 30% of the oleate in 12 hours. Removal of isopropyl xanthate was more efficient in the presence of both fully-grown culture and the metabolites than with cells alone. For example, the acidic metabolites (pH 3-4) could degrade 150 ppm of the xanthate in about 10 hours where as it took only 5 hours for such removal with the bacterial culture. It is well established that xanthate collectors are unstable in acidic solutions.
TABLE 3 Biodegradation of different collector reagents under various conditions of treatment
Percent removal after 12 hours Collector reagents Cells alone (10 l° cells/ml) DAA DA-16 Sodium oleate Isopropyl xanthate
100% (in 6h) 100% (in 8h) 75% 40%
10 'U cells/ml with medium and metabolite (fully grown culture) 100% (in 2 h) 100% (in 2 h) 80%* 100% (in 5h) complete oleate removal.
Metabolite alone 45% 45% 30% 100% (in 10 h)
Thus the direct role of bacterial cells as well as the indirect role of their metabolites in the biodegradation of the collector reagents could be seen. Moreover, cationic collectors such as amines also exhibit enhanced bioadsorption onto cell surfaces through electrostatic and chemical forces. The surface-adsorbed amines were also subsequently utilised by the bacteria for their metabolism. Under the circumstances, higher rates of biodegradation and removal of amines could be expected. In fact, it has been observed that the cell-adsorbed amines on continuous growth of the cells in a medium were degraded as attested to by surface analysis by FTIR spectroscopy.
Biological stripping of adsorbed collectors from mineral surfaces Another practical application of the results of this study is biological stripping of adsorbed collector reagents from mineral surfaces. Presence of adsorbed collector reagents in the mineral concentrate poses problems in subsequent processing, such as pelletisation in case of iron ores. Surface stripping of hydrophobic reagents is also essential if the concentrate need be further beneficiated through flotation or flocculation using a different reagent. With this in mind, the utility of Bacillus polymyxa in the removal of adsorbed collectors from mineral surfaces was also examined. Three typical examples are illustrated below, so as to demonstrate the potential of the bioprocess.
a) b) c)
Bioremoval of adsorbed DA- 16 from hematite Bioremoval of adsorbed oleate from calcite and Bioremoval of adsorbed xanthate from pyrite.
In these tests, the collectors were initially adsorbed on the respective mineral surfaces and then subjected to bacterial treatment using cells of Bacillus polymyxa for different periods of time. The mineral surfaces after collector adsorption and subsequent biotreatment were examined through FTIR spectroscopy. Typical FTIR spectra for the above systems are illustrated in Figures 6 to 8.
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N. Deo and K. A. Natarajan
58
1II
30 4000
I
3OOO
I
I
2000
I
1000
WAVE N U M B E R ( cm - 1 )
54
B
t~J t.> Z #..-
Z IZ p-
34 I
4000
I
3000
I
I
I
2000 WAVENUMBER
I
1000
( cm -1 )
Fig.6 F T I R spectra o f a) D A - 16 alone; b) H e m a t i t e interacted with D A - 1 6 .
Biological removalof flotation collector reagents
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c
LU Z I.I,-
X z
nrI-.-
I
461 400O
I
I
3000
~
2000
I
1000
WAVENUMBER (cm °1)
150 D
LU (3 Z
I-I--
Z Ln z
i--
70 l
4( ) 0 0
I
3000
l
I
2000 WAVENUMBER
Fig.6
i
I
1000
( cm -1)
PTIR spectra of c) Amine adsorbed hematite after treatment with cells of Bacillus polymyxa d) Hematite alone.
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N. Dee and K. A, Natarajan
Figure 6a represents the FTIR spectra of DA-16. The aminium ion, displays strong broad absorption in the 3300 cm -~ region due to NH-stretching vibrations. A broad band between 3000 to 2800 cm -~ appears due to asymmetrical and symmetrical stretching of NH3÷ group. The band at about 1578 cm -~ is due to asymmetrical and symmetrical bending of NH3÷. The band at about 1472 cm -~ is due to NH4÷ bending, whereas the band at about 1378 cm -~ is due to C - N stretching. The strong band at about 1200 cm-' is O II
attributed to C - C - C stretching vibrations. The medium broad absorption band between 900--660 cm -1 is due to NH wagging vibrations. The FTIR spectrum of hematite after interaction with DA-16 is shown in Figure 6b. The broad band at about 3300 cm -~ is due to NH stretching vibrations. Absorption peaks between 2800-3000 cm -~ due to asymmetrical and symmetrical stretching of NH3÷ groups and at about 1600 cm -~ due to those of NH3÷ bending could also be seen. The band at about 1478 cm -~ assignable to O rl
NH4÷ bending vibration and another one at about 1120 cm -~ due to C-C--C stretching also could be observed. The bands between 400-600 cm -~ are due to shifts from the original ones (660 cm -1) after interaction with the amine. When the amine-treated hematite was subjected to interaction with bacteria, the amine bands are seen to be removed completely (Figure 6c) with the resulting spectra becoming similar to that of fresh hematite (Figure 6d). Similar FTIR studies were also made with DAA-treated hematite and complete bioremoval of surfaceadsorbed amine was observed. Figures 7a, b and c represent the FTIR spectra of sodium oleate, calcite treated with sodium oleate and of oleate-interacted calcite after treatment with bacterial cells. Figure 7a represents the FTIR spectrum of sodium oleate. The bands between 2840-2950 cm -t are due to C-H stretching mode of the CH 2 group. The band at 1713 cm -l is due to C=O absorption group of aliphatic acids. The carboxylate anions have two strongly coupled C'-K) bands with bond strengths intermediate between C=O and C O . The bands at 1561 and 1440 em -~ are due to strong asymmetrical stretching and symmetrical stretching of the above groups. The band at around 722 cm -~ is attributed to C-H bending vibrations. Figure 7b represents the FTIR spectrum of calcite interacted with sodium oleate. From the Figure it is clear that after interaction with sodium oleate many new peaks have appeared and shifting of old peaks were also observed. The peaks between 2950-2860 cm -l are due to C - H stretching mode of the CH 2 group. The narrow strong peak due to CO32- ion at around 1430 cm -I got broadened due to chemical interaction of calcite with sodium oleate (combination of 1430 cm -~ band of calcite and 1560 cm -~ , 1447 and 1713 cm -~ bands of oleate). A broad strong band appeared centered around 1400 cm -~ . The typical calcite bands at around 887 cm -~ and 720 cm -~ were shifted from 887 cm -~ to 870 cm -1 and from 720 cm -~ to 710 cm -~ mainly due to chemical interaction with sodium oleate. The FTIR spectra of sodium oleate interacted calcite after treatment with bacterial cells is shown in Figure 7c. From the Figure it is clear that after interaction with cells, the peaks due to oleate disappeared from the mineral surface and several new peaks emerged due to subsequent interaction with cells. This indicates that sodium oleate can be degraded or can be utilized by the bacterial cells during interaction. The bacterial signatures left behind on the mineral surface (Figure 7d) after removal of the adsorbed collector corresponds to OH and fatty acid groups formed due to bacterial metabolism. Figure 8a represents the FTIR spectrum of sodium isopropyl xanthate. The band at around 3300 c m - l is due to the presence of CH group. The bands between 3000-2800cm -1 are due to the presence of aliphatic hydrocarbons (C-H stretching). The band at around 2110 cm -~ is due to unsaturation. The band at around
1612 cm -I is due to the presence of C=O group, HC-S--C--O-. The bands between 1370-1450 cm -~ are due to the presence of aliphatic hydrocarbon. The band at around 1271 cm -~ is due to C--O-C bond. The bands between 1300-700 cm -1 are attributed to ×anthate group. The bands between 1060 to 1010 cm -~ are due to C=S stretching. The bands between 600-400 cm -~ are due to the rocking vibrations of CH~ groups.
Biological removal of flotationcollector reagents
731
A
LLI Z I'I'--
I Z I-
0 4000
3000
2OO0
1000
50(3
W A V E N U M B E R ( c m- 1 )
B
A
.-ev
IJJ t.; Z pliD Z IZ I--
0
4OOO
3000
2000
1000
500
WAVENUMBER (cm- 1 ) Fig.7 FTIR spectra of a) Sodium oleate alone; b) Calcite interacted with sodium oleate.
732
N. Deo and K. A. Natarajan
80 IJJ (J Z I.I.--
~E z <(
Iz !'-
20 ,
t
4000
,
I
,
t
,
3000
I
l
i
I
2000
I
I
1000
50O
WAVENUMBER (cm-1)
70 D
J A
U.I U
z.<[ I-I--
~r
u~ z
10 I 4000
I
I 3000
I
I 2000
iV
I 1000
II 500
WAVENUMBER ( cm-1 ) Fig.7
FTIR spectra of c) Oleate adsorbed calcite after treatment with cells of Bacillus polymyxa; d) Calcite alone.
Biological removalof flotationcollectorreagents
733
100 A
IJJ o Z
3E
tf) Z .,~ tY I,-
O
-
4000
3000
2000
1000
500
WAVENUMBER (cm-1)
8O
Z
I,.-
m
5C ,
4000
,
I
I
3000
I
i
~
1
i
2000
I
~
I
1000
I
I
500
WAVENUMBER (cm-1 ) Fig.8 FTIR spectra of a) Isopropyl xanthate alone; b) Pyrite interacted with isopropyl xanthate.
734
N. Deo and K. A. Natarajan
100
v
LLI z I--
~E
u') z <( n.i--
60
4000
3000
2000 WAVENUMBER
60
1000
500
(cm -1 )
D
;z LLI (D Z I'-
VE
U') Z <{ rr" I'-
20 I
4000
I
,
I
,
,
I
3OOO WAVENUMBER
Fig.8
I
,I
2OOO
,
,
I
IOO0
,
I
5OO
( cm- 1 )
FTIR spectra of c) Xanthate adsorbed pyrite after treatment with cells of Bacillus polymyxa; d) Pyrite alone.
Biologicalremovalof flotationcollectorreagents
735
Figure 8b represents the F U R spectrum of pyrite, after interaction with xanthate. The bands between 2800-3000 cm -x are due to CH 2 groups. The bands at around 1384 cm -l are attributed to aliphatic group. The broad bands between 1300-800 cm -1 are due to the combination of xanthate and Fe-S band. From the Figure it is clear that xanthate is chemically interacted with pyrite. Figure 8c represents the F H R spectrum of xanthate-interacted pyrite after treatment with bacterial cells. From the Figure it is clear that after bacterial interaction, all the xanthate peaks disappeared, and the resulting spectra resembled those from fresh unreacted pyrite (Figure 8d). Probable m e c h a n i s m s o f biodegradation
From the above results, it is evident that Bacillus polymyxa efficiently utilise the various organic collector reagents for their metabolism and generate organic acids, proteins and polysaccharides in the process. The progress of the bioremoval of the various flotation collectors along with the generation of newer reaction products was monitored through UV-visible spectra. In the presence of the amines, bacterial interaction resulted in the generation of polysaccharides and organic acids (200-210 nm) as well as proteins and nucleic acids (260-280nm). In the presence of sodium oleate also, bacterial degradation resulted in the simultaneous generation of large quantities of polysaccharides and organic acids (210-220nm) with lower amounts of proteinaceous compounds. With isopropyl xanthate, the characteristic xanthate ion peaks (226 and 301nm) disappeared with time, with the generation of newer peaks corresponding to CS 2, carbohydrates and some fatty acids. Based on the above experimental evidences, the following possible mechanisms for the biodegradation of various collector reagents are proposed. O tl B-oxidation R--CH2--CH2---C--OH ............ ~ R--COOH + CH3COOH The acetate then directly enter into the TCA (Tricarboxylic acid) cycle and synthesise essential products such as carbohydrates, nucleic acids, peptidoglycan, DNA. RNA etc. [8]. With respect to biodegradation of sodium oleate, the following sequences may take place. Bacterial attack on the weak double bond region. CH 3 (CH2)TCH=CH(CH2)TCOO CH3(CH2)TCHO + CH 3 (CH2)7 COOH
$
CH 3 (CH2)7 COOH B-oxidation of the so formed acids takes place in the presence of bacteria leading to further degradation to lower aldehydes and fatty acids. Thus, the oleates are converted to carbohydrates, which on further bacterial action ultimately decomposed to CO 2 and H20. Bacteria are able to satisfy their complete nitrogen requirements by utilising ammonia and amines [9]. Infact, both carbon and nitrogen requirements can be satisfied by utilisation of amines. Different reaction sequences involving decarboxylation, deamination, hydrolysis and oxidation are brought to play depending on the environmental conditions. However, the first essential step is always degradation of an amino acid and others being typical metabolic reactions of the products formed from the amino acid. Probable mechanisms in the biodegradation of the two types of amines used in this study involve the following steps.
736
N. Deo and K. A. Natarajan CH3 - (CH2)9-.~H2-CH3
CH3-(CH2)9-CH2+NH2-CH2-CH2-NH2+H202
/
O
0
I I
I
H20-t-O2 Bacteria
CH 2
CH2
I
CH2-NH
CttO
I I CI-I2 I CI-I2 I
(aldehyde)
Ctt2
NH2 Further oxidation NI-I2-CH2-CH2-NH2. . . . . . . . . . . . . . . . . . . _.~ NH2-CH2-CHO+NH3+H202 H20+O2, bacteria Further oxidation NH2-CH2--CHO. . . . . . . . . . . . . . . . . . __~ CH3--COOH + NH 3 + H202 H20+O2, bacteria NH 3 can be utilised by the bacteria as the nitrogen source and CH3--COOH can enter directly into the TCA cycle and can substitute as carbon source. Similarly, biodegradation of the DAA involves the following stages: O
II
H20
CH3-(CH2)II-NH-C-O"
CH3-(CH2)II-NH2 + HCOOH + H202 bacteria
H20 Bacteria
CH3-(CH2)10-CHO+NH3+H2CO3+H202 I ~. CO2+H20
oxidation
CH3-(CH2)Io-CHO
~ CH3-(CH2)lo-COOH ~
~-oxidation
R-COOH+CH3COOH The acetic acid enters directly into the central metabolic pathway.
Biologicalremovalof flotationcollectorreagents
737
There is evidence that lead to the conclusion that an aldehyde is formed when amines are oxidised. A precipitate was formed when the reaction mixture (from which protein was removed by precipitation with trichloroacetic acid) was poured into a cold saturated solution of 2:4 dinitrophenyl hydrazine in 2N HC1 and allowed to stand on ice for an hour. Most amine oxidases studies have shown to form H202 as a product [10]. It was suspected that H202 was being formed, but however, destroyed by endogenous catalase. catalase H20 2 ......... ~ H20 + 1/2 02 The role of the enzyme in microbial metabolism is not clear, but it does serve to detoxify the amine. In case of solutions containing isopropyl xanthate, the roles of both bacterial metabolism and acidic metabolites need be considered to explain its degradation and decomposition. The decomposition behaviour of xanthates in solution at different pH levels has been the subject of numerous investigations [3,11-14]. Xanthates are unstable in acidic solutions. The production of xanthic acid and the subsequent decomposition to alcohol and CS 2 at low pH regions have been established [12-14]. The acid metabolites generated by Bacillus polymyxa can thus lead to rapid decomposition of the xanthate in solution. UV-visible spectra of the xanthate solution in the presence of bacterial metabolite indicated the gradual reduction and disappearance of the xanthate peaks and emergence of CS 2 peak. Moreover, the bacteria were seen to utilize the xanthate reagent for their metabolism as well. From the UV-visible spectra taken during bacterial utilisation of xanthate solution, it became evident that carbohydrates and fatty acids were generated. Similar to carbon and nitrogen, sulphur is also an important element in bacterial metabolism. As pan of the carbon cycle, microbes degrade organic sulphur compounds [15-16]. Bacillus sp. are known to derive energy from the oxidation of H2S and organic sulphur [17]. Possible mechanisms in the biodegradation of isopropyl xanthate involve the following stages. CH3
S
I
II
Bacteria H2S + CH3 CO CH3 + CO2
CH3 - C - O - C - S"
Bacteria CH3 CO COOH
CH3 CHO + CO2 Bacteria
Bacteria CH3 COOH The CH 3 COOH can directly enter the TCA cycle and satisfy the carbon requirements. H2S formed can be further biooxidised to sulphur and sulphate [ 18].
CONCLUSIONS The following major conclusions could be made based on this study. 1.
Bacillus polymyxa, a heterotrophic soil bacterium indigenously associated with several ore deposits was found to be capable of biodegradation of several organic flotation collectors such as dodecyl and di-amine, isopropyl xanthate and sodium oleate.
2.
The bacteria could utilize the above organic reagents for their carbon and nitrogen requirements
738
N. Deo and K. A. Natarajan
and grow very well, generating polysaccharides, proteins and fatty acids during metabolism. .
The bacteria can also be utilized to strip adsorbed collector reagents from mineral surfaces.
4.
Biodegradation mechanisms include both direct bacterial metabolism as well as contributions from the acidic metabolites.
.
A biological route for detoxification of mineral processing effluents containing residual organic reagents is demonstrated.
REFERENCES .
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Phalguni, A., Modak, J.M. & Natarajan, K.A., Biobeneficiation of bauxite using Bacillus polymyxa; Calcium and iron removal, Int. J. Miner. Process; 48, 51--60 (1996). Siggia, S., Quantitative Organic Analysis via Functional Groups, John Wiley, NY, 20-26 (1949). Leja, J., Surface Chemistry of Froth Flotation, Plenum Press, N.Y. 228 -258 (1983) Snell, F.D. & Snell, C.T., Colorimetric Methods of Analysis, D. Van Nostrand Co. N.Y. (1954). Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantifies of protein utilizing the principle of protein-dye binding; Anal, Biochem., 72, 248-254 (1976). Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. & Smith, F., Colorimetric method for the determination of sugars and related substances, Ana. Chem., 28, 350-356 (1956). Taggart, A.F., Hand Book of Mineral Dressing, John Wiley, N.Y. (1967). Prave, P., Faust, U., Sittig, W. & Sukatsch, D.A., Fundamentals of Biotechnology, VCH, Weinheim, 67-132 (1987). Gibson, D.T., Microbial Degradation of Organic Compounds, Marcel Dekker Inc., N.Y., 43-129 (1984). Yamada, H., Adachi, O. & Ogata, K., Amine Oxidases of microorganisms, Biol. Chem., 29, 117-123 (1965). Iwasaki, I. & Cooke, S.R.B., Absorption spectra of some sulfyhydral compounds, Trans. SME (AMIE), 208, 1267-1268 (1957). Iwasaki, I. & Cooke, S.R.B., Dissociation constant of xanthic acid as determined by spectrophotometric method, J. Phys. Chem., 63, 1321-1322 (1959). Pomianowski, A. & Leja, J., Spectrophotometric study of xanthate and dixanthogen solutions, Can. J. Chemistry, 41, 2219-2228 (1963). Jones, M.H. & Woodcock, J.T., Decomposition, of alkyl dixanthogens in aqueous solutions, Int. J. Miner. Process, 10, 1-24 (1983). Ehrlich, H.L., Geomicrobiology, Second Edition, Marcel Dekker, Inc., N,Y. (1990). Freney, J.R., Sulphur-containing organics, in McLaren, A.D. and Petersen, G.H. (Eds.), Soil Biochemistry, Marcel Dekker, Inc., N.Y. 229-259 (1967). Strohl, W.R. & Larkin, J.M., Enumeration, isolation and characterization of Beggiatoa from fresh water sediments, App. Environ. Microbiol., 36, 755-770 (1978). Pringsheim, E.G., Die mixotrophie von Beggiatoa, Arch. Mikrobiol, 59, 247-254 (1967).
C o r r e s p o n d e n c e on papers published in Minerals Engineering is invited, preferably by email to [email protected], or b y Fax to + 4 4 - ( 0 ) 1 3 2 6 - 3 1 8 3 5 2
Pergamon 0892-6875(98)00058-2
BIOLOGICAL REMOVAL FROM AQUEOUS
© 1998 Elsevier Science Lid All fights reserved 0892-6875/98/$ - see front matter
OF SOME FLOTATION COLLECTOR REAGENTS SOLUTIONS AND MINERAL SURFACES
N. DEO and K.A. NATARAJAN Department of Metallurgy, Indian Institute of Science, Bangalore - 560 012, India Email: kan @metalrg.iisc.ernet.in
(Received 31 January 1998; accepted 20 May 1998)
ABSTRACT
The utility of BaciUus polymyxa, a heterotrophic bacterium indigenous to ore deposits in the degradation of several flotation collectors has been demonstrated. Dodecyl amine, diamine, isopropyl xanthate and sodium oleate could be efficiently removed from alkaline solutions through bioremediation in an environmentally acceptable fashion. The bacterial cells were also found to be capable of stripping adsorbed collector reagents from various mineral surfaces. Mechanisms of such a biodegradation process are illustrated through results obtained from zeta potential, adsorption, bacterial growth and surface analysis studies. © 1998 Elsevier Science Ltd. All rights reserved. Keywords Flotation collectors; bacteria; biotechnology; environmental; mineral processing
INTRODUC~ON Froth flotation is extensively used the world over for the beneficiation of sulfide and oxide ores. Alkyl xanthates are used as collector reagents in sulfide ore flotation, while amines and fatty acids find application in iron ore flotation and the beneficiation of calcite, fluorite and quartzite. Although these collector reagents react preferentially with the concerned minerals in the treated ore pulp, excess and unreacted concentrations of these organic surfactants end up in the mill process effluents. Actual concentrations of various organic flotation collectors remaining in the effluent and process waters of mineral process operations are not readily available. It has been known that even smaller concentrations of these reagents in water streams are toxic to water life, besides their deleterious influence on end stream processes during recycling. Bioremediation has long been recognized as a cheap, flexible and environmentally benign technique in waste water treatment. Organic flotation reagents are also amenable to biological degradation. However, no systematic study on the potential of biological processes to detoxify flotation plant effluents has so far been reported. In this paper, the utility of a biological process for the efficient removal of some typical organic flotation collectors from aqueous solutions is illustrated. Two types of amines, namely, Deodecylammonium acetate (a primary amine) and a diamine, DA-16 which are extensively used in iron ore flotation were taken as examples. Further, sodium isopropyl xanthate, a universal sulfide collector reagent and sodium oleate a fatty
717
718
N. Deo and K. A. Natarajan
acid collector also were chosen. Also, the utility of a biological process to strip residual adsorbed collector reagents from mineral surfaces is demonstrated. A soil bacterium, namely, Bacillus polymyxa, which occurs indigenously, associated with several ore deposits was used in this work. M A T E R I A L S AND M E T H O D S
Bacterial strain and growth conditions A pure strain of Bacillus polymyxa NCIM 2539 obtained from national collection of industrial microoranisms, National Chemical Laboratory, Pune, India. was used in all the studies. A 10% v/v of an active inoculum was added to Bromfield medium [1] and incubated at 30°C on a rotary shaker at 240 rpm. The bacterial growth was studied by microscopic counting using a Petroff-Hausser counter under a phase contrast microscope and also by colony counting after plating, pH changes during bacterial growth was monitored and enough cell mass (>109 cells/ml) was generated by continuous growth for 8 hours. The culture was filtered thorough whatman No. 1 filter paper to remove precipitates and centrifuged at 15000 rpm for 15 minutes. The cell pellet was washed several times and resuspended in deionised double distilled water (pH 7 and specific conductivity less than 1.5 la mhos/cm).
Biodegradation of flotation collector reagents Biodegradation of sodium oleate, sodium isopropyl xanthate, isododecyl oxypropyl aminoprypyl amine (DA16) and dodecyl ammonium acetate (DAA) was studied under different conditions and concentrations. All the above collector reagents were of analytical grade and prepared in double distilled deionised water at the desired pH. The various conditions for biodegradation studies were: (a) (b) (c) (d)
During bacterial growth, In presence of metabolite alone In presence of cells only, and In presence of fully-grown culture (cells + metabolite + medium)
(a)
To 100 ml of each previously autoclaved Bromfield medium (without sucrose), different concentrations (50, 100, 150 ppm) of xanthate, oleate, DA-16 and DAA were added and the pH was adjusted to 9.0 and 10% of active inoculum was added to each flask. Two of each, uninoculated control flasks were maintained separately.
(b)
To 100 ml of each cell-free culture filtrate (the culture which reached stationary phase), different concentrations (50, 100, 150 ppm) of xanthate, oleate, DA-16 and DAA were added.
(c)
To 100 ml of each cell suspension (6 x 109 cells/ml in 10-3 M KNO 3, solution) different concentrations (50, 100, 150 ppm) of xanthate, oleate, DA-16 and DAA were added and the pH was adjusted to 9.0. Two of each cell free control flasks were also maintained separately.
(d)
To 100 ml of each active culture (the culture at mid-exponential phase) different concentrations (50, 100, 150 ppm) of xanthate, oleate, DA-16 and DAA were added.
All flasks were incubated at 30"C on a rotary shaker at 240 rpm for different periods of time. After each interval of time, the pH was checked and the residual concentrations of oleate [2], xanthate [3] DA16, and DAA [4] were measured in the cell-free supernatant by acid-titration and by U.V-visible spectrophotometry. The stages during biodegradation of the various flotation reagents, namely, oleate, xanthate, DA-16, and DAAwere also monitored through UV-visible and FTIR spectra. For this purpose, an initial blank spectrum for the different reagents was obtained. The progress of degradation and disappearance from the solutions of the various collector reagents in the presence of cells, metabolite, and cells + metabolites, under the four
Biologicalremovalof flotationcollectorreagents
719
different conditions mentioned above was then monitored through the disappearance, shifting of the initial absorption peaks and also the appearance of new peaks. Growth of Bacillus polymyxa in presence of oleate, xanthate, DA-16 and DAA To 100 ml of each previously autoclaved inorganic medium (without sucrose in 500 ml Erlenmeyer flasks) 100 ppm of xanthate, oleate, DA-16 and DAA were added. Two of each control flasks without the above collector reagents were also taken. The pH of all the flasks was maintained at 9.0. To all these above flasks, 10% of the fully grown culture along with the cells were added. After inoculation, the flasks were incubated on a rotary shaker at 240 rpm at 30°C. The bacterial growth was monitored by microscopic counting using a Petroff-Hausser counter and pH changes during growth were also measured at regular intervals of time. During growth, the protein and polysaccharides produced by the bacteria were also measured frequently from the cell-free supernatant by Bradford method [5] and phenol sulphuric acid method [6] after dialysis. Adsorption of collector reagents on bacterial cells Adsorption of collector reagents onto cell surfaces was estimated at different collector concentrations. A 10.3 M KNO 3 solution was used as the base electrolyte for the purpose. 0.5 g of each wet biomass were interacted with different concentrations (50, 100, 150, 200, 300, 400 ppm) of xanthate, oleate, DA-16, and DAA at pH 9.0 for different intervals of time. After each interval of time, the residual concentrations of collectors were estimated through analysis of the supernatant solution after centrifugation. Electrokinetic studies on bacterial cells after interaction with collector reagents The Zeta potentials of bacterial cells as a function of pH were determined using Zeta meter-3.0. Cell suspensions (109 cells/ml) were interacted with different concentrations of oleate, xanthate, DA-16 and DAA at pH 9.0 for different periods of time. After such interaction, the cell suspension was centrifuged at 15000 rpm for 15 minutes. The cell pellet was washed several times with 10.3 M KNO 3 and then resuspended in 10.3 M KNO 3 solution and adjusted to the required pH for electrokinetic measurements. In this manner, the surface chemical changes on bacterial cell surfaces before and after interaction with various organic reagents could be established as also the probable mechanisms of interaction. Flotation of bacteria after interaction with collectors 4 g of wet biomass was interacted with 1000 ppm of various collector reagents for 5 minutes at pH 9.0. After 5 minutes interaction, the cell suspension was centrifuged and the cell pellets were washed several times with 10.3 M KNO3. Then the cells were suspended in 10.3 M KNO3 solution and the flotation was carried out for five minutes under a nitrogen flow rate of 40 ml/minute using a Leaf and Knoll cell [7]. Biological stripping of adsorbed collectors from mineral surfaces The respective mineral samples, namely, hematite, calcite and pyrite were initially reacted with amine, oleate and xanthate collectors, respectively and subsequently subjected to biotreatment with cells of Bacillus polymyxa. The removal of adsorbed organic reagents from the mineral surfaces was monitored through FTIR spectroscopy.
RESULTS AND DISCUSSION Zeta potential and adsorption studies Surface affinity of the various flotation collectors towards bacterial ceils was assessed initially throgh electrokinetic and adsorption studies. Both the amine reagents, namely, Dodecylammonium acetate (DAA) and Isododecyl oxypropyl aminopropyl amine (DA-16) exhibited enhanced surface adsorption onto cells of
720
N. Deo and K. A. Natarajan
Bacillus polymyxa as shown in Figure 1. For example, the isoelectric point (IEP) of bacterial cells shifted from the initial value at about pH 2 to about pH 2.8 and 3.3, respectively, after one hour interaction with 50 ppm of DAA and DA-16. Similarly, after similar interaction with 100 ppm concentrations of the above amines, the IEP of the cells shifted to pH 3.0 and 4.3, respectively (not shown in the Figure). The IEP shifts were thus found to be a function of amine concentration and indicate chemical interaction between the amine and cell surface components. Also, electrostatic attraction between negatively charged cell surfaces and cationic aminium ions would facilitate biosorption. On the otherhand, interaction with 50-100 ppm of the anionic collectors such as sodium isopropyl xanthate and sodium oleate did not result in any significant surface chemical changes on the bacterial cells as attested by measured zeta potentials. Electrostatic repulsion between negatively charged bacterial cells and anionic collector reagents retard mutual interaction. 20 50 ppm for
1 hr each
0 A
Cells before ht~octlon Atte¢ kltero~tion with
• I"1 •
[
I
I
i
,,
.
')
"
,,
I
DAA IPX Na- Oote
,t
I
DA-16
1
L
I
vE z
~-20
-40
-60
Fig. 1
Zeta potentials as a function of pH for cells of Bacillus polymyxa before and after interaction with various collector reagents.
Adsorption behaviour of various collector reagents onto bacterial cell surfaces was then established through adsorption isotherms at a solution pH of 9.00 as illustrated in Figure 2. Enhanced affinity of the two amine collectors towards bacterial cell surfaces could be readily seen in comparison with the two anionic collectors. For example, the maximum adsorption density for DAA and DA-16 corresponded to about 45 and 36 mg/g, respectively, compared to only about 2.5 mg/g for xanthate and oleate. The isotherms follow a Langmuir model which describes the sorption reaction as m--
MbC l+ bC
or by the linearised form,
C
1
C
m
bM
M
where
m = mass of solute sorbed per unit weight of sorbent, M = mass of solute sorbed per unit weight of sorbent at saturation b = constant accounting for energy of interaction C = measured equilibrium concentration
Biological removal of flotation collector reagents
721
From a plot of C/m vs C, the Langmuir model constants were derived as described in Table 1.
60
~40
"1o .Q
O~
~d •
2o E <~
I
"~ 40
0
J
A
Sodium oleate Xanthate
• O
OAA DA-16
A
I & i i -r I 80 120 150 Residual concentration , mg /L
~-T 200
I 240
Fig.2 Adsorption isotherms for various collector reagents.
TABLE 1 Langmuir adsorption model constants obtained from isotherms Organic reagent DAA D A - 16 Xanthate Oleate
M, mg/g 50.70 38.50 2.90 2.90
b 0.20 0.17 0.12 0.04
Another significant observation was that such amine adsorption rendered the bacterial cell surfaces more hydrophobic as attested to by increased flotability of amine-interacted bacterial cells. For example, 96% and 82% of the cells after 5 minute interaction with DA-16 and DAA could be readily floated and recovered from the aqueous solution at pH 9.0. No such flotation response was observed with xanthate and oleate treated cells.
Bacterial growth in the presence of collector reagents Although only the two amine collectors among the four reagents tested in this work exhibited significant biosorption tendency, all the organic surfactants however promoted bacterial growth in the solution as illustrated in Figures 3a and b. In the absence of any added carbon source such as sucrose, Bacillus polymyxa could grow effectively utilising the carbon present in the amines, sodium oleate and isopropyl xanthate and the nitrogen present in the amines. In such bacterial growth studies, the Bromfiled medium contained all other constituents except sucrose, which is the essential cabon source for normal growth of Bacillus polymyxa in the absence of other organic reagents. Substantial increase in cell population and pH decrease due to organic acid production by bacterial metabolism could be observed. The cell data have been plotted using the Logistic equation, thus enabling direct comparison of rate constants.
o dt
where
-" ~lmax
- Xma x
1
X = cell density (cells/ml)
722
N. Deo and K. A. Natarajan
Xm= = maximum cell density lam~ = growth rate (h -1) X(t~o) = X o
x ] i xo t
Xm~x- X
LXm~x - X o Um"xt
Assuming various values of Xm~ from the experimental data, l a ~ could be estimated from the slope of the linear plot of
lfXm:X] vst The various values of Xm~ and ~tm~x thUS estimated are given in Table 2. From lamx, from Xm~,
DA-16 > oleate > xanthate > DAA oleate > DA-16 > DAA = xanthate
10.0
1011 Oleate
E
1010
8.0
ro
Xanthate
c "1o
7.0 "16 . 0 °"
10 9 Xanthate
E tO
9.0
5.0
lo8 j
Oleate
4.0
E3 0
3.0 107 ~ 0
10
20
30
40
2.0 50
Time (hr) Fig.3a
Logistic curves for growth of Bacillus polymyxa in the Bromfield medium (no sucrose) in the presence of collector reagents--Isopropyl xanthate and sodium oleate.
Biological removalof flotation collectorreagents
723
T A B L E 2 M a x i m u m cell number and growth rate constants Organic reagent Oleate Xanthate D A - 16 DAA
Xm~x(cells/ml) 6.7 x 10'" 6.2 x 10~ 2.65 x 10" 6.4 x 10~
It=,= (h-') 0.2609 0.2188 0.2643 0.1783
~ I:,,.
]1o.o
~/
'°'°F\~
19.o
.--/
[
i
t 'o
I//
° 1°1
,0,ISo 0
10
t,.0
20
30
40
50
Time (hr)
Fig.3b
Logistic curves for growth of Bacillus polymyxa in the Bromfield medium (no sucrose) in the presence of collector reagents--DA-16 and DAA.
Amounts of proteins and polysaccharides generated due to bacterial utilization of the various flotation collectors in a sucrose-free Bromfield medium are illustrated in Figure 4 a and b. It becomes readily apparent that the bacteria could effectively utilize all the organic surfactants and generate polysaccharides '
'
i=
-i
Imthc~'~u
Fig.4a
-~o
TW,*E(h)
Protein and polysaccharide generation by Bacillus polymyxa in the presence of Isopropyl xanthate and sodium oleate.
724
N. Deo and K. A. Natarajan
240
DAA DA16
• •
0 []
21:10
E z
o
E 120 u
8O
4Ol-
I
ot~-mr " L
//
--14o
i 12
I
1
24
36
I Io
48
INTERACTION TiME 3 h
Fig.4b
Protein and polysaccharide generation by Bacillus polymyxa in the presence of DA-16 and DAA.
and proteins as the major metabolic products besides organic acids. Protein production (amino acids) was found to be the highest in the presence of amines, while that of polysaccharides in the presence of xanthate and oleate. This could be expected since the amines containing nitrogen and amino groups are degraded by the cells to generate amino acid containing proteins. It has also been subsequently observed that the amino acids, polysaccharides and fatty acids are further utilised by the bacteria for metabolism and growth leading to complete dissociation of all the collector reagents. The above observation was substantiated by continuous analysis of the solution with time over a period of 4 days.
Biodegradation of collector reagents in aqueous solutions The above results indicate that the bacterium, Bacillus polymyxa can be effectively used to degrade and remove various organic collector reagents from aqueous solutions. Biodegradation of the various collector reagents as a function of time at three initial concentrations under conditions of growth of Bacillus polymyxa was then studied and the results are presented in Figures 5a,b,c and d. In these tests, the initial cell concentrations was low at about 106 cells/ml which gradually increased with time upto 101° cells/ml after full growth in the presence of the collector reagents. Under such conditions, the initial rate of biodegradation was slower, especially at higher concentrations of the collector reagents. As the biomass increased, the rate of removal of the collector reagents also increased as a function of time. Also, the pH decreased from an initial value of 9 to about 5 during the period due to organic acid generated by bacterial metabolism. The following observations are note worthy:
a)
The overall rate of biodegradation of the various collector reagents was found to be influenced by their initial concentrations.
Biological removal of flotation collector reagents
b) c) d)
725
There was an initial lag period for the biodegradation at higher collector concentrations. The amines are biodegraded at a faster rate than oleate and xanthate. With respect to the rate of biodegradation for a given concentration and duration (for example, 30 ppm and 4 hours) the following order holds good. DA16 > DAA > Na- Oleate > Xanthate
r
O
30 ppm
A
100 ppm
O
150 ppm
8C z
40
0
Fig.5a
O
24 36 INTERACTION TIME (h)
12
48
Biodegradation of isopropyl xanthate flotation collector reagent in the presence of Bacillus polymyxa .
.
.
.
O 30 ppm Zl 100 ppm 0 150 ppm E z
Q
i
12
Fig.5b
24
36 48 INTERACTION TIME (h)
GO
72
Biodegradation of sodium oleate flotation collector reagent in the presence of Bacillus polymyxa.
726
N. Deo and K. A. Natarajan
•
30 ppm
I'1 100 ppm
0 150 ppm 12q
! E
0
Fig.5c
-
4 8 INTERACTION TIME (h)
12
Biodegradation of DA-16 flotation collector reagent in the presence of Bacillus polymyxa.
12] 30 pprn A 100 ppm 0
120
150 ppm
2
Z
Q
~ 8c U
40
2
Fig.5d
4
6 8 10 INTERACTION TIME (h)
12
Biodegradation of DAA flotation collector reagent in the presence of Bacillus polymyxa.
Biodegradation of the collector reagents was further evaluated under other experimental conditions as well such as
a) b) c)
in the presence of harvested cells alone (101° cells/ml) Cell-free metabolites and using a fully-grown culture containing bacterial cells (101° cells/ml), medium and metabolites.
Biological removalof flotation collectorreagents
727
Typical results under the above three conditions are illustrated in Table 3. 150 ppm of DAA and DA-16 at an initial solution pH of 9 could be completely removed within about 2 hours in the presence of a fully grown culture, whereas it took between 6-8 hours for their complete degradation in the presence of cells alone. Bacterial metabolites alone could remove only about 45% of both the amines in 12 hours. In the case of sodium oleate, 75-80% could be removed in 12 hours in the presence of either cells alone or fully-grown bacterial culture. Bacterial metabolites alone, on the otherhand, could remove only about 30% of the oleate in 12 hours. Removal of isopropyl xanthate was more efficient in the presence of both fully-grown culture and the metabolites than with cells alone. For example, the acidic metabolites (pH 3-4) could degrade 150 ppm of the xanthate in about 10 hours where as it took only 5 hours for such removal with the bacterial culture. It is well established that xanthate collectors are unstable in acidic solutions.
TABLE 3 Biodegradation of different collector reagents under various conditions of treatment
Percent removal after 12 hours Collector reagents Cells alone (10 l° cells/ml) DAA DA-16 Sodium oleate Isopropyl xanthate
100% (in 6h) 100% (in 8h) 75% 40%
10 'U cells/ml with medium and metabolite (fully grown culture) 100% (in 2 h) 100% (in 2 h) 80%* 100% (in 5h) complete oleate removal.
Metabolite alone 45% 45% 30% 100% (in 10 h)
Thus the direct role of bacterial cells as well as the indirect role of their metabolites in the biodegradation of the collector reagents could be seen. Moreover, cationic collectors such as amines also exhibit enhanced bioadsorption onto cell surfaces through electrostatic and chemical forces. The surface-adsorbed amines were also subsequently utilised by the bacteria for their metabolism. Under the circumstances, higher rates of biodegradation and removal of amines could be expected. In fact, it has been observed that the cell-adsorbed amines on continuous growth of the cells in a medium were degraded as attested to by surface analysis by FTIR spectroscopy.
Biological stripping of adsorbed collectors from mineral surfaces Another practical application of the results of this study is biological stripping of adsorbed collector reagents from mineral surfaces. Presence of adsorbed collector reagents in the mineral concentrate poses problems in subsequent processing, such as pelletisation in case of iron ores. Surface stripping of hydrophobic reagents is also essential if the concentrate need be further beneficiated through flotation or flocculation using a different reagent. With this in mind, the utility of Bacillus polymyxa in the removal of adsorbed collectors from mineral surfaces was also examined. Three typical examples are illustrated below, so as to demonstrate the potential of the bioprocess.
a) b) c)
Bioremoval of adsorbed DA- 16 from hematite Bioremoval of adsorbed oleate from calcite and Bioremoval of adsorbed xanthate from pyrite.
In these tests, the collectors were initially adsorbed on the respective mineral surfaces and then subjected to bacterial treatment using cells of Bacillus polymyxa for different periods of time. The mineral surfaces after collector adsorption and subsequent biotreatment were examined through FTIR spectroscopy. Typical FTIR spectra for the above systems are illustrated in Figures 6 to 8.
728
N. Deo and K. A. Natarajan
58
1II
30 4000
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I
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t~J t.> Z #..-
Z IZ p-
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4000
I
3000
I
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I
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( cm -1 )
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Biological removalof flotation collector reagents
729
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( cm -1)
PTIR spectra of c) Amine adsorbed hematite after treatment with cells of Bacillus polymyxa d) Hematite alone.
730
N. Dee and K. A, Natarajan
Figure 6a represents the FTIR spectra of DA-16. The aminium ion, displays strong broad absorption in the 3300 cm -~ region due to NH-stretching vibrations. A broad band between 3000 to 2800 cm -~ appears due to asymmetrical and symmetrical stretching of NH3÷ group. The band at about 1578 cm -~ is due to asymmetrical and symmetrical bending of NH3÷. The band at about 1472 cm -~ is due to NH4÷ bending, whereas the band at about 1378 cm -~ is due to C - N stretching. The strong band at about 1200 cm-' is O II
attributed to C - C - C stretching vibrations. The medium broad absorption band between 900--660 cm -1 is due to NH wagging vibrations. The FTIR spectrum of hematite after interaction with DA-16 is shown in Figure 6b. The broad band at about 3300 cm -~ is due to NH stretching vibrations. Absorption peaks between 2800-3000 cm -~ due to asymmetrical and symmetrical stretching of NH3÷ groups and at about 1600 cm -~ due to those of NH3÷ bending could also be seen. The band at about 1478 cm -~ assignable to O rl
NH4÷ bending vibration and another one at about 1120 cm -~ due to C-C--C stretching also could be observed. The bands between 400-600 cm -~ are due to shifts from the original ones (660 cm -1) after interaction with the amine. When the amine-treated hematite was subjected to interaction with bacteria, the amine bands are seen to be removed completely (Figure 6c) with the resulting spectra becoming similar to that of fresh hematite (Figure 6d). Similar FTIR studies were also made with DAA-treated hematite and complete bioremoval of surfaceadsorbed amine was observed. Figures 7a, b and c represent the FTIR spectra of sodium oleate, calcite treated with sodium oleate and of oleate-interacted calcite after treatment with bacterial cells. Figure 7a represents the FTIR spectrum of sodium oleate. The bands between 2840-2950 cm -t are due to C-H stretching mode of the CH 2 group. The band at 1713 cm -l is due to C=O absorption group of aliphatic acids. The carboxylate anions have two strongly coupled C'-K) bands with bond strengths intermediate between C=O and C O . The bands at 1561 and 1440 em -~ are due to strong asymmetrical stretching and symmetrical stretching of the above groups. The band at around 722 cm -~ is attributed to C-H bending vibrations. Figure 7b represents the FTIR spectrum of calcite interacted with sodium oleate. From the Figure it is clear that after interaction with sodium oleate many new peaks have appeared and shifting of old peaks were also observed. The peaks between 2950-2860 cm -l are due to C - H stretching mode of the CH 2 group. The narrow strong peak due to CO32- ion at around 1430 cm -I got broadened due to chemical interaction of calcite with sodium oleate (combination of 1430 cm -~ band of calcite and 1560 cm -~ , 1447 and 1713 cm -~ bands of oleate). A broad strong band appeared centered around 1400 cm -~ . The typical calcite bands at around 887 cm -~ and 720 cm -~ were shifted from 887 cm -~ to 870 cm -1 and from 720 cm -~ to 710 cm -~ mainly due to chemical interaction with sodium oleate. The FTIR spectra of sodium oleate interacted calcite after treatment with bacterial cells is shown in Figure 7c. From the Figure it is clear that after interaction with cells, the peaks due to oleate disappeared from the mineral surface and several new peaks emerged due to subsequent interaction with cells. This indicates that sodium oleate can be degraded or can be utilized by the bacterial cells during interaction. The bacterial signatures left behind on the mineral surface (Figure 7d) after removal of the adsorbed collector corresponds to OH and fatty acid groups formed due to bacterial metabolism. Figure 8a represents the FTIR spectrum of sodium isopropyl xanthate. The band at around 3300 c m - l is due to the presence of CH group. The bands between 3000-2800cm -1 are due to the presence of aliphatic hydrocarbons (C-H stretching). The band at around 2110 cm -~ is due to unsaturation. The band at around
1612 cm -I is due to the presence of C=O group, HC-S--C--O-. The bands between 1370-1450 cm -~ are due to the presence of aliphatic hydrocarbon. The band at around 1271 cm -~ is due to C--O-C bond. The bands between 1300-700 cm -1 are attributed to ×anthate group. The bands between 1060 to 1010 cm -~ are due to C=S stretching. The bands between 600-400 cm -~ are due to the rocking vibrations of CH~ groups.
Biological removal of flotationcollector reagents
731
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732
N. Deo and K. A. Natarajan
80 IJJ (J Z I.I.--
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FTIR spectra of c) Oleate adsorbed calcite after treatment with cells of Bacillus polymyxa; d) Calcite alone.
Biological removalof flotationcollectorreagents
733
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734
N. Deo and K. A. Natarajan
100
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Biologicalremovalof flotationcollectorreagents
735
Figure 8b represents the F U R spectrum of pyrite, after interaction with xanthate. The bands between 2800-3000 cm -x are due to CH 2 groups. The bands at around 1384 cm -l are attributed to aliphatic group. The broad bands between 1300-800 cm -1 are due to the combination of xanthate and Fe-S band. From the Figure it is clear that xanthate is chemically interacted with pyrite. Figure 8c represents the F H R spectrum of xanthate-interacted pyrite after treatment with bacterial cells. From the Figure it is clear that after bacterial interaction, all the xanthate peaks disappeared, and the resulting spectra resembled those from fresh unreacted pyrite (Figure 8d). Probable m e c h a n i s m s o f biodegradation
From the above results, it is evident that Bacillus polymyxa efficiently utilise the various organic collector reagents for their metabolism and generate organic acids, proteins and polysaccharides in the process. The progress of the bioremoval of the various flotation collectors along with the generation of newer reaction products was monitored through UV-visible spectra. In the presence of the amines, bacterial interaction resulted in the generation of polysaccharides and organic acids (200-210 nm) as well as proteins and nucleic acids (260-280nm). In the presence of sodium oleate also, bacterial degradation resulted in the simultaneous generation of large quantities of polysaccharides and organic acids (210-220nm) with lower amounts of proteinaceous compounds. With isopropyl xanthate, the characteristic xanthate ion peaks (226 and 301nm) disappeared with time, with the generation of newer peaks corresponding to CS 2, carbohydrates and some fatty acids. Based on the above experimental evidences, the following possible mechanisms for the biodegradation of various collector reagents are proposed. O tl B-oxidation R--CH2--CH2---C--OH ............ ~ R--COOH + CH3COOH The acetate then directly enter into the TCA (Tricarboxylic acid) cycle and synthesise essential products such as carbohydrates, nucleic acids, peptidoglycan, DNA. RNA etc. [8]. With respect to biodegradation of sodium oleate, the following sequences may take place. Bacterial attack on the weak double bond region. CH 3 (CH2)TCH=CH(CH2)TCOO CH3(CH2)TCHO + CH 3 (CH2)7 COOH
$
CH 3 (CH2)7 COOH B-oxidation of the so formed acids takes place in the presence of bacteria leading to further degradation to lower aldehydes and fatty acids. Thus, the oleates are converted to carbohydrates, which on further bacterial action ultimately decomposed to CO 2 and H20. Bacteria are able to satisfy their complete nitrogen requirements by utilising ammonia and amines [9]. Infact, both carbon and nitrogen requirements can be satisfied by utilisation of amines. Different reaction sequences involving decarboxylation, deamination, hydrolysis and oxidation are brought to play depending on the environmental conditions. However, the first essential step is always degradation of an amino acid and others being typical metabolic reactions of the products formed from the amino acid. Probable mechanisms in the biodegradation of the two types of amines used in this study involve the following steps.
736
N. Deo and K. A. Natarajan CH3 - (CH2)9-.~H2-CH3
CH3-(CH2)9-CH2+NH2-CH2-CH2-NH2+H202
/
O
0
I I
I
H20-t-O2 Bacteria
CH 2
CH2
I
CH2-NH
CttO
I I CI-I2 I CI-I2 I
(aldehyde)
Ctt2
NH2 Further oxidation NI-I2-CH2-CH2-NH2. . . . . . . . . . . . . . . . . . . _.~ NH2-CH2-CHO+NH3+H202 H20+O2, bacteria Further oxidation NH2-CH2--CHO. . . . . . . . . . . . . . . . . . __~ CH3--COOH + NH 3 + H202 H20+O2, bacteria NH 3 can be utilised by the bacteria as the nitrogen source and CH3--COOH can enter directly into the TCA cycle and can substitute as carbon source. Similarly, biodegradation of the DAA involves the following stages: O
II
H20
CH3-(CH2)II-NH-C-O"
CH3-(CH2)II-NH2 + HCOOH + H202 bacteria
H20 Bacteria
CH3-(CH2)10-CHO+NH3+H2CO3+H202 I ~. CO2+H20
oxidation
CH3-(CH2)Io-CHO
~ CH3-(CH2)lo-COOH ~
~-oxidation
R-COOH+CH3COOH The acetic acid enters directly into the central metabolic pathway.
Biologicalremovalof flotationcollectorreagents
737
There is evidence that lead to the conclusion that an aldehyde is formed when amines are oxidised. A precipitate was formed when the reaction mixture (from which protein was removed by precipitation with trichloroacetic acid) was poured into a cold saturated solution of 2:4 dinitrophenyl hydrazine in 2N HC1 and allowed to stand on ice for an hour. Most amine oxidases studies have shown to form H202 as a product [10]. It was suspected that H202 was being formed, but however, destroyed by endogenous catalase. catalase H20 2 ......... ~ H20 + 1/2 02 The role of the enzyme in microbial metabolism is not clear, but it does serve to detoxify the amine. In case of solutions containing isopropyl xanthate, the roles of both bacterial metabolism and acidic metabolites need be considered to explain its degradation and decomposition. The decomposition behaviour of xanthates in solution at different pH levels has been the subject of numerous investigations [3,11-14]. Xanthates are unstable in acidic solutions. The production of xanthic acid and the subsequent decomposition to alcohol and CS 2 at low pH regions have been established [12-14]. The acid metabolites generated by Bacillus polymyxa can thus lead to rapid decomposition of the xanthate in solution. UV-visible spectra of the xanthate solution in the presence of bacterial metabolite indicated the gradual reduction and disappearance of the xanthate peaks and emergence of CS 2 peak. Moreover, the bacteria were seen to utilize the xanthate reagent for their metabolism as well. From the UV-visible spectra taken during bacterial utilisation of xanthate solution, it became evident that carbohydrates and fatty acids were generated. Similar to carbon and nitrogen, sulphur is also an important element in bacterial metabolism. As pan of the carbon cycle, microbes degrade organic sulphur compounds [15-16]. Bacillus sp. are known to derive energy from the oxidation of H2S and organic sulphur [17]. Possible mechanisms in the biodegradation of isopropyl xanthate involve the following stages. CH3
S
I
II
Bacteria H2S + CH3 CO CH3 + CO2
CH3 - C - O - C - S"
Bacteria CH3 CO COOH
CH3 CHO + CO2 Bacteria
Bacteria CH3 COOH The CH 3 COOH can directly enter the TCA cycle and satisfy the carbon requirements. H2S formed can be further biooxidised to sulphur and sulphate [ 18].
CONCLUSIONS The following major conclusions could be made based on this study. 1.
Bacillus polymyxa, a heterotrophic soil bacterium indigenously associated with several ore deposits was found to be capable of biodegradation of several organic flotation collectors such as dodecyl and di-amine, isopropyl xanthate and sodium oleate.
2.
The bacteria could utilize the above organic reagents for their carbon and nitrogen requirements
738
N. Deo and K. A. Natarajan
and grow very well, generating polysaccharides, proteins and fatty acids during metabolism. .
The bacteria can also be utilized to strip adsorbed collector reagents from mineral surfaces.
4.
Biodegradation mechanisms include both direct bacterial metabolism as well as contributions from the acidic metabolites.
.
A biological route for detoxification of mineral processing effluents containing residual organic reagents is demonstrated.
REFERENCES .
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Phalguni, A., Modak, J.M. & Natarajan, K.A., Biobeneficiation of bauxite using Bacillus polymyxa; Calcium and iron removal, Int. J. Miner. Process; 48, 51--60 (1996). Siggia, S., Quantitative Organic Analysis via Functional Groups, John Wiley, NY, 20-26 (1949). Leja, J., Surface Chemistry of Froth Flotation, Plenum Press, N.Y. 228 -258 (1983) Snell, F.D. & Snell, C.T., Colorimetric Methods of Analysis, D. Van Nostrand Co. N.Y. (1954). Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantifies of protein utilizing the principle of protein-dye binding; Anal, Biochem., 72, 248-254 (1976). Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. & Smith, F., Colorimetric method for the determination of sugars and related substances, Ana. Chem., 28, 350-356 (1956). Taggart, A.F., Hand Book of Mineral Dressing, John Wiley, N.Y. (1967). Prave, P., Faust, U., Sittig, W. & Sukatsch, D.A., Fundamentals of Biotechnology, VCH, Weinheim, 67-132 (1987). Gibson, D.T., Microbial Degradation of Organic Compounds, Marcel Dekker Inc., N.Y., 43-129 (1984). Yamada, H., Adachi, O. & Ogata, K., Amine Oxidases of microorganisms, Biol. Chem., 29, 117-123 (1965). Iwasaki, I. & Cooke, S.R.B., Absorption spectra of some sulfyhydral compounds, Trans. SME (AMIE), 208, 1267-1268 (1957). Iwasaki, I. & Cooke, S.R.B., Dissociation constant of xanthic acid as determined by spectrophotometric method, J. Phys. Chem., 63, 1321-1322 (1959). Pomianowski, A. & Leja, J., Spectrophotometric study of xanthate and dixanthogen solutions, Can. J. Chemistry, 41, 2219-2228 (1963). Jones, M.H. & Woodcock, J.T., Decomposition, of alkyl dixanthogens in aqueous solutions, Int. J. Miner. Process, 10, 1-24 (1983). Ehrlich, H.L., Geomicrobiology, Second Edition, Marcel Dekker, Inc., N,Y. (1990). Freney, J.R., Sulphur-containing organics, in McLaren, A.D. and Petersen, G.H. (Eds.), Soil Biochemistry, Marcel Dekker, Inc., N.Y. 229-259 (1967). Strohl, W.R. & Larkin, J.M., Enumeration, isolation and characterization of Beggiatoa from fresh water sediments, App. Environ. Microbiol., 36, 755-770 (1978). Pringsheim, E.G., Die mixotrophie von Beggiatoa, Arch. Mikrobiol, 59, 247-254 (1967).
C o r r e s p o n d e n c e on papers published in Minerals Engineering is invited, preferably by email to [email protected], or b y Fax to + 4 4 - ( 0 ) 1 3 2 6 - 3 1 8 3 5 2