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Mine drainage pollution reduction by inhibition of iron bacteria
;latev Research Vol. 9. pp 525 to 52,~ P e r g a m o n Press 1975. Printed in Great Britain.
MINE DRAINAGE P O L L U T I O N REDUCTION BY INHIBITION OF IRON BACTERIA FRASER WALSH and RALPH MITCHELL* Laboratory of Applied Microbiology, Division of Engineering and Applied Physics, Harvard University. Cambridge, MA 02138, U.S.A.
(Received 10 August 1974) Abstract--Perfusion of coal-mine tailings with iron-rich synthetic ground water is shown to significantly reduce rates of total iron and acidity release from the tailings. Observed concurrent decrease in effluent Thiobacillusferrooxidans and Metallogenium populations suggests that the increased influent ferrous iron concentration inhibited the catalytic activity of the iron bacteria by preventing the growth of significant Metallogenium populations. A combination of increasing mine influent ferrous iron concentrations with mine sealing may significantly reduce acidity and total iron releases from working or abandoned coal mines.
INTRODUCTION
The majority of iron and acidity released in coal mine drainage is the result of pyrite degradation by ferric iron (Table 1). Singer and Stumm (1970) showed that the rate of release is dependent upon the rate of ferrous iron oxidation and that, at pH less than 5, abiotic iron oxidation proceeds extremely slowly.
rooxidans activity, and thus a decrease in the rate of acidity and total iron release from pyritic materials. In this report, we describe the effect of increasing influent ferrous iron concentrations on the amount of total iron and acidity released in drainage from coal mine railings. The influent iron concentrations used were in the range observed to inhibit the iron-oxidizing Metallogenium (Walsh and Mitchell, 1972a and 1972b).
Table 1. Chemical reactions in pyrite degradation MATERIALS AND METHODS Overall
reaction:
A coal shale mixture was obtained from a mine tailings pile in West Virginia. This mixture passed a 1/2 in. sieve and contained 2.73% pyritic sulfur, 10.6% Fe203 and 55% ash. Steo react£ons: Eight 3-ft columns were prepared (Baker and Wilshire, 14 Fe ÷2 ~ 7 / 2 0 2 ÷ 14 H÷----~ 14 Fe ÷3 + 7H20 1970) each of which contained 250g of the mixture which FeS 2 + 14 Fe ÷] * 8H20 ~ 15 Fe ÷2 ÷ 2 SO4 -2 + 16 H÷ had been autoclaved at 18 psi for 30 rain on three successive days. The columns were closed with sterile cotton plugs and were perfused slowly (approximately 3 ml h- t) with a synAs pH-neutral ground or surface water flows thetic ground water by gravity feed during the three experimental perfusion periods. through a coal mine or its tailings pile, the water pH The synthetic ground water contained: 50 ppm CaCO3; decreases to less than 3. The rate of decrease is related 25 ppm MgSO,~. 7H20; 5 ppm MnSO,L; 2 ppm (NH,)2SO 4 to the activity of the iron bacteria, such as Thiobacillus and 0.25 ppm KH2PO4. The solution pH was adjusted to ferrooxidans, in that they catalyze pyrite degradation 4.5 with sulfuric acid. This syntbeic ground water was by increasing ferrous iron oxidation rates in those enriched with 0.5, 50.0 or 100.0 mg 1- t ferrous iron (from FeSO,~. 7H.,O). The pH of the enriched water was returned regions of the mine or tailings pile which are at pH less to 4.5 with potassium hydroxide. Eight columns were perthan 5. Once the ferric iron-pyrite reaction has fused: four with 0.5mgl -~ ferrous iron, two with occurred, the sulfur moiety released can then be oxi- 50.0 mg I - t ferrous iron, and two with 100.0 mg 1- ~ ferrous dized (abioticly or with sulfur bacteria mediation) with iron. Inocula of Thiobacillusferrooxidans and of Metallo9enium were added at the beginning of the first perfusion to resultant additional acidity release. six of the columns (seeded) but not to two of the four We previously reported on a pH-dependent succes- columns perfused with 0.5 mg I- t ferrous iron (non-seeded). sion of iron bacteria active at pH less than 5 and The culture of T.ferrooxidans was obtained from the Amerishowed that the activity of the initial species in the suc- can Type Culture Collection. The Memllogenium was isocession (a filamentous Metallogenium) was inhibited by lated from coal mine drainage fWalsh and Mitchell, 1972b). Before perfusion or inoculation, but after autoclaving, the increased ferrous iron concentrations. When the columns were washed rapidly (approximately 10 ml h- t) Metallogenium was inhibited, the rate of pH decrease with autoclaved synthetic ground water until effluent total from iron oxidation was slowed. If this inhibition can i r o n and acidity concentrations were below 30 and 100 mg 1- t (above pH 3.5) respectively. The columns were be affected in coal mines or in tailings piles, there should result a decrease in regions suitable for T. fer- then perfused slowly with the iron-enriched water for 80 days, after which they were again washed rapidly with sterile synthetic ground water until total iron and acidity effluent * To whom correspondence should be addressed. concentrations below 30 and 100 mg I- t respectively were 525 FeS 2 + 7 / 2 02 + H20
~
Fe+2 + 2SO4-2 ÷ 2H*
526
F. WALSHand R. NIITCHELL Table 2. Release of total iron and acidity from mine tailings in 166 days* Seeded
Non-Seeded Influent Total Total
Ferrous
Iron Released, Acidity
Acidity Effluent
0.5
0.5
50.0
6.7
36.L
12.7
9.4
m moles
31.9
128.5
66.7
40.2
Iron, at moles
25.2
92.4
54.0
30.3
3.2
2.5
2.6
2.8
Iron, mg/l m moles
Released,
le~s Ferrous pH
% Reduction % Reduction
in Iron R e l e a s e d ° in A c i d i t y
Released ~
100.0
82
65
74
73
42
67
*These data a~e the s u/~ Of the releases obtained from two p e r f u s i o n s (i st of 80 days, 2 nd of 86 days) and are the average of two columns. T h e y have been c o r r e c t e d for influent ferrous iron and a c i d i t y concentrations.
" % R e d u c t i o n b a s e d on r e l e a s e s o b s e r v e d
again observed. The seeded columns were then inoculated with fresh coal mine drainage and again perfused slowly for a second 86 day period. At the end of this second perfusion, influent concentrations to the seeded 0.5 and I00.0 mg 1-z ferrous iron columns were exchanged. All the columns were then perfused slowly for a third 80 day period. Total effluent iron concentration was measured by atomic absorption spectrometry. Influent and effluent ferrous iron concentration was determined by acid permanganate titration using an o-phenanthroline indicator. Total acidity was measured by titration at room temperature under a nitrogen atmosphere to the phenolphthalene end point. Sulfate was determined by gravimetric analysis using barium chloride (Standard Methods, 1965). An estimate of the precipitated ferric hydroxide remaining in the columns following the three perfusions was made by shaking overnight the coal shale mixture from each column in 500 ml of distilled water acidified to p H I with concentrated sufluric acid and measuring the total iron concentration in the wash water using atomic absorption spectrometry. Effluent iron bacteria populations were monitored by a five tube most probable number technique (Standard Methods, 1965) using our growth medium for Metallogenium (Walsh and Mitchell, 1972bj or the 9 K medium for T.ferrooxidans (Lundgren et al., 1964). Heterotroph populations were estimated by dilution and plating on nutrient agar (Difco) or Sabouraud dextrose agar (Difco).
from seeded
ferrous iron inhibited total iron and acidity release immediately. Lower influent ferrous iron concentrations required longer lag periods belbre inhibition (Fig. 1). The effect of 100 mg 1- t ferrous iron on inhibition of total iron and acidity release is extremely rapid. Table 3 shows the effect of exchanging the influent ferrous iron concentration of the 0.5 and 1 0 0 m g l - ' columns. The reversal in effluent concentrations occurred in less than two weeks. This demonstrates that the iron bacteria are present in all the seeded columns; their activity is inhibited by higher influent ferrous iron concentrations. The effluent populations of the iron bacteria were difficult to measure because they were always at low (less than 10"~ I00 ml- ') levels. On the basis of a most probable number determination, we were able to determine that T. ferrooxiclans was only present at populations of between 102 and 10"~ 100 m l - ' in the effluent from the seeded 0.5 mg 1-z ferrous iron columns (Table 4). Estimation of population levels of Metallogeniurn is difficult due to the filamentous nature of the organism. We observed it only in the effluents of the 0.5 and the 50 mg 1- t columns, but at populations of between 10= and 10'~ 100 m l - t . When the influent concentration to the 100mg 1- ~ columns were changed to 0.5 mg I- ', both iron bacteria were observed in the effluent. No significant heterotrophic growth was observed in the column effluents even during the second perfusion when the columns were seeded with mine drainage. The heterotrophs present were fungi at populations of less than 103 100 ml - t
EXPERIMENTAL
Release of total iron and acidity from 250 g of coal mine tailings was monitored for three eighty day periods. Table 2 shows the effect of seeding and of influent ferrous iron concentration on total iron and acidity release. These data are reported as the total amounts of total iron and acidity observed effluent from the columns less the total amounts of ferrous iron and acidity influent into the columns. They are thus a measure of the amount of iron released from pyrite degradation during the first two perfusion periods. The low release levels from the non-seeded column identify the level of abiotic pyrite degradation. To change the level of abiotic releases, the kinetics of the reactions shown in Table 1 must be changed by altering the concentration of a reactant such as oxygen. Indeed, Baker and Wilshire (1970) showed that aeration increases abiotic reaction rates and, therefore, effluent total iron and acidity concentrations. Increasing the influent ferrous iron concentration decreased the amount of total iron and acidity released from the coal mine tailings. Influent concentration of 100 mg I- t
0.5 mg/l column
Influent
EFe+ 2 ]
\
:
+
+
50-0
0.5
x
x
I00.0
o
o
I0
o
I I0
~
, 30
Time,
-~
" 50
.
~
"tO
doys
Fig. 1. Rate of total iron release from 250 g of mine tailings for the first perfusion. Release rates corrected for release observed in non-seeded 0.5 mg 1- ~ column.
Mine drainage pollution reduction by inhibition of iron bacteria
527
Table 3. Effect of reversing influent ferrous iron concentration on effluent total iron and acidity concentration Seeded 0.5 i00.0
In~luen: [Fe+2] before, mg/l
Effluent [Fe]T
after, mg/l
i00.0
before, mg/i*
193
after, mg/l* Effluent acidity before, mg/l CaCO3" after, mg/l CaCO3*
~Ion-~eeded 0.5
0.5
I00.0
0.5
22
39
12g
21
1069
210
194
868
1440
225
*Results corrected for influent ferrous iron and acidity concentrations.
Table 4. Iron bacteria observed in column effluent during first perfusion Bacterium Influent ferrous iron, mg/l
0.5
T. ferrooxidans
÷÷
Metallo@enium
+~
Heterotrophs
÷
50.0
100.0
(+)* ~
+
~Observed only in first two weeks.
The amount of total iron precipitated in the columns after perfusion also indicates that pyrite degradation was inhibited by the higher influent concentrations of ferrous iron. Table 5 shows that the highest precipitated total iron level occurred in the columns which had the-highest iron release rates, but which had the lowest infiuent ferrous iron levels. These data were obtained following the third perfusion during which the original 0.5 and 100 mg 1-t ferrous iron influents had been exchanged. The increase in precipitated total iron in the seeded 0.5 mg 1-~ columns (the original seeded 100 mg 1- ' columns) must have occurred during the third perfusion, because release levels from such columns were low during the first and second perfusions. Note that the release rate in the third perfusion for the seeded 100 mg 1-1 columns (the original 0.5 m g l - t columns) shown in Table 3 was significantly higher than might have been expected (39 mgl-t). This is probably the result of washout of the iron which had precipitated during the first two perfusions of these columns when the level of pyrite degradation in the columns had been high. A comparison of the total iron and acidity release rates between the non-seeded and seeded 0.5 m g l - t enriched columns shows the catalytic effect of the iron bacteria in increasing the rate of pyrite degradation (Table 2). A further comparison of the releases from the non-seeded 0.5 mg I- l and the seeded 100 mg I- t columns shows that 90°,/0 inhibition of bacterial activity has been achieved by increasing the influent ferrous iron concentration. Increased ferrous iron concentrations should not affect T. ferrooxidans activity if the pH is less than 3.5. but should inhibit Metallogenium
growth if the pH is greater than 3.5 (Walsh and Mitchell, 1972b). in the absence of the Metallogeniu,,, and provided mine water pH remains above that suitable for significant growth of T. ferrooxidans, pyrite degradation rates should be dependent upon abiotic reaction rates. We have not observed directly a decrease in iron bacteria populations due to increased influent ferrous iron concentrations. Our measurements (Table 5) of effluent populations may not, in fact, relate to column populations, but two observations suggest that there is some direct relationship. First. the effluent populations correlate with observed release rates (Fig. 1). Second, the rate of total iron and acidity release in the seeded 0.5 mg 1- ~ column for the first 80 day perfusion was almost twice that in the second perfusion. This was because of a significant lag period of approximately 2 weeks during the second perfusion. There was an even greater decrease in the 50 and 100 mg 1- t columns due again to an increased lag period. The major difference between the two perfusions was the source of the iron bacteria. For the first perfusion, T. ferrooxida,,s and Metallogeniz,,n (less than 10~ of each) were inoculated directly into the columns. For the second perfusion, fresh pH 2.8 mine drainage was inoculated: this contained approximately 10-' cells of T.ferrooxidans and less than 10z fragments of Metallogenium. In that the washing between perfusions was at rates only approximately three times greater than the normal perfusion, if the iron bacteria were strongly held in the coal-shale mixture, the number of iron bacteria in the second inoculation should not have affected release rates. The iron bacteria tested are thus apparently not strongly
Table 5. Precipitated total iron remaining in columns after the third perfusion Column Non-seeded, Seeded,
Precipitated
0.5 m g / l
0.5 m g / l
[Fe +2]
[Fe +2]
Seeded,
50.0 m g / l
Seeded,
i00.0 m g / l
influent
influent
[Fe +2] [Fe +2]
influent influent
328 428 270 122
Fe T ~ m~
52S
F. WALSH and R. MITCHELL
held to the particles of the mine railings mixture: effluent populations should provide some measure of active populations. The decreases observed in total iron and acidity releases in mine tailings drainage, following increases in influent ferrous iron concentrations, suggest a new approach to the control of mine or tailings pile drainage pollution. Drainage, which is rich in ferrous iron and which has been partially neutralized to pH 4-4.5. could be reintroduced to the coal mine or to the tailings pile to provide the necessary influent ferrous iron concentrations. This should inhibit Merallogenium growth and reduce the catalytic activity of the succession of iron bacteria. Such a recycling system should be completely successful in those mine or tailings pile regimes in which water residence time is not so long as to permit acidity formation abioticly or due to the activity of sulfur bacteria to result in pH levels suitable for T. ferrooxidans activity. SU,~,IMARY A new a p p r o a c h to reduction of the release of total iron a n d acidity in tailings pile drainage has been
shown to be successful under laboratory conditions. The m e t h o d may be applicable to field conditions, provided influent ferrous iron concentrations can be raised a n d influent pH is greater than 3.5 but less than 5. REFERENCES
Baker R. A. and Wilshire A. G. [1970) Microbial factors in acid mine drainage formation: a pilot plant study. Envir. Sci. Techn. 4, 401-407. Lundgren D. G., Anderson K. J.. Remsen C. C. and Mahoney R. P. (1964) Culture. structure and physiology of the chemoautotreph Ferrobacillus ferrooxidans. Det's. ind. M icrobiol. 6, 250-259. Singer P. and Stumm W. (1970) Acidic mine drainage: the rate limiting step. Science, New York 167, 1121-1123. Standard Methods for the Examination of Water and Waste Water (1965) American Public Health Assoc.. New York. Walsh F. and Mitchell R. (1972a)A pH-dependent succession of iron bacteria. Ent'ir. Sci. Techn. 6, 809-812. Walsh F. and Mitchell R. (1972b) An acid-tolerant iron-oxidizing Metalloget,ium. J. gen. Microhiol. 72, 369-374.
MINE DRAINAGE P O L L U T I O N REDUCTION BY INHIBITION OF IRON BACTERIA FRASER WALSH and RALPH MITCHELL* Laboratory of Applied Microbiology, Division of Engineering and Applied Physics, Harvard University. Cambridge, MA 02138, U.S.A.
(Received 10 August 1974) Abstract--Perfusion of coal-mine tailings with iron-rich synthetic ground water is shown to significantly reduce rates of total iron and acidity release from the tailings. Observed concurrent decrease in effluent Thiobacillusferrooxidans and Metallogenium populations suggests that the increased influent ferrous iron concentration inhibited the catalytic activity of the iron bacteria by preventing the growth of significant Metallogenium populations. A combination of increasing mine influent ferrous iron concentrations with mine sealing may significantly reduce acidity and total iron releases from working or abandoned coal mines.
INTRODUCTION
The majority of iron and acidity released in coal mine drainage is the result of pyrite degradation by ferric iron (Table 1). Singer and Stumm (1970) showed that the rate of release is dependent upon the rate of ferrous iron oxidation and that, at pH less than 5, abiotic iron oxidation proceeds extremely slowly.
rooxidans activity, and thus a decrease in the rate of acidity and total iron release from pyritic materials. In this report, we describe the effect of increasing influent ferrous iron concentrations on the amount of total iron and acidity released in drainage from coal mine railings. The influent iron concentrations used were in the range observed to inhibit the iron-oxidizing Metallogenium (Walsh and Mitchell, 1972a and 1972b).
Table 1. Chemical reactions in pyrite degradation MATERIALS AND METHODS Overall
reaction:
A coal shale mixture was obtained from a mine tailings pile in West Virginia. This mixture passed a 1/2 in. sieve and contained 2.73% pyritic sulfur, 10.6% Fe203 and 55% ash. Steo react£ons: Eight 3-ft columns were prepared (Baker and Wilshire, 14 Fe ÷2 ~ 7 / 2 0 2 ÷ 14 H÷----~ 14 Fe ÷3 + 7H20 1970) each of which contained 250g of the mixture which FeS 2 + 14 Fe ÷] * 8H20 ~ 15 Fe ÷2 ÷ 2 SO4 -2 + 16 H÷ had been autoclaved at 18 psi for 30 rain on three successive days. The columns were closed with sterile cotton plugs and were perfused slowly (approximately 3 ml h- t) with a synAs pH-neutral ground or surface water flows thetic ground water by gravity feed during the three experimental perfusion periods. through a coal mine or its tailings pile, the water pH The synthetic ground water contained: 50 ppm CaCO3; decreases to less than 3. The rate of decrease is related 25 ppm MgSO,~. 7H20; 5 ppm MnSO,L; 2 ppm (NH,)2SO 4 to the activity of the iron bacteria, such as Thiobacillus and 0.25 ppm KH2PO4. The solution pH was adjusted to ferrooxidans, in that they catalyze pyrite degradation 4.5 with sulfuric acid. This syntbeic ground water was by increasing ferrous iron oxidation rates in those enriched with 0.5, 50.0 or 100.0 mg 1- t ferrous iron (from FeSO,~. 7H.,O). The pH of the enriched water was returned regions of the mine or tailings pile which are at pH less to 4.5 with potassium hydroxide. Eight columns were perthan 5. Once the ferric iron-pyrite reaction has fused: four with 0.5mgl -~ ferrous iron, two with occurred, the sulfur moiety released can then be oxi- 50.0 mg I - t ferrous iron, and two with 100.0 mg 1- ~ ferrous dized (abioticly or with sulfur bacteria mediation) with iron. Inocula of Thiobacillusferrooxidans and of Metallo9enium were added at the beginning of the first perfusion to resultant additional acidity release. six of the columns (seeded) but not to two of the four We previously reported on a pH-dependent succes- columns perfused with 0.5 mg I- t ferrous iron (non-seeded). sion of iron bacteria active at pH less than 5 and The culture of T.ferrooxidans was obtained from the Amerishowed that the activity of the initial species in the suc- can Type Culture Collection. The Memllogenium was isocession (a filamentous Metallogenium) was inhibited by lated from coal mine drainage fWalsh and Mitchell, 1972b). Before perfusion or inoculation, but after autoclaving, the increased ferrous iron concentrations. When the columns were washed rapidly (approximately 10 ml h- t) Metallogenium was inhibited, the rate of pH decrease with autoclaved synthetic ground water until effluent total from iron oxidation was slowed. If this inhibition can i r o n and acidity concentrations were below 30 and 100 mg 1- t (above pH 3.5) respectively. The columns were be affected in coal mines or in tailings piles, there should result a decrease in regions suitable for T. fer- then perfused slowly with the iron-enriched water for 80 days, after which they were again washed rapidly with sterile synthetic ground water until total iron and acidity effluent * To whom correspondence should be addressed. concentrations below 30 and 100 mg I- t respectively were 525 FeS 2 + 7 / 2 02 + H20
~
Fe+2 + 2SO4-2 ÷ 2H*
526
F. WALSHand R. NIITCHELL Table 2. Release of total iron and acidity from mine tailings in 166 days* Seeded
Non-Seeded Influent Total Total
Ferrous
Iron Released, Acidity
Acidity Effluent
0.5
0.5
50.0
6.7
36.L
12.7
9.4
m moles
31.9
128.5
66.7
40.2
Iron, at moles
25.2
92.4
54.0
30.3
3.2
2.5
2.6
2.8
Iron, mg/l m moles
Released,
le~s Ferrous pH
% Reduction % Reduction
in Iron R e l e a s e d ° in A c i d i t y
Released ~
100.0
82
65
74
73
42
67
*These data a~e the s u/~ Of the releases obtained from two p e r f u s i o n s (i st of 80 days, 2 nd of 86 days) and are the average of two columns. T h e y have been c o r r e c t e d for influent ferrous iron and a c i d i t y concentrations.
" % R e d u c t i o n b a s e d on r e l e a s e s o b s e r v e d
again observed. The seeded columns were then inoculated with fresh coal mine drainage and again perfused slowly for a second 86 day period. At the end of this second perfusion, influent concentrations to the seeded 0.5 and I00.0 mg 1-z ferrous iron columns were exchanged. All the columns were then perfused slowly for a third 80 day period. Total effluent iron concentration was measured by atomic absorption spectrometry. Influent and effluent ferrous iron concentration was determined by acid permanganate titration using an o-phenanthroline indicator. Total acidity was measured by titration at room temperature under a nitrogen atmosphere to the phenolphthalene end point. Sulfate was determined by gravimetric analysis using barium chloride (Standard Methods, 1965). An estimate of the precipitated ferric hydroxide remaining in the columns following the three perfusions was made by shaking overnight the coal shale mixture from each column in 500 ml of distilled water acidified to p H I with concentrated sufluric acid and measuring the total iron concentration in the wash water using atomic absorption spectrometry. Effluent iron bacteria populations were monitored by a five tube most probable number technique (Standard Methods, 1965) using our growth medium for Metallogenium (Walsh and Mitchell, 1972bj or the 9 K medium for T.ferrooxidans (Lundgren et al., 1964). Heterotroph populations were estimated by dilution and plating on nutrient agar (Difco) or Sabouraud dextrose agar (Difco).
from seeded
ferrous iron inhibited total iron and acidity release immediately. Lower influent ferrous iron concentrations required longer lag periods belbre inhibition (Fig. 1). The effect of 100 mg 1- t ferrous iron on inhibition of total iron and acidity release is extremely rapid. Table 3 shows the effect of exchanging the influent ferrous iron concentration of the 0.5 and 1 0 0 m g l - ' columns. The reversal in effluent concentrations occurred in less than two weeks. This demonstrates that the iron bacteria are present in all the seeded columns; their activity is inhibited by higher influent ferrous iron concentrations. The effluent populations of the iron bacteria were difficult to measure because they were always at low (less than 10"~ I00 ml- ') levels. On the basis of a most probable number determination, we were able to determine that T. ferrooxiclans was only present at populations of between 102 and 10"~ 100 m l - ' in the effluent from the seeded 0.5 mg 1-z ferrous iron columns (Table 4). Estimation of population levels of Metallogeniurn is difficult due to the filamentous nature of the organism. We observed it only in the effluents of the 0.5 and the 50 mg 1- t columns, but at populations of between 10= and 10'~ 100 m l - t . When the influent concentration to the 100mg 1- ~ columns were changed to 0.5 mg I- ', both iron bacteria were observed in the effluent. No significant heterotrophic growth was observed in the column effluents even during the second perfusion when the columns were seeded with mine drainage. The heterotrophs present were fungi at populations of less than 103 100 ml - t
EXPERIMENTAL
Release of total iron and acidity from 250 g of coal mine tailings was monitored for three eighty day periods. Table 2 shows the effect of seeding and of influent ferrous iron concentration on total iron and acidity release. These data are reported as the total amounts of total iron and acidity observed effluent from the columns less the total amounts of ferrous iron and acidity influent into the columns. They are thus a measure of the amount of iron released from pyrite degradation during the first two perfusion periods. The low release levels from the non-seeded column identify the level of abiotic pyrite degradation. To change the level of abiotic releases, the kinetics of the reactions shown in Table 1 must be changed by altering the concentration of a reactant such as oxygen. Indeed, Baker and Wilshire (1970) showed that aeration increases abiotic reaction rates and, therefore, effluent total iron and acidity concentrations. Increasing the influent ferrous iron concentration decreased the amount of total iron and acidity released from the coal mine tailings. Influent concentration of 100 mg I- t
0.5 mg/l column
Influent
EFe+ 2 ]
\
:
+
+
50-0
0.5
x
x
I00.0
o
o
I0
o
I I0
~
, 30
Time,
-~
" 50
.
~
"tO
doys
Fig. 1. Rate of total iron release from 250 g of mine tailings for the first perfusion. Release rates corrected for release observed in non-seeded 0.5 mg 1- ~ column.
Mine drainage pollution reduction by inhibition of iron bacteria
527
Table 3. Effect of reversing influent ferrous iron concentration on effluent total iron and acidity concentration Seeded 0.5 i00.0
In~luen: [Fe+2] before, mg/l
Effluent [Fe]T
after, mg/l
i00.0
before, mg/i*
193
after, mg/l* Effluent acidity before, mg/l CaCO3" after, mg/l CaCO3*
~Ion-~eeded 0.5
0.5
I00.0
0.5
22
39
12g
21
1069
210
194
868
1440
225
*Results corrected for influent ferrous iron and acidity concentrations.
Table 4. Iron bacteria observed in column effluent during first perfusion Bacterium Influent ferrous iron, mg/l
0.5
T. ferrooxidans
÷÷
Metallo@enium
+~
Heterotrophs
÷
50.0
100.0
(+)* ~
+
~Observed only in first two weeks.
The amount of total iron precipitated in the columns after perfusion also indicates that pyrite degradation was inhibited by the higher influent concentrations of ferrous iron. Table 5 shows that the highest precipitated total iron level occurred in the columns which had the-highest iron release rates, but which had the lowest infiuent ferrous iron levels. These data were obtained following the third perfusion during which the original 0.5 and 100 mg 1-t ferrous iron influents had been exchanged. The increase in precipitated total iron in the seeded 0.5 mg 1-~ columns (the original seeded 100 mg 1- ' columns) must have occurred during the third perfusion, because release levels from such columns were low during the first and second perfusions. Note that the release rate in the third perfusion for the seeded 100 mg 1-1 columns (the original 0.5 m g l - t columns) shown in Table 3 was significantly higher than might have been expected (39 mgl-t). This is probably the result of washout of the iron which had precipitated during the first two perfusions of these columns when the level of pyrite degradation in the columns had been high. A comparison of the total iron and acidity release rates between the non-seeded and seeded 0.5 m g l - t enriched columns shows the catalytic effect of the iron bacteria in increasing the rate of pyrite degradation (Table 2). A further comparison of the releases from the non-seeded 0.5 mg I- l and the seeded 100 mg I- t columns shows that 90°,/0 inhibition of bacterial activity has been achieved by increasing the influent ferrous iron concentration. Increased ferrous iron concentrations should not affect T. ferrooxidans activity if the pH is less than 3.5. but should inhibit Metallogenium
growth if the pH is greater than 3.5 (Walsh and Mitchell, 1972b). in the absence of the Metallogeniu,,, and provided mine water pH remains above that suitable for significant growth of T. ferrooxidans, pyrite degradation rates should be dependent upon abiotic reaction rates. We have not observed directly a decrease in iron bacteria populations due to increased influent ferrous iron concentrations. Our measurements (Table 5) of effluent populations may not, in fact, relate to column populations, but two observations suggest that there is some direct relationship. First. the effluent populations correlate with observed release rates (Fig. 1). Second, the rate of total iron and acidity release in the seeded 0.5 mg 1- ~ column for the first 80 day perfusion was almost twice that in the second perfusion. This was because of a significant lag period of approximately 2 weeks during the second perfusion. There was an even greater decrease in the 50 and 100 mg 1- t columns due again to an increased lag period. The major difference between the two perfusions was the source of the iron bacteria. For the first perfusion, T. ferrooxida,,s and Metallogeniz,,n (less than 10~ of each) were inoculated directly into the columns. For the second perfusion, fresh pH 2.8 mine drainage was inoculated: this contained approximately 10-' cells of T.ferrooxidans and less than 10z fragments of Metallogenium. In that the washing between perfusions was at rates only approximately three times greater than the normal perfusion, if the iron bacteria were strongly held in the coal-shale mixture, the number of iron bacteria in the second inoculation should not have affected release rates. The iron bacteria tested are thus apparently not strongly
Table 5. Precipitated total iron remaining in columns after the third perfusion Column Non-seeded, Seeded,
Precipitated
0.5 m g / l
0.5 m g / l
[Fe +2]
[Fe +2]
Seeded,
50.0 m g / l
Seeded,
i00.0 m g / l
influent
influent
[Fe +2] [Fe +2]
influent influent
328 428 270 122
Fe T ~ m~
52S
F. WALSH and R. MITCHELL
held to the particles of the mine railings mixture: effluent populations should provide some measure of active populations. The decreases observed in total iron and acidity releases in mine tailings drainage, following increases in influent ferrous iron concentrations, suggest a new approach to the control of mine or tailings pile drainage pollution. Drainage, which is rich in ferrous iron and which has been partially neutralized to pH 4-4.5. could be reintroduced to the coal mine or to the tailings pile to provide the necessary influent ferrous iron concentrations. This should inhibit Merallogenium growth and reduce the catalytic activity of the succession of iron bacteria. Such a recycling system should be completely successful in those mine or tailings pile regimes in which water residence time is not so long as to permit acidity formation abioticly or due to the activity of sulfur bacteria to result in pH levels suitable for T. ferrooxidans activity. SU,~,IMARY A new a p p r o a c h to reduction of the release of total iron a n d acidity in tailings pile drainage has been
shown to be successful under laboratory conditions. The m e t h o d may be applicable to field conditions, provided influent ferrous iron concentrations can be raised a n d influent pH is greater than 3.5 but less than 5. REFERENCES
Baker R. A. and Wilshire A. G. [1970) Microbial factors in acid mine drainage formation: a pilot plant study. Envir. Sci. Techn. 4, 401-407. Lundgren D. G., Anderson K. J.. Remsen C. C. and Mahoney R. P. (1964) Culture. structure and physiology of the chemoautotreph Ferrobacillus ferrooxidans. Det's. ind. M icrobiol. 6, 250-259. Singer P. and Stumm W. (1970) Acidic mine drainage: the rate limiting step. Science, New York 167, 1121-1123. Standard Methods for the Examination of Water and Waste Water (1965) American Public Health Assoc.. New York. Walsh F. and Mitchell R. (1972a)A pH-dependent succession of iron bacteria. Ent'ir. Sci. Techn. 6, 809-812. Walsh F. and Mitchell R. (1972b) An acid-tolerant iron-oxidizing Metalloget,ium. J. gen. Microhiol. 72, 369-374.