A pH-Dependent Succession of Iron Bacteria Fraser Walsh' and Ralph Mitchell Laboratory of Applied Microbiology, Division of Engineering and Applied Physics, Harvard University, Cambridge, Mass. 021 38
The role of a n acid-tolerant, filamentous iron bacterium in a succession of pH-dependent events affecting the rate of iron oxidation is identified. This organism significantly catalyzes iron oxidation in the p H range 4.5-3.5. At p H greater than 4.5, abiotic iron oxidation proceeds rapidly. At p H less than 3.5, Thiobacillus ferrooxidans significantly catalyzes iron oxidation. The activity of the filamentous iron bacteria in this succession of events may directly affect the rate of acidity production in coal mine waters. H
T
he rate of acidity production from the degradation of iron pyrites found in coal deposits has been shown by Singer and Stumm (1970) to depend on the rate of ferrous iron oxidation. The rate of abiotic ferrous iron oxidation is directly related to solution p H (Stumm and Lee, 1961) with the relationship above p H 4.5 being: -d[Fe dt
=
k[Fe z+]p[02][OH-]
Materials and Methods
where k = 8.0 X 1013I. mol-2 atm-l min-l at 25°C. At p H between 4.5 and 3.5, chemical ferrous iron oxidation proceeds at a rate indirectly p H dependent (Singer and Stumm, 1970). The oxidation rate a t p H less than 3.5 is approximately 7 x mg/l.-day for a n initial ferrous iron concentration of 250 mg/l. This rate remains relatively constant at lower pH. Table I shows the relationship between p H and the rate of abiotic ferrous iron oxidation over the meso-acidic p H range (pH 3.5-5.5). The ferric iron released can then oxidize iron pyrite even in the absence of oxygen. Singer (1969) showed that the half time of reaction between ferric iron and pyrite in the absence of oxygen is 25 min for a pyrite concentration of 5 gram/l. The chemical equations of the pyrite degradation reactions are shown in Table 11. The acidity in coal mine drainage waters is produced either by the formation of ferric hydroxides (Equation 4) o r by the oxidation of the sulfur moiety in pyrite degradation (Equation 3). I n a coal mine, pyrite degradation may occur in the selfaccelerating cycle identified in Table I1 with pyrite being oxidized by ferric iron. Of the two ferric iron-producing equations shown in Table I1 (Equations 1 and 2), Singer and Stumm (1970) showed that only ferrous iron oxidation is a significant source of ferric iron. Assuming that influent mine waters have an average total iron concentration of 0.5 mg/l., that ferrous iron oxidation is the only ferric iron source, and that no ferric iron is hydrolyzed, 93 turns of the pyrite degradation cycle (Equations 2 and 3, Table 11) must occur to result in the release of 300 mg/l. total iron commonly observed in mine drainage. If average mine water residence time is 10 months, each pyrite degradation cycle can thus take n o longer than 3.2 days. Ferric iron hydrolysis and precipitation ~
To whom correspondence should be addressed.
(Equation 4) will naturally increase the number of cycles required and thus decrease the theoretical time available per cycle. The iron oxidation-pyrite degradation cycle identified in Table I1 will proceed slowly at p H < 4.5 in the absence of catalysis. This is because at p H < 4.5, the cycle is rate-limited by the ferrous iron oxidation rate (Singer and Stumm, 1970). Catalysis of ferrous iron oxidation can be biologically o r abiotically mediated. Singer (1969) examined the effect of chemical and physical catalysts on abiotic ferrous iron oxidation and he showed that their effect was a catalysis factor of less than 30. The iron bacterium, Thiobacillus ,ferrooxidans, significantly catalyzes iron oxidation with catalysis factors greater than 300 under laboratory conditions. (Schniiitman, et al., 1969). However, optimal activity of this organism is at p H < 3.5 (Silverman, 1967). Thus, there is no mechanism for rapid catalysis of the iron oxidation-pyrite degradation cycle in the p H range 4.5-3.5. I n this report, we describe the effect of a filamentous iron bacterium on catalyzing the formation of the environment necessary for optimal T. ferrooxidans activity and thus for pyrite degradation.
~~~
Samples of sediment and mine water were obtained from five widely separated sources: a coal mine in Illinois, a n iron-bearing stream in New Hampshire, a zinc mine in New Jersey, a coal mine in West Virginia, and an iron-bearing stream in Vermont. These samples were obtained during different seasons (August, September, December, May). From all of these samples we isolated a n acid-tolerant filamentous iron bacterium of the genus Metallogenium, using a medium containing the following constituents: ( N H 4 ) S 0 4 ,
Table I. Rate of Ferrous Iron Oxidation over the Meso-Acidic pH Range. Abiotic Fez+ PH oxidation rate, mg/l.-dayh 3.5 0.007 4.0 0.25 4.5 0.70 5.0 5.76 5.5 57.6 5
h
From Singer and Stumm, 1970. [Fe*-]o = 250 mg/l.
Table 11. Chemical Reactions in Iron Pyrite Degradation
Pyrite oxidation: FeS2(s) 7/202HzO -+ Fez+
+ 2SOd2- + 2Hi Pyrite degradation cycle: Fez+ + 1/402 + H+ Fe3+ + l/pH20 FeS2(s) + 14Fe3++ 8 H 2 0+ 15Fe2++ 2S042- + 16H+ Ferric iron deposition: Fe3+ + 3 H z 0 e Fe(OH)3(s) + 3H+ +
+
+
(1) (2)
(3)
(4)
Volume 6, Number 9, September 1972 809
Table 111. Rate of Fez+ Oxidation in the Presence of the Iron-Oxidizing Metallogenium~~~ Fez+oxidation, mg/l.-day Initial Fez+ concn, mg/l. pH 3.6 pH 4 . 1 pH 5.0 0 0 0 0 50 3 5 0 250 12 58 4 400 7 24 2 1,250 5 14 0 a Rates reported are differences between rates observed in inoculated and in control flasks. b I n the presence of 0.4 % KH phthalate.
0.1 %; C a C 0 3 , 0.01 %; MgS04, 0.02%; K H 2 P 0 4 , 0.001 %; K H phthalate, 0.4%; and 250 ppm ferrous iron from a n acidified F e S 0 4 .7 H 2 0 solution (Wdlsh, 1971). Phthalate was used to buffer the growth medium in the p H 4 range. Initial isolation required addition of 0 . 4 x formalin to 100 ml of the isolating medium in a 250-mI Erylenmeyer flask (Nunley and Krieg, 1968). After three transfers, each following four days of growth, pure cultures were obtained in nine of ten flasks. Culture purity was based on the absence of contaminant growth on solid and liquid media used to culture heterotrophs and other iron or sulfur bacterid (Walsh, 1971). The extent of iron oxidation catalysis by this acid-tolerant stalked iron bacterium was measured by comparing the change in the ferrous iron concentration in solution in an inoculated flask and in a control flask. Ferrous iron concentration was measured by acid permanganate titration of a 1-ml sample (diluted to 50 ml) using a n o-phenanthroline indicator. T o identify the pH-dependent succession of iron bacteria, T. ferrooxidans and the iron-oxidizing Metallogenium, as well as eight heterotrophs isolated from acidic coal mine drainage, were inoculated into a 500-ml solution with ion concentrations similar to those observed in some influent mine waters [CaC03, 50 mg/l.; MgS04’7H20, 25 mg/l.; MnS04, 5 mg/l.; (NH4)2S04,2 mg/l.; K H 2 P 0 4 ,0.25 mg/l.l. Initial p H was adjusted to 5.0. Ferrous iron concentration was initially 20 mg/l. with an additional 20 mg/l. added every two weeks. Sterile distilled water was added weekly to maintain the initial volume of 500 ml. This medium was circulated in a 10-ft Tygon tube in. diam) using a nitrogen gas lift pump. The total population of the heterotrophs in the medium was followed by colony counting on the eight solid media used to isolate the bacteria. T.ferrooxidans and Metallogenium populations were followed using a five-tube most probable
Figure 1. Electron micrograph of the iron-oxidizing Metallogenium washed in 0.057, oxalic acid (X27,OOO) number (MPN)technique (“Standard Methods for the Examination of Water and Waste Water,” 1965). T.ferrooxidans was tested on the 9 K medium (Dugan and Lundgren, 1964); the iron-oxidizing Metallogenium was tested on our liquid growth medium. Because of the unusual morphology of the Metallogenium, care was taken not to break the filaments during dilution in the MPN procedure. A wide-tipped pipet was used, and shaking of the dilution tubes was kept to a minimum. A control system was run in which bacterial populations were also followed. The same liquid medium was used in this system, and it was inoculated with the heterotrophs and T. ferrooxidans but not with the Metallogenium. Again heterotrophic populations were counted on solid media and those of T . ferrooxidans by the MPN technique. A second control system was run which was buffered at p H 4.1 with phthalate. This system was inoculated with T. ferrooxidans, the Metallogenium, and the eight heterotrophs. Experimental Cultures of the filamentous iron bacterium were isolated from five widely separated sources and appeared morphologically to belong to a single species. The organism grew in multibranching colonies of interweaving ferric iron encrusted stalks (0.1-0.4 p diam) without a conventional cell body. Macroscopically, these stalks appeared as a rusty precipitate, but when this precipitate was washed carefully with 0.05 oxalic acid, the stalks were visible under the electron microscope, still partially encrusted with iron (Figure 1). This organism is morphologically similar to the manganeseoxidizing bacterium Metallogenium symbioticum described by Zavarzin (1961, 1964) and Dubinina (1969, 1970). It differs from the stalked iron bacterium, Gallionella ferruginia, in the
Table IV. Bacterial Populations Observed in the Mine Water System No Metallogenium Mine water inoculated with Metallogenium Time, days 2 5 10
20 30 45 4
pH 5.0 4.3 4.3 3.8 3.2 2.6
T. ferrooxidansa MetalIogeniuma Heterotrophs“
... ... ... ... 2.0 4.0
Log number of cells/ml.
810 Environmental Science & Technology
...
5.1
2.7 4.1 4.0 1.2
7.8
...
8.3
8.5 4.7 2.7
PH 5.0 5.0 4.9 4.5 4.1 4.0
T.
ferrooxidans” Metallogeniuma Heterotrophsa ... ... 4.0 ... ... 5.5 ... ... 7.1 ... ... 7.2 ... ... 7.0 ... ... 6.3
absence of a conventional cell body and in its aicd tolerance (Walsh, 1971). The iron bacterium we have isolated does not catalyze manganese oxidation but we consider it t o be a n acid-tolerant, iron-oxidizing strain of Metallogenium. This is based on morphological similarities and o n the inhibition of both organism strains by high initial concentrations of their metal ion substrates (Walsh, 1971). Although we have measured a base pair ratio (gc) of 72-73 % for the iron-oxidizing strain of Metallogenium, there are n o similar data available in the literature for comparison with Gallionella o r other Metallogenia. Initial studies showed that the iron-oxidizing strain of Metallogenium is active in the p H 3.5-5.0 range with a n activity optimum at p H 4.1 (Walsh, 1971). Initial ferrous iron concentration directly affected activity. Concentrations over 40 mg/l. increasingly inhibited growth uith a n upper tolerance limit of 150 mg/l. ferrous iron. The phthalate buffer in the growth medibm permitted growth in a medium of higher initial ferrous iron content (Table 111). This effect was probably the result of the formation of a ferrous iron phthalate complex. We investigated the changes in populations of acid mine drainage heterotrophs, of T . ferrooxidans, and of the ironoxidizing Metallogenium in a synthetic mine water system. Table IV shows the changes in bacterial populations in the test system. As the p H decreased from 5.0 to 3.3, the Metallogenium population increased from less than l O 3 / l O O ml at p H 5 to almost 107/100ml at p H 4.2 and then decreased again to less than 103/100ml a t p H 3.5. There was heavy bacterial growth o n the tube surface when the p H declined to 4.3. When the p H decreased from 3.5 to 2.5, the T. ferrooxidans population increased from less than l o 3cells/100 ml to almost 108 cells/100 ml. Table IV also shows a comparison of populations between the test and a control tube system. This control system had not been inoculated with the Metallogenium. The p H of the control system remained above 4.0. The population of T. ferrooxidans remained below l o 3 cells/100 ml and only a minor decline in the heterotrophic population was observed after 35 days. This decrease was significantly less than that observed in the test system. The final characteristics of the test system after 45 days are very similar t o those reported by Tuttle et al., (1968) for a gob pile drainage. Table V shows that the final characteristics of the control system are not similar to those of the gob pile o r the test system. A second control system was run to further demonstrate the effect of p H on the distribution of iron bacteria populations in the column. In this control, the p H was buffered a t 4 with phthalate. No decline in the Metallogenium population and no increase in the T. ferrooxidans population was observed after 45 days. The heterotrophic population again remained high as in the previous control system.
Table V. Comparison of Characteristics of Gob Pile Drainage. with Test or Control Mine Water System9 T. Hetero[ Fe]T , ferrooxidans, t rophs, mg/l. cells/rnl cells/ml G o b pile drainage 2.5-3.0 100-500 30-3,500 1-65 Test system 2.6 80 10,000 500 Control system 4.0 80
