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Microbial refinement of kaolin by iron-reducing bacteria
Applied Clay Science 22 (2002) 47 – 53 www.elsevier.com/locate/clay
Microbial refinement of kaolin by iron-reducing bacteria Eun Young Lee a,b, Kyung-Suk Cho a,*, Hee Wook Ryu c,d a
National Subsurface Environmental Research Laboratory, Ewha Womans University, 11-1 Daehyon-dong, Seodaemun-gu, Seoul 120-750, South Korea b Department of Environmental Engineering, University of Suwon, Suwon P.O. Box 77, Suwon 440-600, South Korea c Department of Chemical and Environmental Engineering, Soong Sil University, 1-1 Sangdo-dong, Dongjak-gu, Seoul 156-743, South Korea d Research Institute of Biological and Environmental Technology, Biosaint Co. 600-16 Shinsa-dong, Kangnam-gu, Seoul 135-120, South Korea Received 21 May 2001; received in revised form 14 January 2002; accepted 28 February 2002
Abstract Low-grade kaolin contains Fe(III) impurities, which cause the detraction of refractoriness and whiteness of porcelain and pottery. Microbial refinement of low-grade kaolin to remove Fe(III) by iron-reducing bacteria was investigated. The removal of Fe(III) impurities could be performed by the indigenous microorganisms in kaolin, but could be enhanced by the inoculation of iron-reducing bacteria. Maltose, sucrose, and glucose could be used as substrates. The removal efficiency of Fe(III) increased with increasing sugar concentrations in the range of 1 – 5% (w/w, sugar/clay). After the microbial refinement, the whiteness of the kaolin increased from 64.49 to 71.50, and the redness decreased remarkably from 7.41 to 2.55. Fe(III) impurities were selectively removed from the kaolin without changing or losing the mineralogical composition of the kaolin by the microbial refinement. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Microbial iron removal; Iron-reducing bacteria; Clay refinement; Kaolin
1. Introduction Kaolin are essential resources with diverse applications in porcelain and pottery manufacturing, the production of construction materials, nuclear waste treatment, paper manufacturing, and cosmetic chemical industries. The quality of kaolin is measured in terms of iron content because Fe (III) impurities in kaolin cause the reduction of refractoriness of products made of kaolin (Povlov and Meshcheryakova, 1983; Ratzenberger, 1988; Stepkowska and Jefferis,
*
Corresponding author. Tel.: +82-2-3277-2393; fax: +82-23277-3275. E-mail address: [email protected] (K.-S. Cho).
1992; Ryu et al., 1995). High-grade kaolin with low Fe(III) content is being exhausted globally. Therefore, the refinement of low-grade clay is a viable and necessary alternative resource to high-grade clay. To remove Fe(III) impurities from kaolin, physical and chemical methods such as flotation, magnetic separation, and chemical treatments have normally been used (Inoue and Yoshida, 1984; Kimura and Tateyama, 1989; Otsuka et al., 1974). However, there are many disadvantages in utilizing these methods. The removal efficiency of iron is low because the iron in kaolin mineral may be tightly adsorbed or it may be in a captured form that is not easily separated by the first two methods. The third method, chemical treatment, decreases the content of organic material. This is the main factor supporting refractoriness, and
0169-1317/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 1 3 1 7 ( 0 2 ) 0 0 111 - 4
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E.Y. Lee et al. / Applied Clay Science 22 (2002) 47–53
results in the decrease of refractoriness. The most pertinent disadvantages are high-capital and operating costs and low-removal efficiency of iron. Accordingly, biological methods using microorganisms, which oxidize or reduce iron, should be considered as new and alternative methods to remove iron impurities from kaolin (Ryu et al., 1995; Stucki, 1988; Hints et al., 1977). The oxidized form of iron (ferric iron, Fe(III)) is generally insoluble, but its reduced form (ferrous iron, Fe(II)) is soluble (Nealson and Saffarini, 1994). While attempts have been made to remove iron impurities from clay using Pseudomonas sp., Azotobacter sp. and Aspergillus niger, studies have documented disadvantages with these methods as well, including low iron removal efficiency and poor economics (Lee et al., 1997). Recently, it was reported that structural iron reduction in Fe(III)-containing clay minerals has been coupled to carbon oxidation in a pure culture under controlled conditions (Kostka and Nealson, 1996; Kostka et al., 1999a). Kostka and Nealson (1996) showed that the microbial clay reduction has been determined to be an anaerobic process, coupled to carbon metabolism and electron transport in Fe(III)-reducing bacteria. The stoichiometry of microbial clay reduction coupled to organic carbon oxidation has been further documented, and was found to be similar to the reduction of Fe oxide minerals (Kostka et al., 1999a). Furthermore, microbial clay reduction has been shown to have a large impact on the surface chemistry of expandable clay minerals, leading to large changes in surface area and cation exchange capacity (Kostka et al., 1999b). In this study, the removal of Fe(III) impurities from low-grade clay by a mixed iron-reducing culture is described. Factors such as inoculum density, carbon source affecting the removal efficiency of Fe(III) were also investigated. To evaluate the quality of kaolin before and after the microbial treatment to remove iron, the chromaticity of kaolin was compared after firing at 1050 jC.
2. Materials and methods 2.1. Kaolin samples The kaolin used in this research contained 3.22% Fe2O3 and was supplied by the Daemyung San-
cheong-Gun, Kyungnam, Korea. After being dried at room temperature, the kaolin sample was ground to a powder by a bowl mill for use in microbial experiments. The average particle size of the kaolin sample was 0.56 mm in diameter. 2.2. Mixed culture of iron-reducing microorganisms The mixed culture of iron-reducing microorganisms was obtained by acclimation in kaolin samples. Fifty grams of clay, 300 ml of tap water, and 2 g of maltose were combined in 500 ml serum bottles. After the head space had been purged with nitrogen gas, the bottles were sealed tightly with a butyl rubber stopper. The bottles were then incubated at 30 jC. Fermentative gases formed during cultivation were collected with a 30 ml syringe installed on each bottle. When the color of the kaolin samples changed from pink to white, 30 ml of the kaolin sample was inoculated into 270 ml of fresh medium. After this process was repeated 10 times, this acclimated mixed culture broth was used as the inoculum source for subsequent experiments. 2.3. Microbial removal of Fe(III) impurities from kaolin All experiments were carried out in 120 ml serum bottles. The bottles were charged with 10 g of dry kaolin, 50 ml of tap water, 0.4 g of maltose, and 1 ml of mixed culture broth. Incubation methods were performed in the same manner as described in the cultivation of iron-reducing microorganisms. The effect of inoculation was evaluated by comparing the removal efficiencies of Fe(III) impurities, with and without inoculation of the mixed iron-reducing culture. An aseptic autoclaved (121 jC, 15 min) control was also included. To investigate the effect of various maltose concentrations on iron reduction, 1 – 5% (w/w) of maltose was added as a carbon source to the cultures. The Fe(III)-reducing activities of these cultures was supplemented with 4% (w/w, sugar/clay) of glucose, sucrose, galactose, and maltose, each of which was evaluated to demonstrate the effect of the type of carbon source. The effect of inoculum density on the removal efficiencies of Fe(III) impurities in kaolin was inves-
E.Y. Lee et al. / Applied Clay Science 22 (2002) 47–53
tigated by adding different volumes of inocula (0.5, 1, 2, 3, 4 ml) to the medium. That is, when it was defined as inoculation volume per dry clay weight, the inoculum density was 5, 10, 20, 30, and 40% (v/w). In every experiment, the deposited clay samples at the bottom of the bottles were mixed for 1 min daily during the cultivation. Fe(III) reduction was monitored by measuring Fe(II) concentration in the supernatant sampled from each bottle. All experiments were duplicated, and an additional run of the experiment was performed if replicates differed from the mean by more than 10%. 2.4. Methods of analysis The pH and redox potential (Eh) in the culture medium during the reduction reaction were monitored by a pH probe (9157BN, ORION, USA) and a redox probe (9778BN, ORION) attached to a pH meter (420A, ORION). To analyze the total iron content containing the kaolin sample, iron was extracted from the kaolin by heating it for 15 to 20 min in 6N HCl. 10% hydroxylamine hydrochloride was added to this extract to reduce Fe(III) to Fe(II), and then total iron content was determined by the o-phenanthroline method (Furman, 1975). One milliliter of supernatant was periodically sampled from each bottle using a syringe to determine the reduced Fe(II) concentration. The Fe(II) concentration in the supernatant was also determined by the o-phenanthroline method (Furman, 1975). To compare the color of kaolin before and after the microbial treatment, the chromaticity was measured with a colorimeter (ND-300A, Rigaku goniometer, Japan) after firing clay at 1050 jC for 2 h. Color as perceived has three dimensions: hue, chroma, and lightness (Lee et al., 2000). Chromaticity includes hue and chroma (saturation), specified by two chromaticity coordinates. Since these two coordinates cannot describe a color completely, a lightness factor was included to identify a color precisely. Colorimeter measured absolute chromaticity of the specimen in L*a*b* color spaces by the Munsell color system. L* is the lightness factor, a* (red) and b* (yellow) are the chromaticity coordinates. The total elemental analysis of kaolin was made using an energy-dispersive X-ray spectroscopy (EDX 3000, Horiba, Japan).
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3. Results and discussion 3.1. Effect of inoculation with an iron-reducing culture The effect of inoculation with an iron-reducing culture on Fe(III) reduction was investigated (Fig. 1). Two kinds of controls were used: uninoculated control to show what activities indigenous microorganisms already present in the kaolin had; and sterile control, which was used to show the chemical reaction. Reduced Fe(II) was not detected in the uninoculated bottle containing sterilized kaolin. In the uninoculated bottle containing unsterilized kaolin, the reduced Fe(II) began to be observed after 30 h of lag time. This result indicated the existence of inherent Fe-reducing microorganisms in the kaolin. In the bottle inoculated with Fe-reducing bacteria, large amounts of reduced Fe(II) were detected. From these results, it was determined that the removal reaction of Fe(III) from the kaolin was biologically performed. Iron is transformed between the ferrous (Fe(II)) and ferric(Fe(III)) oxidation states by microorganisms. Fe(III) iron precipitates in alkaline environments as
Fig. 1. Effect of iron-reducing bacteria on Fe(III) reduction. o, uninoculated bottle containing sterilized clay; ., uninoculated bottle containing native clay with indigenous bacteria; z, bottle containing native clay inoculated supplemented with iron-reducing bacteria.
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E.Y. Lee et al. / Applied Clay Science 22 (2002) 47–53
Fig. 2. Time profiles of pH, Eh, and Fe(II) concentration during microbial refinement of kaolin clay. ., Eh; o, pH; E, Fe(II) concentration.
ferric hydroxide. The insoluble Fe(III) may be reduced under anaerobic conditions to the more soluble Fe(II) by iron-reducing bacteria and removed from the kaolin. Under alkaline to neutral conditions, Fe(II) is inherently unstable in the presence of O2 and is oxidized to Fe(III) (Atlas and Bartha, 1993). When the iron-reducing bacteria were inoculated into kaolin, it was assumed that gases were produced due to the fermentation of an organic compound (maltose) during the first incubation period. The major components of the gases were hydrogen, carbon dioxide, and a small amount of methane, all of which were detected through gas chromatography analysis (data not shown). The iron reduction reaction in kaolin began in its pink color and gradually faded with the generation of these fermentative gases. When gas generation or color change was no longer observed because of exhaustion of the carbon source, the reduction reaction ceased to progress. The refinement of kaolin was carried out through these processes. The activity of indigenous microorganisms were observed by the production of gas, which increased in the serum bottle, and by the formation of white precipitates in the uninoculated control. However, the rate of Fe(III) reduction in the uninoculated control was slower than that in the inoculated flasks. Also, neither a fermentation reaction of the organic compound nor a chemical iron reduction reaction was detected in the control sterilized by autoclaving at 121 jC for 15 min. This suggested that iron reduction is microbially mediated. For that reason, the kaolin was inoculated with a
mixed laboratory-bred culture consisting of very active iron-reducing bacteria. Fig. 2 shows pH, Eh, and Fe(II) production profiles with cultivation time in the kaolin microbial cultures. Because organic acids such as acetate, butyrate and propionate were accumulated from the fermentation reaction of maltose, the pH of the medium decreased from 6 to 4 (data not shown). During incubation, the initial Eh was 470 mV and then decreased to 210 mV, indicating an anaerobic environment where the reduction reaction performed well. The soluble Fe(II) began to be detected at 22 h when the Eh was 200 mV. Active Fe3+ reduction was observed in an anaerobic environment after the Eh was around 200 mV. This result supports research noting that Fe(III) can generally serve as an electron acceptor in microbial metabolism around 182 mV (Anderson, 1995). 3.2. Effect of carbon source on iron reduction The reduction rates of Fe(III) at each condition where 1 – 5% of maltose was supplemented as a carbon source are shown in Fig. 3. The rate was calculated from the time profiles of the concentration of Fe(II) in the culture media. When no carbon source was added, there was no reduction reaction. The leached Fe(II) concentration and the rate of iron reduction increased as a function of maltose concentration. The iron reduction rates were 1.20 and 1.35
Fig. 3. Effect of maltose concentration on Fe(III) reduction rate.
E.Y. Lee et al. / Applied Clay Science 22 (2002) 47–53
Fig. 4. Effect of sugars on Fe(III) reduction. ., maltose; o, sucrose; z, glucose; q, galactose.
mg Fe2+ g clay 1 day 1 at 4% and 5% of maltose, respectively. Neither a fermentation reaction of maltose nor an iron reduction reaction was observed at maltose concentrations higher than 6%. It is considered that excessive maltose increased the osmotic potential and resulted in the inhibition of activities of iron-reducing bacteria. The characterization of the Fe(III) reduction was compared with that of maltose when substrates other than maltose (glucose, sucrose, and galactose) were used (Fig. 4). Similar results were obtained when glucose was used as the C-source: the soluble reducing Fe(II) began to be detected after 26 h, went on being generated until 46 h, and accumulated to a final concentration of 3.4 mg Fe2+ g clay 1. In a medium supplemented with sucrose as a carbon source, the reduction reaction began 9 h later than that of glucose or maltose, and the accumulated Fe(II) concentration was 3 mg Fe2+ g clay 1. When galactose was used, the reduction reaction began to be observed at 57 h and generated smaller amounts of leached iron than in the other cases. The percentage removal rates of Fe(III) were calculated from the concentration of leached iron based on the total iron in kaolin during the 46-h incubation: the rates were 27%, 28%, 26%, and 11% when maltose, glucose, sucrose, and galactose were
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used as carbon sources, respectively. It has been reported that the reactivity of Fe(III) as an electron acceptor in aquatic sediment is related to its degree of crystallinity (Phillips et al., 1993; Munch and Ottow, 1980; Pronk and Johnson, 1992). The microbial reduction was more active in ferric hydroxide (Fe(OH)3) than in geothite (FeOOH) (Phillips et al., 1993). The last form, hematite (Fe2O3), was difficult to reduce by microorganisms (Phillips et al., 1993). It appears that the lower the degree of crystallization, the higher is the probability of reduction (Phillips et al., 1993). Because Fe(III) impurities in kaolin clay were represented as hematite (Hints et al., 1977), a form in which microorganisms are not as active, the percentage removal of Fe(III) was thought not to exceed 30%. The Fe3+ reduction was understood to performed by the cooperation of microorganisms with different metabolic processes (Nealson and Saffarini, 1994). When organic acids, alcohol, and hydrogen gases were generated from the degradation of sugars during the first incubation period, iron reduction was not distinct as only a small amount of electrons were transferred to Fe(III). However, the reduction reaction supplemented with acetate, the representative fermentation product, and hydrogen gas were shown to produce a large amount of Fe2+ (Lovley and Phillips, 1988; Balashova and Zavarzin, 1980; MacDonell and Colwell, 1985; Caccavo et al., 1992).
Fig. 5. Effect of inoculum density on Fe(III) reduction rate.
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In this study, the active fermentation and inactive iron reduction were observed at the same time during the first incubation period, and the active iron reduction began after a lag period of 24 h (Figs. 2 and 4). This result indicated that the removal of iron impurities from clay might be carried out through two steps: first fermentation and then iron reduction. 3.3. Effect of inoculum density on iron reduction Fig. 5 shows the effect of inoculum density of mixed iron-reducing culture on the iron reduction rates. The iron reduction rates were increased with increasing inoculum density below 20% (v/w). The maximum iron reduction rate was 1.31 mg Fe2+ g clay 1 day 1 at 20% (v/w) of the inoculum density. 3.4. Comparison of chromaticity and composition of kaolin before and after the biological treatment The colors of the raw kaolin sample affect the color of the ceramic product after the coloring process in porcelain and pottery manufacturing. The red color of kaolin gets stronger as the amount of iron oxide increases. This results in the impurities that reduce refractoriness and form cracks during firing (Povlov and Meshcheryakova, 1983; Ratzenberger, 1988; Stepkowska and Jefferis, 1992; Ryu et al., 1995). Therefore, the color of kaolin is the most important factor in determining the quality of the clay. The white clay was classified as high quality (Povlov and Meshcheryakova, 1983; Ratzenberger, 1988; Stepkowska and Jefferis, 1992; Ryu et al., 1995). The chromaticity of the kaolin samples before and after the microbial treatment by iron-reducing bacteria is summarized in Table 1. After the firing at 1050 jC, the brightness of refined kaolin improved with increasing
Table 1 Chromaticity of clay samples
Redness Whiteness Brightness Yellowness
Before firing
After firing
Raw clay
Refined clay
Raw clay
Refined clay
7.41 64.49 70.25 17.93
2.55 71.50 77.37 17.14
12.80 63.65 69.41 14.87
8.58 70.07 75.54 14.95
Table 2 Comparison of mineral compositions of clay before and after microbial treatment (4% (w/w) maltose was supplemented as a carbon source) Weight percentage (%)
Al2O3 SiO2 K2 O CaO TiO2 Fe2O3
Before treatment
After treatment
40.08 53.61 0.46 0.51 0.23 5.11
40.67 54.40 0.52 0.33 0.35 3.73
whiteness and decreasing redness compared with the unrefined raw kaolin. The mineral compositions, which determined the original characteristics of kaolin, did not change after the biological treatment to refine the low-grade kaolin by iron-reducing microorganisms. The mineral compositions of the kaolin samples, before and after the microbial treatment as determined by EDX, are shown in Table 2. The microbial refinement or the firing did not affect the main compositions of elements. The composition of Al2O3 and SiO2 was 40 and 54 wt.%, respectively, which represented a constant value before and after the treatment. These results derived from the substrate specify that Fe(III) impurities from kaolin were selectively leached to the culture medium by iron-reducing bacteria. The microbial refinement did not result in any unfavorable modifications in mineralogical compositions of the kaolin.
4. Conclusions The removal of Fe(III) impurities from low-grade kaolin by a mixed iron-reducing culture was characterized. The insoluble Fe(III) was reduced to soluble Fe(II) by iron-reducing bacteria and removed from the kaolin. The reduction of Fe(III) could have been catalyzed reaction either by fermentative or by dissimilatory Fe-reducing bacteria. After microbial refinement, the brightness of the refined kaolin improved with increasing whiteness and decreasing redness compared with raw clay. The microbial refinement also did not cause any unfavorable modifications in mineralogical compositions of the kaolin.
E.Y. Lee et al. / Applied Clay Science 22 (2002) 47–53
Acknowledgements Funding for this research was provided by the National Research Laboratory Program of the Korean Ministry of Science and Technology.
References Anderson, W.C., 1995. Innovative Site Remediation Technology: Bioremediation. Water Environment Federation, Alexandria, p. 26. Atlas, R.M., Bartha, R., 1993. The iron cycle. Microbial Ecology, Fundamentals and Applications, 3rd edn. The Benjamin/Cummings, Menlo Park, CA, pp. 334 – 335. Balashova, V.V., Zavarzin, G.A., 1980. Anaerobic reduction of ferric iron by hydrogen bacteria. Microbiology 48, 635 – 639. Caccavo Jr., F., Blakemore, R.P., Lovley, D.R., 1992. A hydrogenoxidizing, Fe(III)-reducing microorganism from the Great Bay estuary, New Hampshire. Appl. Environ. Microbiol. 58, 3211 – 3216. Furman, N.H., 1975. Standard Methods of Chemical Analysis, 6th edn. R. E. Krieger Publishing, Huntington, NY. Hints, I., Kiss, S., Papacostea, P., Radulescu, D., Dragan-Bularda, M., 1977. Application of microbiological method for diminution of Fe2O3 content of kaolins. 4th Symposium of Soil Biology. Rumanian National Society for soil Science, Bucharest, pp. 387 – 391. Inoue, K., Yoshida, A., 1984. Iron leaching of Shirasu by acid treatment. J. Ceram. Soc. Jpn. 92, 520 – 524. Kimura, K., Tateyama, H., 1989. Refinement of the low-grade Amakusa pottery stone by hydrothermal treatment. J. Ceram. Soc. Jpn. 97, 439 – 446. Kostka, J.E., Nealson, K.H., 1996. Dissolution and reduction of magnetite by bacteria. Environ. Sci. Technol. 29, 2535 – 2540. Kostka, J.E., Haefele, E., Viehweger, R., Stucki, J.W., 1999a. Respiration and dissolution of Fe(III)-containing clay minerals by bacteria. Environ. Sci. Technol. 33, 3127 – 3133. Kostka, J.E., Wu, J., Nealson, K.H., Stucki, J.W., 1999b. The impact of structural Fe(III) reduction by bacteria on the surface chemistry of clay minerals. Geochim. Cosmochim. Acta 63, 3705 – 3713.
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Lee, E.Y., Cho, K.S., Ryu, H.W., Bae, M., 1997. Effect of carbon sources on biological removal of iron impurities from kaolinite. Korean J. Appl. Microbiol. Biotechnol. 25, 552 – 559. Lee, D.Y., Kim, D.J., Song, Y.S., 2000. Chromaticity, hydrothermal stability, and mechanical properties of t-Zr2/Al2O3 composites doped with yttrium, niobium, and ferric oxides. Mater. Sci. Eng., A 289, 1 – 7. Lovley, D.R., Phillips, E.J.P., 1988. Manganese inhibition of microbial iron reduction in anaerobic sediments. Geomicrobiol. J. 6, 145 – 155. MacDonell, M.T., Colwell, R.R., 1985. Phylogeny of the Vibrionaceae, and recommendation for two new genera, Listonella and Shewanella. Syst. Appl. Microbiol. 6, 171 – 182. Munch, J.C., Ottow, J.C.G., 1980. Preferential reduction of amorphous to crystalline iron oxides by bacterial activity. Soil Sci. 129, 15 – 21. Nealson, K.H., Saffarini, D., 1994. Iron and manganese in anaerobic respiration: environmental significance, physiology, and regulation. Annu. Rev. Microbiol. 48, 311 – 343. Otsuka, N., Hayashi, T., Okanishi, K., Shiraki, Y., 1974. The removal of iron oxide from clay by sodium dithionite-sulfuric acid system (III). Nendo Kagaku 14, 45 – 57. Phillips, E.J.P., Lovley, D.R., Roden, E.E., 1993. Composition of non-microbially reducible Fe(III) in aquatic sediments. Appl. Environ. Microbiol. 59, 2727 – 2729. Povlov, V.F., Meshcheryakova, V., 1983. Reducing the coloring effects of iron oxides in porcelain bodies. Glass Ceram. 40, 50 – 152. Pronk, J.T., Johnson, D.B., 1992. Oxidation and reduction of iron by acidophilic bacteria. Geomicrobial. J. 10, 153 – 171. Ratzenberger, H., 1988. The influence of the mineralogical composition of structural ceramics and heavy clay materials on kiln scumming and efflorescence. Ziegelind Int. 41, 99 – 105. Ryu, H.W., Cho, K.S., Chang, Y.K., Kim, S.D., Mori, T., 1995. Refinement of low-grade clay by microbial removal of sulfur and iron compounds using Thiobacillus ferrooxidans. J. Ferment. Bioeng. 80, 46 – 52. Stepkowska, E.T., Jefferis, S.A., 1992. Influence of microstructure on firing color of clays. Appl. Clay Sci. 6, 319 – 342. Stucki, J.W., 1988. Structural iron in smectites. In: Stucki, J.W., Goodman, B.A., Schwertmann, U. (Eds.), Iron in Soils and Clay Minerals. D. Reidel Publishing, Boston, pp. 625 – 675.
Microbial refinement of kaolin by iron-reducing bacteria Eun Young Lee a,b, Kyung-Suk Cho a,*, Hee Wook Ryu c,d a
National Subsurface Environmental Research Laboratory, Ewha Womans University, 11-1 Daehyon-dong, Seodaemun-gu, Seoul 120-750, South Korea b Department of Environmental Engineering, University of Suwon, Suwon P.O. Box 77, Suwon 440-600, South Korea c Department of Chemical and Environmental Engineering, Soong Sil University, 1-1 Sangdo-dong, Dongjak-gu, Seoul 156-743, South Korea d Research Institute of Biological and Environmental Technology, Biosaint Co. 600-16 Shinsa-dong, Kangnam-gu, Seoul 135-120, South Korea Received 21 May 2001; received in revised form 14 January 2002; accepted 28 February 2002
Abstract Low-grade kaolin contains Fe(III) impurities, which cause the detraction of refractoriness and whiteness of porcelain and pottery. Microbial refinement of low-grade kaolin to remove Fe(III) by iron-reducing bacteria was investigated. The removal of Fe(III) impurities could be performed by the indigenous microorganisms in kaolin, but could be enhanced by the inoculation of iron-reducing bacteria. Maltose, sucrose, and glucose could be used as substrates. The removal efficiency of Fe(III) increased with increasing sugar concentrations in the range of 1 – 5% (w/w, sugar/clay). After the microbial refinement, the whiteness of the kaolin increased from 64.49 to 71.50, and the redness decreased remarkably from 7.41 to 2.55. Fe(III) impurities were selectively removed from the kaolin without changing or losing the mineralogical composition of the kaolin by the microbial refinement. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Microbial iron removal; Iron-reducing bacteria; Clay refinement; Kaolin
1. Introduction Kaolin are essential resources with diverse applications in porcelain and pottery manufacturing, the production of construction materials, nuclear waste treatment, paper manufacturing, and cosmetic chemical industries. The quality of kaolin is measured in terms of iron content because Fe (III) impurities in kaolin cause the reduction of refractoriness of products made of kaolin (Povlov and Meshcheryakova, 1983; Ratzenberger, 1988; Stepkowska and Jefferis,
*
Corresponding author. Tel.: +82-2-3277-2393; fax: +82-23277-3275. E-mail address: [email protected] (K.-S. Cho).
1992; Ryu et al., 1995). High-grade kaolin with low Fe(III) content is being exhausted globally. Therefore, the refinement of low-grade clay is a viable and necessary alternative resource to high-grade clay. To remove Fe(III) impurities from kaolin, physical and chemical methods such as flotation, magnetic separation, and chemical treatments have normally been used (Inoue and Yoshida, 1984; Kimura and Tateyama, 1989; Otsuka et al., 1974). However, there are many disadvantages in utilizing these methods. The removal efficiency of iron is low because the iron in kaolin mineral may be tightly adsorbed or it may be in a captured form that is not easily separated by the first two methods. The third method, chemical treatment, decreases the content of organic material. This is the main factor supporting refractoriness, and
0169-1317/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 1 3 1 7 ( 0 2 ) 0 0 111 - 4
48
E.Y. Lee et al. / Applied Clay Science 22 (2002) 47–53
results in the decrease of refractoriness. The most pertinent disadvantages are high-capital and operating costs and low-removal efficiency of iron. Accordingly, biological methods using microorganisms, which oxidize or reduce iron, should be considered as new and alternative methods to remove iron impurities from kaolin (Ryu et al., 1995; Stucki, 1988; Hints et al., 1977). The oxidized form of iron (ferric iron, Fe(III)) is generally insoluble, but its reduced form (ferrous iron, Fe(II)) is soluble (Nealson and Saffarini, 1994). While attempts have been made to remove iron impurities from clay using Pseudomonas sp., Azotobacter sp. and Aspergillus niger, studies have documented disadvantages with these methods as well, including low iron removal efficiency and poor economics (Lee et al., 1997). Recently, it was reported that structural iron reduction in Fe(III)-containing clay minerals has been coupled to carbon oxidation in a pure culture under controlled conditions (Kostka and Nealson, 1996; Kostka et al., 1999a). Kostka and Nealson (1996) showed that the microbial clay reduction has been determined to be an anaerobic process, coupled to carbon metabolism and electron transport in Fe(III)-reducing bacteria. The stoichiometry of microbial clay reduction coupled to organic carbon oxidation has been further documented, and was found to be similar to the reduction of Fe oxide minerals (Kostka et al., 1999a). Furthermore, microbial clay reduction has been shown to have a large impact on the surface chemistry of expandable clay minerals, leading to large changes in surface area and cation exchange capacity (Kostka et al., 1999b). In this study, the removal of Fe(III) impurities from low-grade clay by a mixed iron-reducing culture is described. Factors such as inoculum density, carbon source affecting the removal efficiency of Fe(III) were also investigated. To evaluate the quality of kaolin before and after the microbial treatment to remove iron, the chromaticity of kaolin was compared after firing at 1050 jC.
2. Materials and methods 2.1. Kaolin samples The kaolin used in this research contained 3.22% Fe2O3 and was supplied by the Daemyung San-
cheong-Gun, Kyungnam, Korea. After being dried at room temperature, the kaolin sample was ground to a powder by a bowl mill for use in microbial experiments. The average particle size of the kaolin sample was 0.56 mm in diameter. 2.2. Mixed culture of iron-reducing microorganisms The mixed culture of iron-reducing microorganisms was obtained by acclimation in kaolin samples. Fifty grams of clay, 300 ml of tap water, and 2 g of maltose were combined in 500 ml serum bottles. After the head space had been purged with nitrogen gas, the bottles were sealed tightly with a butyl rubber stopper. The bottles were then incubated at 30 jC. Fermentative gases formed during cultivation were collected with a 30 ml syringe installed on each bottle. When the color of the kaolin samples changed from pink to white, 30 ml of the kaolin sample was inoculated into 270 ml of fresh medium. After this process was repeated 10 times, this acclimated mixed culture broth was used as the inoculum source for subsequent experiments. 2.3. Microbial removal of Fe(III) impurities from kaolin All experiments were carried out in 120 ml serum bottles. The bottles were charged with 10 g of dry kaolin, 50 ml of tap water, 0.4 g of maltose, and 1 ml of mixed culture broth. Incubation methods were performed in the same manner as described in the cultivation of iron-reducing microorganisms. The effect of inoculation was evaluated by comparing the removal efficiencies of Fe(III) impurities, with and without inoculation of the mixed iron-reducing culture. An aseptic autoclaved (121 jC, 15 min) control was also included. To investigate the effect of various maltose concentrations on iron reduction, 1 – 5% (w/w) of maltose was added as a carbon source to the cultures. The Fe(III)-reducing activities of these cultures was supplemented with 4% (w/w, sugar/clay) of glucose, sucrose, galactose, and maltose, each of which was evaluated to demonstrate the effect of the type of carbon source. The effect of inoculum density on the removal efficiencies of Fe(III) impurities in kaolin was inves-
E.Y. Lee et al. / Applied Clay Science 22 (2002) 47–53
tigated by adding different volumes of inocula (0.5, 1, 2, 3, 4 ml) to the medium. That is, when it was defined as inoculation volume per dry clay weight, the inoculum density was 5, 10, 20, 30, and 40% (v/w). In every experiment, the deposited clay samples at the bottom of the bottles were mixed for 1 min daily during the cultivation. Fe(III) reduction was monitored by measuring Fe(II) concentration in the supernatant sampled from each bottle. All experiments were duplicated, and an additional run of the experiment was performed if replicates differed from the mean by more than 10%. 2.4. Methods of analysis The pH and redox potential (Eh) in the culture medium during the reduction reaction were monitored by a pH probe (9157BN, ORION, USA) and a redox probe (9778BN, ORION) attached to a pH meter (420A, ORION). To analyze the total iron content containing the kaolin sample, iron was extracted from the kaolin by heating it for 15 to 20 min in 6N HCl. 10% hydroxylamine hydrochloride was added to this extract to reduce Fe(III) to Fe(II), and then total iron content was determined by the o-phenanthroline method (Furman, 1975). One milliliter of supernatant was periodically sampled from each bottle using a syringe to determine the reduced Fe(II) concentration. The Fe(II) concentration in the supernatant was also determined by the o-phenanthroline method (Furman, 1975). To compare the color of kaolin before and after the microbial treatment, the chromaticity was measured with a colorimeter (ND-300A, Rigaku goniometer, Japan) after firing clay at 1050 jC for 2 h. Color as perceived has three dimensions: hue, chroma, and lightness (Lee et al., 2000). Chromaticity includes hue and chroma (saturation), specified by two chromaticity coordinates. Since these two coordinates cannot describe a color completely, a lightness factor was included to identify a color precisely. Colorimeter measured absolute chromaticity of the specimen in L*a*b* color spaces by the Munsell color system. L* is the lightness factor, a* (red) and b* (yellow) are the chromaticity coordinates. The total elemental analysis of kaolin was made using an energy-dispersive X-ray spectroscopy (EDX 3000, Horiba, Japan).
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3. Results and discussion 3.1. Effect of inoculation with an iron-reducing culture The effect of inoculation with an iron-reducing culture on Fe(III) reduction was investigated (Fig. 1). Two kinds of controls were used: uninoculated control to show what activities indigenous microorganisms already present in the kaolin had; and sterile control, which was used to show the chemical reaction. Reduced Fe(II) was not detected in the uninoculated bottle containing sterilized kaolin. In the uninoculated bottle containing unsterilized kaolin, the reduced Fe(II) began to be observed after 30 h of lag time. This result indicated the existence of inherent Fe-reducing microorganisms in the kaolin. In the bottle inoculated with Fe-reducing bacteria, large amounts of reduced Fe(II) were detected. From these results, it was determined that the removal reaction of Fe(III) from the kaolin was biologically performed. Iron is transformed between the ferrous (Fe(II)) and ferric(Fe(III)) oxidation states by microorganisms. Fe(III) iron precipitates in alkaline environments as
Fig. 1. Effect of iron-reducing bacteria on Fe(III) reduction. o, uninoculated bottle containing sterilized clay; ., uninoculated bottle containing native clay with indigenous bacteria; z, bottle containing native clay inoculated supplemented with iron-reducing bacteria.
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Fig. 2. Time profiles of pH, Eh, and Fe(II) concentration during microbial refinement of kaolin clay. ., Eh; o, pH; E, Fe(II) concentration.
ferric hydroxide. The insoluble Fe(III) may be reduced under anaerobic conditions to the more soluble Fe(II) by iron-reducing bacteria and removed from the kaolin. Under alkaline to neutral conditions, Fe(II) is inherently unstable in the presence of O2 and is oxidized to Fe(III) (Atlas and Bartha, 1993). When the iron-reducing bacteria were inoculated into kaolin, it was assumed that gases were produced due to the fermentation of an organic compound (maltose) during the first incubation period. The major components of the gases were hydrogen, carbon dioxide, and a small amount of methane, all of which were detected through gas chromatography analysis (data not shown). The iron reduction reaction in kaolin began in its pink color and gradually faded with the generation of these fermentative gases. When gas generation or color change was no longer observed because of exhaustion of the carbon source, the reduction reaction ceased to progress. The refinement of kaolin was carried out through these processes. The activity of indigenous microorganisms were observed by the production of gas, which increased in the serum bottle, and by the formation of white precipitates in the uninoculated control. However, the rate of Fe(III) reduction in the uninoculated control was slower than that in the inoculated flasks. Also, neither a fermentation reaction of the organic compound nor a chemical iron reduction reaction was detected in the control sterilized by autoclaving at 121 jC for 15 min. This suggested that iron reduction is microbially mediated. For that reason, the kaolin was inoculated with a
mixed laboratory-bred culture consisting of very active iron-reducing bacteria. Fig. 2 shows pH, Eh, and Fe(II) production profiles with cultivation time in the kaolin microbial cultures. Because organic acids such as acetate, butyrate and propionate were accumulated from the fermentation reaction of maltose, the pH of the medium decreased from 6 to 4 (data not shown). During incubation, the initial Eh was 470 mV and then decreased to 210 mV, indicating an anaerobic environment where the reduction reaction performed well. The soluble Fe(II) began to be detected at 22 h when the Eh was 200 mV. Active Fe3+ reduction was observed in an anaerobic environment after the Eh was around 200 mV. This result supports research noting that Fe(III) can generally serve as an electron acceptor in microbial metabolism around 182 mV (Anderson, 1995). 3.2. Effect of carbon source on iron reduction The reduction rates of Fe(III) at each condition where 1 – 5% of maltose was supplemented as a carbon source are shown in Fig. 3. The rate was calculated from the time profiles of the concentration of Fe(II) in the culture media. When no carbon source was added, there was no reduction reaction. The leached Fe(II) concentration and the rate of iron reduction increased as a function of maltose concentration. The iron reduction rates were 1.20 and 1.35
Fig. 3. Effect of maltose concentration on Fe(III) reduction rate.
E.Y. Lee et al. / Applied Clay Science 22 (2002) 47–53
Fig. 4. Effect of sugars on Fe(III) reduction. ., maltose; o, sucrose; z, glucose; q, galactose.
mg Fe2+ g clay 1 day 1 at 4% and 5% of maltose, respectively. Neither a fermentation reaction of maltose nor an iron reduction reaction was observed at maltose concentrations higher than 6%. It is considered that excessive maltose increased the osmotic potential and resulted in the inhibition of activities of iron-reducing bacteria. The characterization of the Fe(III) reduction was compared with that of maltose when substrates other than maltose (glucose, sucrose, and galactose) were used (Fig. 4). Similar results were obtained when glucose was used as the C-source: the soluble reducing Fe(II) began to be detected after 26 h, went on being generated until 46 h, and accumulated to a final concentration of 3.4 mg Fe2+ g clay 1. In a medium supplemented with sucrose as a carbon source, the reduction reaction began 9 h later than that of glucose or maltose, and the accumulated Fe(II) concentration was 3 mg Fe2+ g clay 1. When galactose was used, the reduction reaction began to be observed at 57 h and generated smaller amounts of leached iron than in the other cases. The percentage removal rates of Fe(III) were calculated from the concentration of leached iron based on the total iron in kaolin during the 46-h incubation: the rates were 27%, 28%, 26%, and 11% when maltose, glucose, sucrose, and galactose were
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used as carbon sources, respectively. It has been reported that the reactivity of Fe(III) as an electron acceptor in aquatic sediment is related to its degree of crystallinity (Phillips et al., 1993; Munch and Ottow, 1980; Pronk and Johnson, 1992). The microbial reduction was more active in ferric hydroxide (Fe(OH)3) than in geothite (FeOOH) (Phillips et al., 1993). The last form, hematite (Fe2O3), was difficult to reduce by microorganisms (Phillips et al., 1993). It appears that the lower the degree of crystallization, the higher is the probability of reduction (Phillips et al., 1993). Because Fe(III) impurities in kaolin clay were represented as hematite (Hints et al., 1977), a form in which microorganisms are not as active, the percentage removal of Fe(III) was thought not to exceed 30%. The Fe3+ reduction was understood to performed by the cooperation of microorganisms with different metabolic processes (Nealson and Saffarini, 1994). When organic acids, alcohol, and hydrogen gases were generated from the degradation of sugars during the first incubation period, iron reduction was not distinct as only a small amount of electrons were transferred to Fe(III). However, the reduction reaction supplemented with acetate, the representative fermentation product, and hydrogen gas were shown to produce a large amount of Fe2+ (Lovley and Phillips, 1988; Balashova and Zavarzin, 1980; MacDonell and Colwell, 1985; Caccavo et al., 1992).
Fig. 5. Effect of inoculum density on Fe(III) reduction rate.
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In this study, the active fermentation and inactive iron reduction were observed at the same time during the first incubation period, and the active iron reduction began after a lag period of 24 h (Figs. 2 and 4). This result indicated that the removal of iron impurities from clay might be carried out through two steps: first fermentation and then iron reduction. 3.3. Effect of inoculum density on iron reduction Fig. 5 shows the effect of inoculum density of mixed iron-reducing culture on the iron reduction rates. The iron reduction rates were increased with increasing inoculum density below 20% (v/w). The maximum iron reduction rate was 1.31 mg Fe2+ g clay 1 day 1 at 20% (v/w) of the inoculum density. 3.4. Comparison of chromaticity and composition of kaolin before and after the biological treatment The colors of the raw kaolin sample affect the color of the ceramic product after the coloring process in porcelain and pottery manufacturing. The red color of kaolin gets stronger as the amount of iron oxide increases. This results in the impurities that reduce refractoriness and form cracks during firing (Povlov and Meshcheryakova, 1983; Ratzenberger, 1988; Stepkowska and Jefferis, 1992; Ryu et al., 1995). Therefore, the color of kaolin is the most important factor in determining the quality of the clay. The white clay was classified as high quality (Povlov and Meshcheryakova, 1983; Ratzenberger, 1988; Stepkowska and Jefferis, 1992; Ryu et al., 1995). The chromaticity of the kaolin samples before and after the microbial treatment by iron-reducing bacteria is summarized in Table 1. After the firing at 1050 jC, the brightness of refined kaolin improved with increasing
Table 1 Chromaticity of clay samples
Redness Whiteness Brightness Yellowness
Before firing
After firing
Raw clay
Refined clay
Raw clay
Refined clay
7.41 64.49 70.25 17.93
2.55 71.50 77.37 17.14
12.80 63.65 69.41 14.87
8.58 70.07 75.54 14.95
Table 2 Comparison of mineral compositions of clay before and after microbial treatment (4% (w/w) maltose was supplemented as a carbon source) Weight percentage (%)
Al2O3 SiO2 K2 O CaO TiO2 Fe2O3
Before treatment
After treatment
40.08 53.61 0.46 0.51 0.23 5.11
40.67 54.40 0.52 0.33 0.35 3.73
whiteness and decreasing redness compared with the unrefined raw kaolin. The mineral compositions, which determined the original characteristics of kaolin, did not change after the biological treatment to refine the low-grade kaolin by iron-reducing microorganisms. The mineral compositions of the kaolin samples, before and after the microbial treatment as determined by EDX, are shown in Table 2. The microbial refinement or the firing did not affect the main compositions of elements. The composition of Al2O3 and SiO2 was 40 and 54 wt.%, respectively, which represented a constant value before and after the treatment. These results derived from the substrate specify that Fe(III) impurities from kaolin were selectively leached to the culture medium by iron-reducing bacteria. The microbial refinement did not result in any unfavorable modifications in mineralogical compositions of the kaolin.
4. Conclusions The removal of Fe(III) impurities from low-grade kaolin by a mixed iron-reducing culture was characterized. The insoluble Fe(III) was reduced to soluble Fe(II) by iron-reducing bacteria and removed from the kaolin. The reduction of Fe(III) could have been catalyzed reaction either by fermentative or by dissimilatory Fe-reducing bacteria. After microbial refinement, the brightness of the refined kaolin improved with increasing whiteness and decreasing redness compared with raw clay. The microbial refinement also did not cause any unfavorable modifications in mineralogical compositions of the kaolin.
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Acknowledgements Funding for this research was provided by the National Research Laboratory Program of the Korean Ministry of Science and Technology.
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