Clay Minerals
(2005) 40, 481±491
How kaolinite plays an essential role in remediating oil-polluted seawater S. K. CHAERUN 1 , 2 , * AND K. TAZAKI 3 1
Indonesia Agrogeology Development Center, Jenderal Soedirman University, Jl. Dr. Bunjamin No. 708, Grendeng, 2
Purwokerto 53122, Central Java, Indonesia, Graduate School of Natural Science and Technology, Kanazawa 3
University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan, and Department of Earth Sciences, Faculty of Science, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan
(
Received 8 June 2005; revised 19 August 2005
)
ABSTRACT: An investigation was carried out on the bioavailability of kaolinite and the role it plays in remediating oil-polluted seawater, since kaolinite is known to enable hydrocarbon-degrading bacteria to grow well. Experimental results revealed that significant amounts of Al and Si dissolved from kaolinite were not observed (( > 0.05) in comparison with a control sample which contained no kaolinite) in the aqueous phase during the ~24 day experimental period. Transmission electron microscope observations and energy-dispersive spectroscope data revealed that some altered kaolinite particles appeared, connected to intact kaolinite and bacterial cells. Bacterial cells were associated and encrusted with intact and/or altered kaolinite clay particles, where mixed (C, O, Na and Si)-precipitates of kaolinite clays were formed on the surface of hydrocarbon-degrading bacterial cells. However, the uptake of Si (from kaolinite) by bacterial cells appeared to be more prevalent than Al, and there were no significant changes in basal spacings of kaolinite due to these altered kaolinite particles. Separate studies showed that hydrocarbon-degrading bacteria have a high resistance to Si. Thus, the present data suggest that Si from kaolinite may facilitate hydrocarbondegrading bacterial growth as shown in our previous study (Chaerun ., 2005), and the C-O-Na-Si complexes on the surfaces of bacterial cell walls may be the stimulator for hydrocarbon-degrading bacterial growth in seawater contaminated with oil spill. P
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KEYWORDS: kaolinite, C-O-Na-Si complexes, oil spill, marine environment, hydrocarbon-degrading bacteria, TEM-EDS. Clay minerals are abundant and ubiquitous in the natural environment, and are particularly important because they are highly reactive (Kostka ., 1999) and account for a large proportion of Al-Sicontaining minerals in nature. All clays are dominated by silica and, along with microorganisms, provide some of the most catalytic surfaces in the environment (Kostka ., 2002; Shelobolina ., 2004). In aquatic and marine environments, silicate or clay minerals make up the majority of minerals where Si is required for growth of siliceous microbes such as diatoms and radiolaria et al
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* E-mail:
[email protected] DOI: 10.1180/0009855054040185
(Asada , 2002; Coale ., 2004). Also, amorphous silica is a dominant component in marine surface sediments, most of which are generated by the activity of living organisms (Kastner, 1981; Asada & Tazaki, 2001; Inagaki, 2003). Thus, clay minerals may have potential for use in the growth of bacteria in natural environments: they are thought to have a significant impact on nutrient cycles and the environmental fate of pollutants, e.g. oil spills (petroleum hydrocarbons). Kaolinite is the second most abundant clay mineral in ocean sediments (Coles & Yong, 2002) and is an important industrial mineral and geological indicator (Fialips, 2003). Our previous investigation of the interactions between kaolinite, et al.
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482 microorganisms and heavy oil in polluted marine and coastal environments has shown that kaolinite is capable of stimulating microbial growth in combination with biofilm formation (i.e. the kaolinites act as microbial growth-support materials) (Chaerun ., 2005). However, it is still not completely understood why kaolinite acts as it does. Hence, the primary goal here was to conduct a further laboratory-scale investigation of the role played by kaolinite in seawater polluted with oil spill (e.g. the oil spill) with emphasis on the elemental roles of kaolinite in direct interaction with hydrocarbon-degrading bacterial cells. These matters have important implications for oil-polluted marine environments, where kaolinite clay minerals are abundant and Si-bearing materials are available to microorganisms.
S. K. Chaerun and K. Tazaki
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MATERIALS AND METHODS Organisms and media
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Experimental design and procedure
Pseudomonas
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The hydrocarbon-degrading bacterium, affiliated to the bacterium (98% similarity), based on the result of 16S rDNA sequencing analysis, was indigenous to the oil spill and was isolated from Atake Seashore, Ishikawa Prefecture, Japan (Chaerun ., 2004a,b). This bacterium was capable of degrading heavy oil and was employed in this study in combination with other hydrocarbon degraders inhabiting the heavy oil and seawater (collected from the Sea of Japan) (Chaerun ., 2005). The growth medium used was described previously (Chaerun ., 2005); it comprised two solutions (volume ratio of 1:4 for solutions A and B, respectively). Solution A contained 25 g of NH4NO3, 0.5 g of FeC6H5O7´ H2O (ferric citrate), and 0.5 g of K2HPO4 per litre of distilled water, while solution B was made of natural seawater without filtration (collected from the Sea of Japan). The elemental composition of natural seawater was Na (8.27 wt.%), Mg (1.49 wt.%), Si (0.26 wt.%), S (2.94 wt.%), Cl (79.94 wt.%), K (2.99 wt.%), Ca (3.57 wt.%), Co (0.05 wt.%), Br (0.42 wt.%) and Sr (0.06 wt.%) (Chaerun ., 2005). Pseudomonas
Japan). Kaolinite was used in batch experiments without pretreatment (Chaerun ., 2005). The chemical composition is given in Table 1. . Five batch bacterial experiments were conducted in sterile 300 ml Erlenmeyer flasks containing 150 ml of growth medium under aerobic condition. The growth medium comprising two solutions (A and B) was supplemented with 1 g/l of yeast extract and adjusted to pH 7.8ÿ8 using 1 N NaOH solution. The kaolinite (0.5, 2, 10 and 30 g/l) was added to four of the flasks, but not to that for the biotic control (0 g/l kaolinite). Before introducing the inoculation, which originated in the heavy oil, and the natural seawater (solution B), into the flasks, all five flasks, together with their contents, were autoclaved, allowed to cool to room temperature (24ëC), and then inoculated with solution B, the stock liquid culture of bacterial strain (10% v/v), and heavy oil (collected from the oil spill) to a final concentration of ~25 g/l, serving as the sole carbon and energy source. Cultures were incubated for 566 h (~24 days) at room temperature (24ëC) with shaking at 125 rpm, allowing bacteria to grow. The experimental period of ~24 days was selected as the sampling time for two reasons: (1) the last of the exponential growth phase was reached after ~6ÿ8 days of incubation, and so the cultures had reached the long stationary phase thereafter; and (2) heavy oil used as the substrate was absent from the solutions (observed visually) by this stage. The pH in solution of each culture was monitored periodically. After 566 h (~24 days), separate sets of samples were made up and prepared for analysis
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Kaolinite
The kaolinite standard JCSS-1101 (from Kanpaku, Japan) used in this study was purchased from The Clay Science Society of Japan (Okayama,
TABLE 1. Elemental composition of kaolinite analysed by ED-XRF. Element Al Si P S K Ca Ti Mn Fe Ga Sr
Wt.% 31.83 59.75 1.28 2.32 0.53 0.33 2.10 0.03 0.72 0.08 1.01
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by energy dispersive X-ray fluorescence spectroscopy (ED-XRF), X-ray powder diffraction analysis (XRD), and transmission electron microscopy (TEM). In an attempt to investigate the effect of the major elemental compositions of kaolinite used here on hydrocarbon-degrading bacterial growth, a separate experiment was undertaken with those elements at concentrations of 0.01, 0.1, 1, 5, 10, 25, 100 and 500 mg/l, as described below. Since some elements such as Al and Si are present in trace amounts and are rarely required for bacterial growth, they areÿ6 generally required in concentrations of 0.05). Although dissolved Si was not found in the kaolinite solutions after ~24 days, it is reasonable to suggest that hydrocarbon-degrading bacteria are very resistant to soluble Si which may, in turn, facilitate enhanced growth of hydrocarbon-degrading bacteria at high concentrations of heavy oil, as shown previously (Chaerun ., 2005). This is also in agreement with TEM/EDS results obtained here (Figs 4, 5a, 6), showing that altered crystals of kaolinite
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TABLE 2. Effect of Al and Si on bacterial growth. Concentration (mg/l) 0.00b 0.01 0.1 1 5 10 25 100 500
Al 100Ô0.00 102Ô1.33 101Ô1.92 96Ô1.99 90Ô0.46 77Ô7.30 59Ô8.30 43Ô5.26 11Ô3.07
%Va
Si 100Ô0.00 99Ô0.62 102Ô3.81 99Ô0.49 99Ô2.00 99Ô1.99 91Ô2.05 80Ô1.61 52Ô4.90
%V = growth with added elements 7 growth in controls lacking elements6100 b Control lacking added element The values are means Ôstandard deviations based on triplicate experiments a
surrounding the cells are assumed to be amorphous silica due to high elemental Si and O contents. Both the soluble Si and Al experiments and TEM/EDS imaging suggested that the bacteria were apparently able to access Si more easily than Al. Such behaviour is expected since Al is toxic to many forms of life (e.g. Maurice ., 2000). However, the hydrocarbon-degrading bacteria used in this study are apparently uninhibited by the presence of et al
Al, at least when it is supplied in mineralogical form (i.e. in kaolinite). Silica dissolved in geothermal hot water (a relevant environment under extreme conditions) may be a significant component in the maintenance of position and survival of microorganisms in limited niches (Inagaki , 2003), and the rapid formation of biosilica may be utilized to remove dissolved toxic compounds (such as Al, As, Hg, Fe, Pb, Se, etc.) from polluted environments (e.g. Ichikuni, 1970; Ferris ., 1986; Cook & Stakes, 1995; Inagaki ., 2003). Thus, it can be suggested that mixed (C, O Na and Si)precipitates of kaolinites formed on the surfaces of hydrocarbon-degrading bacterial cells may participate (and help) in reduction of the toxic effects of oil spilled in the marine environment. et al.
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CONCLUSIONS
The present data suggest that kaolinite present in an oil-polluted marine environment was capable of stimulating hydrocarbon-degrading bacterial growth, as shown in our earlier work (Chaerun ., 2005), probably because: (1) Si from kaolinite facilitates bacterial usage of hydrocarbon; (2) C-ONa-Si complexes on the surfaces of bacterial cell walls are a stimulator for bacterial growth in the presence of hydrocarbon.
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TABLE 3. Chemical composition a (molar basis) of the solution at ~24 days in all the experimental cultures. Chemical composition (%) [Na] [Mg] [Al] [Si] [P] [S] [Cl] [K] [Ca] [Fe] [Br]
0 g/l K
0.5 g/l K
2 g/l K
10 g/l K
30 g/l K
20.70 (1.91) b 17.31 (0.77) 0.20 (0.14) 0.49 (0.17) 0.25 (0.04) 10.20 (0.67) 40.26 (0.21) 6.15 (0.67) 4.40c (0.58) n.d. 0.06 (0.01)
19.16 (5.64) 15.66 (2.83) 0.15 (0.04) 0.37 (0.08) 0.19 (0.03) 8.65 (2.56) 41.99 (3.76) 5.85 (0.84) 4.59 (0.95) n.d. 0.06 (0.01)
20.13 (2.55) 15.17 (1.23) 0.11 (0.12) 0.38 (0.06) 0.25 (0.06) 8.26 (0.67) 43.99 (2.26) 7.66 (1.14) 3.98 (0.46) n.d. 0.07 (0.01)
17.94 (0.65) 18.31 (2.04) 0.02 (0.03) 0.60 (0.15) 0.22 (0.03) 10.21 (1.31) 40.49 (2.93) 7.80 (0.67) 4.37 (0.29) n.d. 0.06 (0.01)
19.96 (2.85) 17.24 (0.90) 0.16 (0.09) 0.79 (0.13) 0.16 (0.04) 8.81 (1.06) 42.20 (2.49) 6.02 (0.82) 4.58 (0.43) 0.03 (0.01) 0.06 (0.01)
Based on quantitative ED-XRF analyses ( = 3) Standard deviation Not detected K: Kaolinite The dissolved Al and Si in the kaolinite solutions (0.5, 2, 10 and 30 g/l K) . the biotic control (0 g/l K) are not statistically different ( > 0.05), except for dissolved Si in the 30 g/l kaolinite solution. a b c
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ACKNOWLEDGMENTS
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We thank Dr Hiroaki Watanabe for helping with the TEM/EDS work, Dr Koichi Shiraki for helping with the XRD work, Dr Ryuji Asada for his help, and all the students of the Tazaki laboratory and the Okuno laboratory of Kanazawa University for their cooperation. We also thank the anonymous reviewers for their constructive comments. SKC was supported by a MONBUKAGAKUSHO scholarship.This work was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology to KT. REFERENCES
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