in Aquatic Sediments by Bacteria Fig. 1. Se K -edge spectra of culture after 0, 12, 21, 28, 37, 43, 53 and 66 hours from seeding S. barnesii , along with Se, SeO 2 and CaSeO 4 for reference. The implementation of Pollutant Release and Transfer Register (PRTR) obliged us to report the total amount of chemical substances, such as toxic metals, released into the atmosphere, hydrosphere and soil. The total amount of these substances that have been released into the environment due to human activity is now becoming clear. However, the chemical state changes of toxic metals have not been clarified. We should clarify the behavior of toxic metals in the environment to ensure safety against damage due to toxic metal contamination, because the toxicity of a metal depends on its valence or combined states. In many cases, microorganisms have assumed a vital role in the chemical state changes of metals. The use of microorganisms is expected to lead to the development of environmental restoration technologies by revealing the biological reaction pathway of metals. Recently, a selenate reduction bacterium, Sulfurospirillum barnesii [1] , was isolated from a freshwater sediment. S. barnesii has the ability to reduce selenic acid [S e( V I) ] under anaerobic conditions, using organic acid as an electron donor. We present here the chemical state changes observed for selenium by X- ray absorption fine structure ( XAFS ), focusing our attention on the reduction ability of S. barnesii to reveal the behavior of selenium in a biological reactive process. We used a DSM 10660 strain [2] ( = A T CC 700032 strain) for S. barnesii , cultivated in a 500 ml sealed bottle with Sulfurospirillum II medium containing 83 mg / l S e( V I). C ultivation was carried out at constant temperature ( 30 C ) under anaerobic conditions with the in j ection of inert gases ( N 2 /CO 2 ). XAFS measurement was carried out at beamline BL01B1 with a two - crystal S i( 111 ) monochromator. XAFS spectra of S. barnesii culture were collected in the fluorescence mode using a G e 19- element solid - state detector. F igure 1 shows S e K - edge XANES spectra of S. barnesii culture and reference materials. A pea k due to metallic selenium [S e( 0 ) ] was clearly observed in the spectra after 28 , 37 , 43 , 53 and 66 hours. A lso , a pea k due to tetravalent selenium [S e(I V ) ] was observed in the same spectra. This result shows that the reduction pathway from S e( V I) to S e( 0 ) by S. barnesii goes through S e(I V ). T o estimate the chemical composition of selenium in each moment, we calculated the ratio of each chemical states by the curve - fitting method using the spectra of the reference materials. The result is shown in F ig. 2 . The change of the chemical composition of selenium indicates that S e( V I) constantly decreases with increasing S. barnesii growth rate. In this case, although a rate - limiting step of the reduction pathway from S e(I V ) to S e( 0 ) occurred for 20 hours after seeding, the reduction of S e( V I) to S e(I V ) proceeded constantly. Thus, from the above finding, it is assumed that the reduction of S e( V I) to S e(I V ) by S. barnesii is faster than that of S e ( I V ) t o S e ( 0 ) u n d e r s u c h a h i g h s e l e n i u m concentration ( 83 mg / l selenium). Normalized Absorption 66 hours 53 hours 43 hours 37 hours 28 hours 21 hours 12 hours 0 hour SeO 2 Se CaSeO 4 Photon Energy (eV) 12640 12660 12680 Changes of Chemical States of Toxic Metals 97 Shigeki Fujiwara a , Norizo Saito b and Yasuhiro Konishi b (a) JFE Engineering Corporation (b) Osaka Prefecture University E-mail: fujiwara-shigeki@jfe-eng.co.jp Fig. 2. Change in chemical composition o f s e l e n i u m w i t h t i m e . ( R e s u l t o f calculation by the curve-fitting method using the spectra of reference materials). References [1] R.S. Oremland et al. : A ppl. Environ. M icrobiol. 65 (1 999 ) 4385 . [ 2 ] D S MZ L ist of M edia: (http: // www.dsmz.de / media / med 77 1.htm). T he results also indicate that selenium released in to the hydrosphere is immobilized as metallic selenium by anaerobic bacteria, such as S. barnesii , which have the ability to reduce selenium. Figure 3 shows pattern diagrams of dynamics of selenium in a q uatic sediments. I f we can accelerate the growth of selenium reduction bacteria, and keep them in high density, we will be able to effectively render selenium harmless. Fig. 3. Pattern diagrams of dynamics of selenium in aquatic sediments. Se(VI) Se(IV) Se(0) 0 20 40 60 80 Time after seeding of S. barnesii (hours) Ratio (-) 1.0 0.8 0.6 0.4 0.2 0.0 S. barnesii Se(0) organic acid oxidation reduction sediment (anaerobic) Se(VI) hydrosphere (aerobic) organic acid Se(IV) Se(VI) 98
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