Electron Microscopy and X-ray Analysis of Cr-Containing Precipitates Synthesized by Newly Isolated Actinobacterium, Flexivirga alba ST13T

Abstract

Chromium(Cr) precipitate synthesized by Cr(VI)-reducing bacterium Flexivirga alba ST13 T was examined using transmission electron microscopy (TEM) and the energy dispersive X-ray (EDX). The strain showed altered-morphology after exposing to Cr(VI) in minimal medium. The resultant precipitate included bacterial pellet and needle-like structure which was similar to the structure made from Cr(OH) 3 precipitate. Cr was observed in bacterial cells using TEM-EDX. Bacteria with high electron density showed the precipitation of Ca in addition to Cr. The isolated strain would be useful to precipitate Cr from Cr(VI)-containing environment.Keywords Chromium Á Electron microscopy Á Bacterium Á Actinobacterium Chromium(Cr) is an important element in industrial processes, i.e. electroplating and leather tanning. However a valence state of Cr, hexavalent Cr (Cr(VI)), is the toxic and harmful chemical form. Therefore it is important to develop technologies for the prevention of Cr(VI) from effusing into natural environment and for the detoxification of Cr(VI) by transforming the valence state into trivalent Cr (Cr(III)). In many bioreduction experiment, the most of Cr(III) was detected in water soluble form but not in insoluble precipitate. It is needed to understand Cr(III) precipitation mechanisms in Cr(VI)-reducing bacteria.Here we describe TEM and EDX analysis of Cr-containing precipitates which were synthesized by newly isolated actinomyces, Flexivirga alba ST13 T (=NBRC 107850 T = DSM 24460 T ) as a model of Cr(III)-accumulating bacterium [1]. The strain reduced Cr(VI) efficiently in minimal medium supplemented with molasses and formed water insoluble Cr(OH) 3 compound [2].Bacteria were grown aerobically in minimal medium (6 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , 1 g/L NH 4 Cl, 0.5 g/L NaCl, 0.246 g/L MgSO 4 Á7H 2 O, 0.01 g/L CaCl 2 , 0.4 % (w/w) molasses; pH 6.8) in a rotating bioshaker (Taitec, Saitama, Japan) at 200 rpm at 27°C. For the preparation of Cr(VI)-containing medium, 0.4 mM CrO 3 (Wako, Osaka, Japan) was added to the medium. Hexavalent and total Cr concentration were measured as described previously [2]. Strain ST13 T growth in medium containing CrO 3 was centrifuged (5,4009g, 5 min) to separate the culture supernatant and the cell pellet. The supernatant was used for Cr(VI) measurement. Briefly, 1 mM diphenylcarbazide (Wako) in 1.2 N H 2 SO 4 was mixed with an equal volume of supernatant for 10 min. The absorbance at 540 nm was determined using a spectrophotometer 3100 (GE Healthcare, Buckinghamshire, England). The Cr(VI) concentration in the sample was estimated by a standard curve generated with known amounts of Cr(VI). About 48 and 5 % Cr(VI) remained in medium on days 5 and 13, respectively. To examine the effect of Cr(VI) on bacterial morphology, gram staining was performed using Fiber-G (Nissui Pharmaceutical Co., Tokyo, Japan) and observed