March 14, 2003 16:43 GMB TJ693-05
Geomicrobiology Journal, 20:143–156, 2003
Copyright C° 2003 Taylor & Francis
0149-0451/03 $12.00 + .00
DOI: 10.1080/01490450390193255
Use of Ferric and Ferrous Iron Containing Minerals
for Respiration by Desulfitobacterium frappieri
EVGENYA S. SHELOBOLINA
Department of Microbiology
University of Massachusetts
Morrill Science Center IVN
Amherst, Massachusetts, USA and
Institute of Microbiology
Russian Academy of Sciences
Moscow, Russia
CATHERINE GAW VANPRAAGH
DEREK R. LOVLEY
Department of Microbiology
University of Massachusetts
Morrill Science Center IVN
Amherst, Massachusetts, USA
The potential of Desulfitobacterium frappieri strain G2, which was isolated from sub-
surface smectite bedding, to participate in iron redox reactions was investigated. Strain
G2 can use poorly crystalline Fe(III) oxide, soluble forms of Fe(III) and Fe(III) in the
structure of phyllosilicate minerals as electron acceptors. It can also oxidize Fe(II)-NTA
or Fe(II) in the structure of phyllosilicate minerals with nitrate as the electron acceptor.
These results suggest for the first time that strains of Desulfitobacterium frappieri may
play an important role in iron cycling in sedimentary environments.
Keywords Desulfitobacterium, iron cycling, poorly crystalline ferric oxide,
phyllosilicates
Introduction
Iron is the most abundant element in the earth as a whole and the fourth most abundant
element in the earth’s crust. In sedimentary environments the average total Fe content is
approximately 3.9 mass%, a value close to the average Fe concentration in the earth’s
crust (5.09 mass%), and the average Fe(III)/Fe(II) ratio is 1.35 (Murad and Fischer 1988).
Received 29 January 2002; accepted 7 May 2002.
We would like to thank E.L. Blunt for her technical assistance, S.M. Pickering for advice and help with
collecting smectite samples, and Dry Branch Kaolin Co., GA, for access to its mines. We thank D. Bond for
his thermodynamic suggestions and M. Coppi for critically reviewing the manuscript. We also thank L. Yin of
the University of Massachusetts microscopy facility, which is supported by NSF Grant # BBS 8714235. This
work was supported by NSF NATO (grant DGE-9902554) and by the Natural and Accelerated Bioremediation
Research (NABIR) program, Biological and Environmental Research (BER), U.S. Department of Energy (grant
DE-FG02-97ER62475 and DE-FG02-00ER62985).
Address correspondence to Derek R. Lovley, Department of Microbiology, University of Massachusetts,
Morrill Science Center IVN, Amherst, Massachusetts 01003, USA. E-mail: dlovley@microbio.umass.edu
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March 14, 2003 16:43 GMB TJ693-05
144 E. S. Shelobolina et al.
Microorganisms can gain energy from both Fe(III) reduction and Fe(II) oxidation. Nu-
merous Fe(III)-reducing bacteria have been isolated and characterized during the past two
decades (Lovley 2000). The role of nitrate-reducing bacteria in the oxidative part of the
iron cycle has also been the subject of a number of recent studies (Emerson 2000; Straub
et al. 2001). By regenerating Fe(III), anaerobic microbial Fe(II) oxidation may enhance the
mineralization of organic material in sediments by Fe(III)-reducing bacteria.
In sedimentary environments, iron can be found as discrete iron minerals (Fe sul-
fides, carbonates, oxides) or in the form of structural iron, iron bound within the lattice of
phyllosilicates. Structural iron can constitute a significant portion of the iron present in the
clay fraction. The clay fraction, which consists of particles less than 2 „m in diameter,
has been found to be the most chemically active inorganic fraction of sediments and soils
(Dixon 1998).
As has been outlined by Konig et al. (1997), Ernstsen et al. (1998), and Konig et al.
(1999), a significant fraction of the iron present in the lattice of phyllosilicates is redox
sensitive and can undergo many redox cycles without moving or mobilizing. Thus structural
iron can store, and accumulate redox energy in situ, making it unique among the various
iron species present in sedimentary environment.
Anaerobic enrichment cultures (Wu et al. 1988; Gates et al. 1998; Kostka et al. 1999),
mixed cultures of Pseudomonas spp (Gates et al. 1993, 1998), a bacterial strain P1 that was
isolated from SWa-1 clay (Stucki et al. 1987), as well as strains of Shewanella putrefaciens
(Kostka et al. 1996, 1999) and Geobacter sp. (Lovley et al. 1998; Kostka et al. 1999) have
all been shown to be capable of reducing structural Fe(III) in smectite.
In this paper we describe the isolation and characterization of Desulfitobacterium frap-
pieri strain G2, which can not only reduce poorly crystalline Fe(III) oxides, but can also
participate in reversible redox reactions of iron within the lattice of phyllosilicates.
Materials and Methods
Source of the Organism
Strain G2 was enriched from subsurface smectite bedding of the Twiggs Clay Formation
of Late Eocene Age. The sample used for enrichment cultures was taken from an active
Dry Branch Kaolin Company mine in Georgia, sent by overnight delivery service to the
laboratory, and placed in an anaerobic chamber filled with nitrogen.
Media and Cultivation
Strict anaerobic techniques (Miller and Wolin 1974; Balch et al. 1979) were used through-
out. An anaerobic basal bicarbonate-buffered freshwater medium (Lovley et al. 1993) was
dispensed into anaerobic pressure tubes or 160-ml serum bottles under N2–CO2 (80:20).
The tubes or bottles were capped with butyl rubber stoppers and sterilized by autoclaving.
Reducing agents were FeCl2 (1.3 mM) for Fe(III)-containing media and cysteine (0.5 mM)
for all other media and were added after the medium was autoclaved.
Enrichment cultures were initiated in 160 ml serum bottles containing 90 ml of fresh-
water medium and 10 g of clay from the site. Strain G2 was recovered from an enrichment
culture established with H2 as the electron donor and Fe(III)-NTA as the electron acceptor.
The enrichment was transferred onto anaerobic agar plates solidified with 1.2% purified
agar (BBL Agar, Becton Dickinson, Cockeysville, MD) containing lactate (20 mM) as the
electron donor and Fe(III)-NTA (10 mM) as the electron acceptor. Agar plates were in-
cubated in an anaerobic chamber under an atmosphere of N2:CO2:H2 (83:10:7) at 30–C.
Individual colonies were picked and restreaked multiple times to ensure purity. A single