Published in GCA in 1998 as a tripartite discussion with Brad Tebo (Scripps) and Jim Moffett (WHOI).

1. PII S0016-7037(98)00012-X
COMMENT
Comment on ‘‘Oxidation of cobalt and manganese in seawater via a common microbially
catalyzed pathway’’ by J. W. Moffett and J. Ho
ANGELINA W. M. G. SOUREN*
Institute of Earth Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Moffett and Ho (1996) reported the results of investigations
concerning cobalt and manganese uptake onto suspended par-
ticles in seawater from Waquoit Bay and the Sargasso Sea. The
authors conclude that in Waquoit Bay water cobalt uptake is
controlled by microbial oxidation and strongly correlated with
manganese oxidation and propose that these oxidations occur
via a common enzymatic pathway. Manganese and cobalt up-
take in Sargasso Sea water samples are reported to be de-
coupled, with nonoxidative and biologically mediated Co up-
take, and almost exclusively oxidative Mn uptake. Moffett and
Ho (1996) also point out the striking similarities with microbial
Ce oxidation.
In this comment, I present specific fungal processes and a
particular enzyme as a possible explanation for the oxidations
of Co, Mn, and Ce and their transition from the dissolved to the
particulate phase, in addition to oxidation by notably, spores
and spore coats of marine Bacillus sp. (De Vrind et al., 1986;
Lee and Tebo, 1994; Mandernack et al., 1995; Mann et al.,
1988). I will also discuss specific issues addressed by Moffett
and Ho (1996). It should be noted that in this comment the
ligninolytic processes are described in a highly simplified man-
ner, for the sake of brevity. Interested readers are urged to
consult the mycological and biotechnological literature.
(1) Moffett and Ho (1996) state that in general Mn ‘‘is
present as Mn2ϩ
(aq) and is not significantly organically com-
plexed’’ in seawater. However, Kostka et al. (1995) have
already drawn attention to the fact that dissolved Mn(III) could
be an important oxidant in marine and in freshwater environ-
ments. In addition, Luther et al. (1994) cautioned that the
widely used method of differential pulse polarography to de-
termine Mn(II) may be misleading: ‘‘the reduction of Mn(II) to
Mn(0) which has been used to measure Mn in natural waters is
not specific for the Mn(II) redox state. This reduction peak
which occurs near Ϫ1.5 V measures total soluble manganese.’’
This means that the popular opinion regarding Mn in seawater
needs to be reevaluated. Also, Luther et al. (1994) studied
manganese speciation in Chesapeake Bay and found that Mn
can in fact be complexed by organic ligands, especially in areas
with a large degree of organic matter breakdown, such as
Waquoit Bay. In their own experiments, Moffett and Ho (1996)
measured total dissolved and particulate Mn by AAS and by
radiotracer counting, respectively, while they attempted to es-
tablish the oxidation state of particulate Mn in an indirect
manner. As I will show in this comment, oxidation of Mn(II) to
Mn(III) can be accomplished microbially, in particular by the
widely studied white rot fungi (Glenn et al., 1986; Harris et al.,
1991; Kishi et al., 1994; Wariishi et al., 1992).
(2) Moffett and Ho (1996) state that ‘‘microbial Mn oxida-
tion has not yet been attributed to a specific enzyme,’’ whereas
microbial Mn oxidation has in fact already been ascribed spe-
cifically to the microbial enzyme manganese peroxidase or
MnP (Glenn et al., 1986; Wariishi et al., 1992). MnPs are
extracellular enzymes secreted by certain wood-degrading
fungi. (According to Mann et al. (1988), bacterial components
that catalyze Mn oxidation have been identified as well.)
I propose that wood-degrading fungi may very well be major
mediators of the oxidations of Co, Mn, and Ce, especially in
organic-rich and near-shore environments, and may hence also
be a major cause of aquatic Ce anomalies. As a consequence I
present MnP as a prime candidate for the enzyme in the
‘‘common enzymatic pathway’’ (Ho and Moffett, 1994; Mof-
fett, 1994a; Moffett and Ho, 1996) along which Co, Mn, and Ce
are oxidized (Fig. 1). Table 1 shows consistencies between
findings of Moffett and Ho (1996) and other workers and
findings for wood-degrading fungi and MnP.
In the context of marine biogeochemical metal cycles, the
terms ‘‘microorganisms’’ and ‘‘microbes’’ have become al-
most synonymous with ‘‘bacteria.’’ Fungi, however, are mi-
crobes as well. There appears to be a widespread belief that
there are only a few marine fungi (see for instance Madigan et
al., 1997) occurring perhaps at a few exceptional locations. In
fact, more than 500 fungi have been documented in the marine
environment (Kohlmeyer and Kohlmeyer, 1979), including the
open ocean, the deep sea, and polar areas (Kohlmeyer and
Kohlmeyer, 1979; Moss, 1986). New marine fungi are reported
on a regular basis, especially from mangrove areas such as in
Florida, Australia, and Malaysia. Most marine fungi are asso-
ciated with wood (Kohlmeyer and Kohlmeyer, 1979) which is
abundant in coastal and estuarine environments, as are other
decaying materials. However, marine fungi also live as para-
sites on marine algae, copepods, and other organisms (Webster,
1977), and can cause severe epidemics in fish (Austin, 1988).
The fact that for instance the ratio of fungal:microalgal:bacte-
rial mass in decaying saltmarsh grass leaves can be as high as
600:4:1 (Newell et al., 1996) clearly indicates that the role of
fungi in marine biogeochemical cycles must be taken into
account (Gon˜i et al., 1993; Haddad et al., 1992; Hedges et al.,
* Present address: Department of Chemistry, Geobiochemistry
Group, Rijks Universiteit Leiden, P. O. Box 9502, 2300RA Leiden,
The Netherlands.
Pergamon
Geochimica et Cosmochimica Acta, Vol. 62, No. 2, pp. 351–355, 1998
Copyright © 1998 Elsevier Science Ltd
Printed in the USA. All rights reserved
0016-7037/98 $19.00 ϩ .00
351

2. 1988; Pracejus and Halbach, 1996; A. W. M. G. Souren,
unpubl. data).
Firstly, marine fungi effectuate the transition of metals from
the dissolved phase to particulates, for instance through adsorp-
tion. Chitin and chitosan, the major cell wall components of
fungi, scavenge metals so effectively (Gonzalez-Davila and
Millero, 1990; Gonzalez-Davila et al., 1990; Hawke et al.,
1991) that chitosan has even been proposed as a means of
extracting metals from seawater (Muzarelli and Sipos, 1971).
Secondly, many fungi display redox activities and actively
dissolve or precipitate minerals and degrade organic materials
(unpubl. data A. W. M. G. Souren). For the study of the
oxidations of Co, Mn, and Ce, the ligninolytic wood-degrading
fungi are particularly interesting: (a) these fungi represent an
impressive oxidative force and (b) these fungi are known to
oxidize Mn(II) and Co(II).
Ligninolytic or white rot fungi cause white rot, during which
the highly refractory compound lignin is degraded (Kleman-
Leyer et al., 1992). Manganese peroxidases (MnPs), laccases,
and lignin peroxidases (LiPs) are the major extracellular fungal
enzymes involved in white rot, but ongoing research keeps
shedding different lights on the exact actions of these and other
enzymes, especially in the breakdown of xenobiotics
(A. W. M. G. Souren, unpubl. data).
MnP is a glycoprotein containing one heme group (Fig. 2)
and occurring as several isoenzymes (Wariishi et al., 1992).
The purpose of MnP is to oxidize Mn(II) to Mn(III) which in
turn oxidizes lignin and other organic substrates (Fig. 1). Ox-
idation of Mn(II) to Mn(III) has also been reported to occur
occasionally through laccases and LiPs (Daniel et al., 1994;
Popp et al., 1990) but MnP is completely dependent on Mn(II)
for the reduction of MnP compound II (Fig. 1) (Aitken and
Irvine, 1990), whereas laccases and LiPs are not obligate
Mn(II) oxidizers.
Stabilization of Mn(III) which tends to disproportionate into
Mn(II) and Mn(IV) is achieved through complexation by ox-
alate, malonate, and other organic acid chelators, also produced
by these wood-degrading fungi. These organic acid chelators
facilitate the release of Mn(III) from MnP as well (Glenn et al.,
1986; Kishi et al., 1994; Perez and Jeffries, 1992; Wariishi et
al., 1992). A great advantage of the metal complex is its
relatively small size, which enables it to interact at sites that are
Fig. 1. Simplified reaction scheme for the reactions involving Mn
and MnP. H2O2 can be produced in various ways, notably through
fungal enzymes such as pyranose oxidase and glyoxal oxidase. Super-
imposed on this scheme are reduction of Mn(III) by H2O2 (H2O2
ϩ 2Mn3ϩ
3 2Hϩ
ϩ O2 ϩ 2Mn2ϩ
) and reduction of Mn(IV) through
the mediation of humic acids and light (Spokes and Liss, 1995).
Table 1. Oxidation of manganese, cobalt, and cerium: similarities between findings in marine biogeochemical studies in ‘mycological’
research.
Marine biogeochemical studies Mycological research
● The microbially mediated oxidations of Mn, Co and Ce
are linked via a common enzymatic pathway (Emerson
et al., 1986; Ho and Moffett, 1994; Lee and Fisher,
1993; Lee and Tebo, 1994; Moffett 1994a,b; Moffett
and Ho, 1996; Sholkovitz, 1993)
● The extracellular enzyme MnP oxidizes Mn and Co (Glenn et al.,
1986; Harris et al., 1991; Wariishi et al., 1992) and is expected to
oxidize Ce as well (this comment)
Fungicides or tests for the presence of fungi or fungal
enzymes were not applied in the associated research
Fungi are also (eucaryotic) microbes
● Co oxidation is much slower than Mn oxidation
(Moffett and Ho, 1996)
● Co oxidation is much slower than Mn oxidation (Glenn et al., 1986;
Harris et al., 1991)
● Competitive inhibition between Co and Mn oxidation
(Moffett and Ho, 1996)
● Competitive inhibition between Co and Mn (Harris et al., 1991)
● Microbial Mn and Co oxidation appears to be a two-
step process in which the second reaction step is rate-
determining (Moffett and Ho, 1996)
● Mn and Co precipitation via MnP is a two-step process. Also, MnP
compound II is rate-determining in the catalytic cycle of MnP (Kishi
et al., 1994).
● Sodium azide prevents microbial oxidation of Mn and
Ce (Moffett, 1990, 1994a,b; Moffett and Ho, 1996)
● Sodium azide inhibits MnP (DePillis et al., 1989; Harris et al., 1991)
● Co enhances sodium azide’s inhibition of microbial Mn
(Moffett and Ho, 1996)
● Co enhances sodium azide’s inhibition of Mn oxidation by MnP
(Harris et al., 1991)
● Mn, Co and Ce precipitation and oxidation are
catalyzed by Mn solids (Bidoglio et al., 1992; Hem,
1978; Lee and Tebo, 1994; Murray, 1975; Murray and
Dillard, 1979)
● Mn solids are formed during wood decay and in experiments with
MnP and with wood-degrading fungi (Blanchette, 1984; Buswell et
al., 1995; Glenn et al., 1986; Harris et al., 1991; Perez and Jeffries,
1992)
● Negative Ce anomalies appear to be formed mainly in
estuarine areas but occur in rivers as well (Moffett,
1994; Sholkovitz, 1993)
● Estuarine areas and rivers are usually rich in decaying plant
materials and hence in fungi. The ratio fungal:microalgal:bacterial
mass in decaying salt marsh grass leaves can be 600:4:1 (Newell et
al., 1996)
352 A. W. M. G. Souren

3. inaccessible for larger compounds such as enzymes (Kleman-
Leyer et al., 1992). The complexed Mn(III) is still able to
oxidize phenolic substrates after which, ideally, Mn(III) returns
to Mn(II). However, precipitation of solid Mn phases does
occur and has been found in decaying wood (Blanchette, 1984)
as well as in experiments (Buswell et al., 1995; Glenn et al.,
1986; Harris et al., 1991; Perez and Jeffries, 1992).
Thus, fungal ligninolysis through MnP provides two mech-
anisms for Co and Ce to be precipitated and oxidized along
with Mn: (a) catalyzation through MnO2 and other solid Mn
phases (Bidoglio et al., 1992; Hem, 1978; Lee and Tebo, 1994;
Murray, 1975; Murray and Dillard, 1979) and (b) oxidation by
MnP of Co(II) to the barely soluble Co(III) and of Ce(III) to the
insoluble Ce(IV). In addition, Ce may very well be precipitated
as insoluble complex, such as Ce oxalate or oxalate hydrate.
MnP has already been reported to oxidize Co(II) albeit at a
much lower rate than for Mn(II), and more or less inadvertently
(Glenn et al., 1986; Harris et al., 1991), in concordance with the
results of Moffett and Ho (1996). Metals with lower reduction
potentials (Ni, Cu, and Fe) interact with MnP but are not
oxidized by it (Aitken and Irvine, 1990; Glenn et al., 1986;
Paszczyn´ski et al., 1986). To my knowledge, no experiments
have yet been conducted to investigate the interactions of Ce
with MnP. However, the standard reduction potential of the Ce
couple, at ϩ1.61 V, lies between those of the Mn(II)/Mn(III)
couple (ϩ1.51 V) and the Co couple (ϩ1.81 V) (Atkins, 1990).
Complexation of Mn(III) is expected to lower the couple’s
reduction potential slightly, as it stabilizes Mn(III). One obvi-
ous conclusion is that Ce(III) is likely to be oxidized by MnP
as well, along with Co(II) and Mn(II).
Sodium azide is often used in killed controls and was also
applied by Moffett and Ho (1996) in order to determine microbial
mediation of the occurring oxidations. Whenever inhibition of the
oxidations of Co, Mn, and Ce is the case, this tends to be inter-
preted as an indication of bacterial mediation (Emerson et al.,
1982; Ho and Moffett, 1994; Lee and Tebo, 1994; Moffett, 1990,
1994a,b; Moffett and Ho, 1996). Moffett and Ho (1996) used
ascorbate to investigate whether Co(III) is actually formed as
ascorbate reduces Co(III). Although ascorbate would not affect
adsorbed Co(II) and Mn(II) directly, it also reduces Mn(IV) and
would release incorporated Co(II) as well (Moffett and Ho, 1996).
In Waquoit Bay, both ascorbate as well as azide were effec-
tive. In Sargasso Sea waters, azide inhibited uptake but ‘‘ascor-
bate had no effect.’’ In general, Co and Mn appeared to
compete for the same site, and Co(II) appeared to slightly
enhance sodium azide’s inhibition of Mn oxidation (Moffett
and Ho, 1996). The same has also been reported by Harris et al.
(1991) for MnP. The structure of MnP and its interactions with
azide and metals offer a possible explanation of the findings of
Moffett and Ho (1996). Figure 3 shows a model of the heme
group of MnP. Azide, which is applied in studies to probe the
sites of MnP, combines with MnP to form an azidyl radical
(DePillis et al., 1989; Harris et al., 1991). Azide inactivates
MnP very efficiently, usually within one to two minutes, but the
inactivation rate is concentration-dependent and requires H2O2
(Harris et al., 1991). Azide prevents oxidation of Mn and Co
but does not block access of these metals to the MnP metal site
(Harris et al., 1991). Sodium azide inactivates LiPs as well, but
not as efficiently. A combination of azide, Co and Mn would
lead to occupation of two sites on MnP, for one of which Co
and Mn compete. As a result, Co(II) appears to enhance azide’s
inhibition of Mn(II) oxidation.
In the Sargasso Sea samples, Co apparently was not oxi-
dized, whereas Mn uptake was mainly oxidative. Many factors
influence fungal metabolism and their enzymatic processes but
the most obvious explanation is that there is significantly less
fungal activity in the Sargasso Sea due to a much smaller
amount of degradable organic matter. Cobalt uptake would be
mainly by other organisms.
The lag shown by Co compared to Mn in the experiments of
Moffett and Ho (1996) appears to indicate that some equilibra-
tion process is necessary to render Co(II) more reactive and ties
in with the strong time-dependence of Co oxidation by MnP
(Harris et al., 1991). Possibly, a change in Co complexation
precedes interaction between Co and MnP, as both Co(II) and
Co(III) from strong complexes.
Fig. 2. Model of the manganese peroxidase heme group and its active
sites; the Mn(II) site is located at the ␦-meso edge. After Harris et al.
(1991) and DePillis and Ortiz de Montellano (1989).
Fig. 3. Side view of the MnP heme group and its sites, as proposed
by Harris et al. (1991). The azidyl radical (see text for explanation) is
located above the heme group plane. The metal site is situated at the
bottom, and the azidyl radical would not block access of small entities
to the metal site, but would prevent the oxidation of Mn(II).
353Discussion: Comment

4. Although this may not have been of crucial importance
during the experiments if Moffett and Ho (1996), where ascor-
bate lead to reduction and dissolution of precipitates, it should
be kept in mind that ascorbate also inactivates MnP 100%
(Paszczyn´ski et al., 1986).
Finally, the issue of pH needs to be addressed. Most micro-
bial Mn oxidations take place around neutral pH values (Glenn
et al., 1986), but the optimal pH values for MnPs are roughly
between 4 and 6 (Kishi et al., 1994; Ru¨ttiman-Johnson et al.,
1994) which is considerably lower than an average seawater pH
value of 8.2. On the other hand, to my knowledge there has not
yet been any research into MnPs acting in marine conditions,
and therefore no data about their optimal pH conditions are
known yet, and different fungi have distinct types of enzymes
with distinct optimal conditions. In addition, the pH of coastal
and estuarine waters is lower than 8.2, due to mixing with
terrestrial runoff. MnP activity is not extremely dependent on
pH and has been documented up to a pH of about 7 although
the reaction rates appear to be considerably lower at the higher
pH (Glenn et al., 1986). Even more interestingly, the white rot
fungus Phanerochaete chrysosporium has been found to in-
crease pH from 4.5 to 8.2 and 8.5 in liquid cultures, with
maximum pyranose oxidase production (H2O2 generation) at
the higher pH (Daniel et al., 1992). Fungi are notorious for
changing pH in experimental conditions (Ru¨ttiman-Johnson et
al., 1994) and the type of organic acid chelator the wood-
degrading fungi produce probably also has a large influence on
the pH dependence of MnP. Wood-degrading fungi can easily
create and control a microenvironment within organic matter or
within the sheaths often surrounding the hyphae. That extra-
cellular enzymes can be stable and active in marine environ-
ments was already established by Kim and ZoBell (1974).
In conclusion, it appears reasonable to expect that MnPs are
also active in the marine environment, particularly in coastal
areas, and that wood-degrading fungi and their enzymes play a
major role in the biogeochemical cycles of Co, Mn, and Ce but
further research is obviously needed to clarify the details. In
fact, manganese oxidation by fungi (and bacteria) was already
studied at the beginning of this century (Beijerink, 1913) and
was also reported for the Bay of Biscay and for the North Sea
more than 25 years ago (Krumbein, 1971). Examples of marine
white rot fungi are Nia vibrissa, Digitatispora marina´, and
halocyphina villosa (Leightley, 1980; Mouzouras, 1986). Al-
though MnP is extracellular, its activity appears to be mostly
associated with mycelium (Paszczyn´ski et al., 1986) and extra-
cellular membranes (Moen and Hammel, 1994). A closer ex-
amination of the ‘‘polymeric material’’ Moffett and Ho (1996)
observed in their experiments might therefore lead to further
interesting results. Future investigations might also be signifi-
cant in the context of manganese nodule and crust formation
and for cerium’s use as a paleoenvironmental indicator (Ger-
man and Elderfield, 1990; Liu and Schmitt, 1988).
Acknowledgments—I am particularly indebted to Drs. F. F. Beunk of
the Vrije Universiteit in Amsterdam and M. Visser of the Universiteit
Utrecht, both in The Netherlands, for highly appreciated support and
comments, and to Dr. R. H. Byrne of the University of South Florida
for being a catalyst. Also to Dr. K. E. Hammel of the University of
Wisconsin, to Dr. S. Y. Newell of the University of Georgia (Sapelo
Island), and of course to Dr. B. M. Tebo and two anonymous reviewers
for valuable input and reviews.
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