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VIII. Free Radicals in Redox Processes
REDOX CHEMISTRY OF SOILS
+ 3e- + 3H+ H,O + OH (hydroxyl free radical)
0, + 4e- + 4H+
HO; + H,O,
H,O + 0, + OH.
OH. + H 2 0 2 - H 2 0 + H Q
2H202 a 2 H 2 0 + 0,
2HO; x H Z O Z 0,
+ Mn3? + 2H,O
With one electron, a hydrogen atom is a free radical, a single protonated
electron in frantic search for an electron mate. It is the simplest and most
reactive (least stable) free radical. As H,, however, it is quite stable, because the electrons are paired.
Catalases or fresh recently oxidized Mn oxides at pH > 6 (Bartlett,
I98 la) will catalyze the oxidation of one of the oxygens in H,Oz by the
other and thereby will destroy the peroxide by dismutation [Eq. (34)]. In
acid media, oxidized Mn will oxidize H,O,. The dismutation of HO; to
H,O, and 0, by superoxide dismutase (SOD) enzymes, Eq. (33), followed
by H,O, dismutation to 0, and H,O or oxidation of H,O, to O,, make
aerobic life possible (Fridovich, 1975; Halliwell, 1974) by preventing the
formation of the biodestructive hydroxyl free radical by Eq. (3 1). Reforming HO; by Eq. (32) is prevented.
Oxygen free radicals are much more reactive than 0,. Probably free
radical mechanisms explain why kinetically very slow and seemingly unlikely redox transformations sometimes occur readily. The hydroxyl free
radical, (OH the superoxide free radical, -OF, and the supermanganese
free radical ( Mn33are close to being the most powerful oxidizing agents in
soil systems, and superoxide and supermanganese also are the most powerful reducing agents (Table I, Fig. 3, and see Sections VI,A and VI,C)
(Bartlett, 198 I a).
Oxygen free radicals are among the few species having the thermodynamic capability for oxidizing Mn(I1). This means that Mn is one of the
few elements that is capable of scavenging these radicals and protecting life
RICHMOND J. BARTLETT AND BRUCE R. JAMES
forms from their biotoxicity. By scavenging free radicals, Mn disrupts the
tendency toward thermodynamic equilibrium between 0, and soil organic
matter (or living roots), allowing the persistence of metastable humus and
roots in an oxidative environment.
Equation (35) shows the MnO, oxidation of hydroquinone by a single
electron step to form two free radicals, the semiquinone free radical and
the supermanganese free radical, Mn? It seems that free radicals could be
the latchkeys to the linking together of reducing phenolic compounds into
organic polymers that are stable in the presence of 0,. For example, a
single OH free radical initiates the linking process in the formation of a
polyethylene chain. The free radical attacks and breaks the 71 bond of an
ethylene molecule, forming a new free radical, which then attacks another
ethylene molecule and forms another new free radical, and so on, until
thousands of molecules are joined (Zumdahl, 1986).
IX. MANGANESE A N D IRON
A. LIVINGEARTHR E D ~ X
The redox elements, oxygen and carbon, are basic to the building and
functioning of soils and all living systems, and basic to the functioning of
oxygen and carbon in life’s overall redox scheme are Mn and Fe (Figs. 7
and 8). As the key that unlocks oxygen from water in the process of
photosynthesis, Mn is responsible for the presence of the oxygen in the
atmosphere of the planet Earth. Soil Mn also is a protector of life, the
scavenger of death-dealing oxygen free radicals.
Manganese and iron together provide the key to the establishment of the
organic mantle, the humified soil top layer that covers the surface of the
earth and serves as the nurturing home for the roots of all plants and
carbon-recycling microorganisms. It is the sole provider of food for a
variety of Earth’s creatures, including people.
Iron is vital in the life systems of all plants, animals, microbes, and soils.
In biological systems, Fe appears to play many roles similar to those of Mn.
In animals, on the other hand, Fe is supreme, and Mn, although essential,
displays some toxicity tendencies that may prevent its predomination of
Fe. Iron is the chief camer of oxygen in blood, and it is a camer of
electrons to oxygen in both plants and animals. Except in the soil redox
scheme, Fe appears to have a bigger role than Mn in many living redox
systems. In redox of soils, Fe plays second fiddle to Mn, but the melody of
soil processes requires both metals.
REDOX CHEMISTRY OF SOILS
0 f r e e radical
0 free radicals
Figure 7. The manganese redox system: rather than a simple redox cycle or even a series
of cycles, the Mn system appears to be more a complex web of interacting redox transformations.
Fe(I1) in Primary Minerals
[Low Solubility Fe(lI1) Minerals]
0 free radicals
Organic f r e e radicals
Hemoglobin- Fe ( II)
Met hemoglo bin- Fe( III)
pe, pH, darkness, H
Per o x i d a s e s - Fe( III)
Figure 8. Iron redox cycling: though not depicted here as a circular scheme, the reversibility of the various Fe reactions shown here indicates that Fe is involved in cycling, once it
moves out of its primary mineral origins.
RICHMOND J. BARTLE1-T AND BRUCE R. JAMES
Direct oxidation of soil organic compounds by atmospheric 0, is a rare
occurrence. The majority of apparently spontaneous oxidation reactions
are catalyzed by microbial enzymes, metal oxides, peroxides, or free radicals. Such species serve as electron camers, that is, substances that will
oxidize reduced substances and will in turn be oxidized by more highly
oxidized species. Proteins containing metals-usually Fe, Cu, or Mnserve as redox enzymes in microbial systems carrying electrons from reduced carbon to 0,. These enzymes are essential links in the respiration of
all aerobic organisms. Flavoproteins without metals can transfer electrons
to O,, but the rate is too slow to account for aerobic respiration.
The Fe in hemoglobin must be in the ferrous form in order for it to
combine reversibly with O2and act as the all important carrier of 0, in the
blood of animals (Fruton and Simmonds, 1961). If the Fe is oxidized to
Fe( III), methemoglobin is formed, which does not combine with 0,. The
danger of nitrate in drinking water, spinach, or forage plants is that nitrite,
formed from nitrate by reduction in an infant’s intestine, or in the rumen
of a cow, can oxidize hemoglobin to methemoglobin, causing methemoglobinemia, the inability of the blood to carry oxygen. In the heme structure
of cytochrome c, the Fe is reversibly oxidized and reduced, but in catalases
and peroxidases, also heme compounds, the Fe remains trivalent.
In the enzyme cytochrome oxidase, Fe(II1) is reduced by an organic
compound that is oxidized in the process. The Fe(I1) formed then
“cames” an electron to atmospheric 0,, which oxidizes it spontaneously
back to Fe(II1). Names of redox enzymes are sometimes confusing. An
enzyme carrying an electron from an electron donor to O2 is called an
oxidase because the donor is oxidized. But an enzyme that oxidizes a
carbon compound by transfemng an electron to an oxidized substance
other than O2 or peroxide usually will be referred to as a reductase. An
oxidase that removes both a proton and an electron from an electron-donating substance is called a dehydrogenase, because a proton and an electron
comprise a hydrogen atom. Peroxidase is a peroxide reductase (pe = -4.6
at pH 7; Table I ) usually refined from horseradish. A molecule (about
40,000 g mol-’) contains one atom of Fe(I1) (Fruton and Simmonds,
196 I). To pass along an electron to H,O,, the Fe(II1) borrows an electron
from a carbon atom to form, for an instant, an atom of Fe(I1). The Fe( 111)
is restored during that same instant as the H 2 0 2is reduced.
Because it is so readily oxidized by O,, Fe( 11) can catalyze the oxidation
of a phenolic compound that complexes Fe( 111). The phenolic is oxidized
to a quinone when it reduces the Fe( 111) to Fe( II), which is reoxidized by
0, (Stumm and Morgan, 1981). The Fe(II1) formed is complexed and
REDOX CHEMISTRY OF SOILS
reduced by the excess phenolic compound, and so on. Trace levels of Fe or
other metals can catalyze the oxidation and spoilage of foods by similar
mechanisms. Citric acid is added to prepared food to tie up the Fe( 111) and
prevent its complexation and reduction by the easily oxidized electrondonating food. This reaction occurs commonly in soils in the absence of
light. More difficult oxidations that apparently do not occur in darkness
frequently do take place when the Fe and reduced carbon are exposed to
energy from sunlight (see Section XI).
Because its reoxidation is so much more difficult than that of Fe, Mn is
less effective than Fe in catalyzing complete oxidation of organics. Manganese is more likely to oxidize organic residues partially (making free radicals), setting them up for further microbial breakdown. It is interesting to
note that of the three trivalent redox cations, Cr(II1) can hydrolyze or it
can oxidize, Mn( 111) can only oxidize, and Fe( 111) can only hydrolyze.
1 . Ultimate Electron Acceptor
The outstanding contribution of manganese to life on Earth appears as a
simple entry in the upper right-hand comer of Fig. 7. Both Mn(II1) and
Mn(IV) are powerful oxidants in the soil redox system, especially the free
radical supermanganese Mnw ion, which has the thermodynamic capability of oxidizing 202- to 0, gas. It does this in the leaves of green plants
exposed to radiation from the sun in the process of photosynthesis and
thereby is responsible for creating the oxygen in the atmosphere. However,
the mechanism for this profound redox transformation is only partially
understood (Brudvig and Crabtree, 1989; Thorp and Brudvig, 1991).
It is possible that a singlet chlorine free radical is the direct electron
acceptor, and that the Mn role is one of accepting an electron from C1- to
form (CIS). Marschner (1986) discusses the evidence that chloride acts as a
cofactor in the Mn-containing 0,-evolving system.
2. Proportionation and Disproportionation
Mn2+ is a soluble or exchangeable cation. It forms by reduction of
Mn(II1) or Mn(1V) by a multiplicity of easily oxidizable, reduced organics,
often microbial by-products. The reductive dissolution of Mn oxides by
microbial metabolites has been studied extensively by Stone and Morgan
( 1984). Simultaneous formation of Mn( 11) and Mn( IV) can take place by
the thermodynamically spontaneous disproportionation, or dismutation,
RICHMOND J. BARTLETT AND BRUCE R. JAMES
of two Mn( 111) ions. One Mn( 111) loses an electron to the other to become
Mn(IV), while the electron-accepting Mn( 111) forms Mn( 11). In a reverse of
this dismutation reaction, two molecules of free radical Mn( 111) are constituted when a Mn( 11) gives up an electron to Mn( IV) as follows:
+ 2H20 + 2H+
This reverse dismutation would not be thermodynamically spontaneous
if it were not for the energy of formation of the organic acid-Mn(II1)
complex. An organic acid that readily couples with Mn(II1) and also is
easily oxidized by it can drive the reverse dismutation equation to the right.
With low-molecular-weight organic acids, the Mn( 111) complexes are soluble and range in color from yellow to yellowish brown to red. To be readily
oxidized by Mn( III), the acid must have an oxygen on a carbon adjacent to
a carboxyl group. Oxalic, citric, and tartaric acids are examples, but succinic acid, which chelates Fe( 111) and Al( III), does not complex Mn( 111)
and will not drive the redox.
Pyrophosphate is another ligand that drives this redox reaction by binding strongly to Mn(II1) to form a violet-pink color (Dion and Mann,
1946). Loss of complex color accompanying the oxidation of dissolved
organic carbon by the Mn(II1) serves as a simple colorimetric method for
measuring dissolved organic carbon (Bartlett and Ross, 1988). Pyrophosphate-bound Mn(II1) is not as powerful an oxidizing agent as Mn3+
Mn(111) -Organic Acids as Reductants
In solution, the gradual fading of the color complex indicates that the
organic acid is being oxidized to CO, and H 2 0 by the bound Mn( 111) while
Mn(111) is being reduced to Mn( 11). The rates of formation of the color
complex and its fading both are inversely proportional to the pH. When
redox decomposition such as this takes place in a soil, base-forming cations
are released, causing the pH to rise and the rate of oxidation of the organic
to be slowed or stopped. Mn(III)-citrate made from K,.,-citrate at pH 4.7
decomposes fairly rapidly for a few hours until the pH reaches 7.6, and
then it remains stable, with no more fading or C02 loss for several months.
It was pointed out in Section VI,C and Fig. 3 that the supermanganese
Mn3+ion can function as a double agent, powerful not only as an oxidizer
but also as a reducer. Mn(II1)-citrate will reduce methylene blue and
tetrazolium blue and oxidize tetramethylbenzidine (see Section XIV,D). In
solutions and in soil in the laboratory, under normal fluorescent lighting,
we found that Mn(II1)-citrate or oxalate, formed by reverse dismutation,
reduced Cr(V1) much more effectively at pH 4-6 than the organic acid
REDOX CHEMISTRY OF SOILS
Figure 9. The marked lowering of net Cr oxidized, shown when Mn(I1) and oxalate ware
added together to soil samples containing different levels of oxidized Mn, demonstrates the
reducing power of Mn(ll1) formed by reverse dismutation.
alone. We also showed that Mn(II1)-citrate reduced Cr(V1) faster than the
These effects were strongly borne out in treated soil samples in which the
chromium net oxidation test (see Section XIV,F) was used to characterize
the oxidative minus the reductive powers of the samples (Bartlett, 1988).
Figure 9 shows that adding Mn2+to soil samples already containing MnO,
decreased the net oxidation of Cr by the soil. Part of the reason may be the
temporary increase in positive charge (Fig. 7) in repelling Cr( 111). But the
most probable reason is the reducing effect of Mn( III), which will form by
reverse dismutation (see Section IX,C,2) when Mn2+ is adsorbed onto
MnO,. Adding citrate alone had less effect than Mn2+alone on reduction
of Cr(VI), as shown by the net test. However, adding both Mn2+ and
citrate together markedly increased reduction of Cr( VI), as indicated by
the net Cr oxidation test. Reduction by Mn(II1) formed by reverse dismutation most surely is the explanation for the huge lowering of the Cr
oxidation net test with the two added together.
RICHMOND J. BARTLETT AND BRUCE R. JAMES
Manganese (111) also has the ability to oxidize an organic compound by
single electron steps to form a reducing organic free radical. For example, a
carboxyl free radical (R-COO.) has a strong disposition for giving up its
remaining odd electron to act as a reducing agent.
4. The Oxymoron: Enhanced Oxidation by Oxygen Restriction
In a slightly reducing environment, the first electron step in the partial
reduction of 0, is the formation of superoxide, the oxidizing/reducing free
radical. Manganese(III), formed in the first electron step in the partial
reduction of MnO, , is another extremely reactive oxidizing/reducing free
radical. Its reoxidation produces highly reactive “fresh” MnO, . This effect
is demonstrated by Mn behavior in oxidizing Cr in soils. For example,
partially restricting aeration by stoppering a flask escalated the Mn oxidizing behavior. Ten times the concentration of Cr(VI) was produced in a soil
suspension incubated 9 days with MnSO, and Cr(OH), in a stoppered
flask, as compared with the same volume of suspension swirled in an open
flask. Vigorous aeration of a high-organic-matter soil increased net Cr
oxidation by Mn oxides, but the same aeration lowered oxidation by a
low-organic-matter soil. Stoppering the low-organic-matter soil increased
net Cr oxidation, whereas stoppering the high-organic-matter soil halted
oxidation entirely. Thus, the redox poise between reactivities of oxidants
and reductants is critical. (R. J. Bartlett, unpublished data). Changing the
balance may reverse the direction of whatever is happening.
Compaction of soil in wheel tracks in turf typically results in dark green
stripes on the surface, not between areas of compaction, but at the site of
compaction. Chemical analysis of the vegetation shows that the extra green
is associated with higher nitrogen. The increased nitrogen is the result of
increased oxidation in the root zones of plants where the soil has been
somewhat compacted. Thus, it appears that partial exclusion of oxygen by
compacting a soil can increase certain oxidative processes in that soil.
The favorable oxidative effects resulting from restricting aeration seem
to be mainly related to the reactivity of Mn oxides. Fresh Mn oxides
provide better aeration than air in an oxidizing soil environment. The
oxides are more ready electron acceptors than oxygen. In paper towels
impregnated with high-Mn-oxide soil compared with those with only nutrient solution, white clover seeds germinated more quickly and had a
higher percentage of germination (R. J. Bartlett, unpublished data).
When aeration and respiration and synthesis of electron donors get out
of balance in the rhizosphere, dangerous-to-life free radicals may form.
The enzymes that scavenge such free radicals depend on metals, generally,
Cu, Mn, and Fe, to effect electron transfers. An example is a superoxide
REDOX CHEMISTRY OF SOILS
dismutase described by Fridovich ( 1979, containing Cu2+and also Zn2+,
as a stabilizer. Reduced Mn acts as a scavenger for oxidizing oxygen free
radicals, and oxidized forms of Mn together with Fe are responsible for
accepting electrons from highly reactive reduced toxic or allelopathic organic compounds and then using these compounds for the synthesis of
benign, nurturing, and stable humic substances. Manganese does these
things in the dark, where plant roots grow and develop in soil. Roots
benefit from the humified materials surrounding them with the right balance between 0, and H,O,while, at the same time, they are being protected by redox metals from toxicity of oxygen free radicals.
5 . Mechanism for Oxidation of Manganese in Soils
Manganese(IV) usually occurs in the soil as a colloidal solid oxide. Often
the negative charges on the oxide surface are occupied by adsorbed Mn(II),
and this gives the overall surface a positive charge (Loganathan et al.,
I977), enabling its adsorption by negatively charged colloidal organic matter (Fig. 7). The change in surface charge of MnO, from negative to
positive is easily observed by a reversal in direction of electrophoretic
mobility (Bartlett, 1988). The positive charges also could arise from adsorbed Mn( 111) ions formed by reverse dismutation of Mn( 11) and MnO,.
It is axiomatic that living plants, animals, and microorganisms supply all
of the soil organic substances that are redox reactive. Soil microorganisms
are indispensable in synthesizing and making available phenolic and aliphatic acids and in influencing pH near reactive surfaces by mineralizing
organic matter and releasing base-forming cations. They also “graze” and
metabolize selectively the most biologically available electron-rich substances, those that would tend to interfere the most with oxidation of Mn.
Because the autooxidation of Mn(I1) by atmospheric 0, cannot be
demonstrated unless the pH is above 8 (Diem and Stumm, 1984), it has
been a common assumption that formation of Mn oxides in most soils
requires specific microbial enzymes, and activities of soil microorganisms
have been studied in this regard [e.g., Ehrlich ( 1 976), Silver et al. (1986),
and Sparrow and Uren (1987)l. Unfortunately, lack of Mn oxide formation after use of a chemical microbial inhibitor has been incorrectly used as
conclusive evidence for dismissing the importance of abiotic mechanisms
of Mn oxidation. Chemical inhibitors (e.g., chloroform or sodium azide)
will reduce MnO, in any soil containing organic acids and will destroy the
soil’s Mn-oxidizing mechanism (Ross and Bartlett, 198I). Using Cr oxidation to evaluate Mn oxides, Ross and Bartlett ( 1 98 I ) , showed that oxidation of added Mn was proportional to existing oxides. Arrhenius plots of
rates of Mn oxidation at different temperatures were indicative of nonbio-
R I C H M O N D J. B A R T L E T T AND BRUCE R. JAMES
logical characteristics for the oxidation. Ross and Bartlett (198 1) hypothesized that the oxidation was autocatalytic and that fresh Mn oxides tended
to form on old oxide surfaces.
Chemical oxidation of Mn(I1) added to an acid (pH 4.4)soil and to a
neutral soil was demonstrated directly in another study (Bartlett, 1988). All
of the oxidation of added MnSO, that was to occur during a 36-hr period
occurred the first 15 min. Obviously the oxidation was dependent on the
biochemical status of electron acceptors already present in the soils at the
moment of addition and not on microbial growth in response to the
Mn(I1) additions. After 5 days, there were marked increases in oxidized
Mn in both soils, suggesting that a biochemical readjustment had taken
place, presumably in response to the newly formed Mn(IV) and/or excess
added Mn(II), and to changes in microbial activities. It is not safe to
assume that Mn not extractable by a neutral salt has been oxidized because
strong inner sphere binding of Mn( 11)by soil organic matter above pH 5 - 6
can prevent its exchangeability (McBride, 1982).
Probably the hypothetical “manganese oxidase” enzyme of Silver ef al.
(1986) in reality is either the hydroxyl free radical (OH - ) or the protonated
superoxide free radical (HO;). These are the most likely electron acceptors
in the oxidation of Mn(I1) to Mn(1V). Fresh, newly formed Mn oxides
usually are found in soil regions where oxygen free radicals are being
formed, at redox interfaces, in rhizospheres, and in regions where atmospheric 0, is in somewhat short supply. Free radicals form, and Mn(I1) is
oxidized in scavenging them. Even if there are not microbes that have
specific roles as manganese oxidizers, microorganisms nevertheless are the
ultimate setters of the scene, and, of course, the oxygen free radicals can be
considered to be indirectly the result of microbial activity. A Mn( 11) ion, in
reducing OH and becoming oxidized in the process, is destroying it and
is preventing a microaerophile, busy setting the scene, from being poisoned
in its own juice.
Atmospheric 0, is the terminal electron acceptor when an oxygen free
radical oxidizes Mn( 11) to Mn( IV). When the soil pH and pe are both high,
as in the presence of free CaCO,, 0, may oxidize Mn(I1) to Mn(1V)
directly. Microbial processes also favor direct oxidation in high-pH microsites as they increase pH by releasing base-forming cations during decomposition of organic residues.
D. MANGANESEAND NITROGEN
There are many bits of circumstantial evidence indicating that Mn is
involved in soil nitrogen redox transformations, but there is little under-
REDOX CHEMISTRY OF SOILS
standing of the processes involved. Manganese oxides are thermodynamically capable of oxidizing NH, and N, to nitrate, but there is no hard
evidence that this happens. Circumstantial evidence consists of good
correlations between nitrate production and content of Mn oxides in
incubated soils. The good correlations may have resulted because soil Mn
oxides retarded or prevented denitrification by oxidizing nitrite back to
nitrate as fast as it formed, or else Mn oxides perhaps prevented denitrification by scavenging readily available organic reducing agents, oxygen free
radicals, or Fe( 11).
Both the oxidation of NH,OH to nitrate and of nitrite to nitrate by
synthetic amorphous MnO, are easily demonstrated in the test tube (Bartlett, 1981b, 1988). Bartlett ( I 98 1b) showed that the amount of nitrate
formed in soils from added nitrite at 0.5”C was directly related to the net
Cr oxidized by the standard oxidation test (see Section XIV,F), that is, to
the net oxidizing ability of the soil Mn oxides. Nitrate formation and
MnO, reduction were stoichiometrically related in the presence or absence
of atmospheric 0,. When MnO,/NO? ratios were high, reduction to
Mn( 111) was mainly observed; when low, Mn( 11) was the reduced product
accompanying nitrate formation.
X. SOIL CHROMIUM CYCLE
The Cr cycle, Fig. 10, begins where it has ended, with Cr in its least
mobile form, Cr( III), precipitated or tightly bound by a variety of ligands,
such as hydroxyls, humates, and phosphates, or, in its most inert forms,
substituting for two atoms of Fe in the magnetite structure, as FeCr204,or
for small amounts of octohedral A1 in clay minerals.
Like Al, Cr( 111) can be mobilized by low-molecular-weight organic acids
such as citrate. The chelated Cr3+ may then interact with negatively
charged MnO, and become oxidized to Cr(VI), the HCrO; ion in the
diagram. Some of the citrate ligands are recycled. If there is a surplus of
citrate, the Mn2+formed when the Cr was oxidized may react with surplus
MnO, and reversely dismutate to two molecules of Mn(II1)-citrate, according to Eq. (36). Highly reducing Mn(II1)-organic, when it forms, will
temporarily interfere with further Cr oxidation.
The next step is “dechromification,” or the reduction of Cr( VI) by
carbon reduced by the sun’s energy through photosynthesis. An intermediate species, such as Fe2+or S2-, reduced by carbon, can serve as the direct
electron donor. Direct sunlight may hasten the process of Cr(V1) reduction. It is theoretically conceivable that dechromification, like denitrifica-