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OH OH
Figure 1. TNT biodegradation p a t h ~ a y s . 6 ~ ' ~ ~ ' ~
tion.'* Ring cleavage occurs through the meta-cleavage toluate, by ring substitution of water to form p-cresol,
pathway using plasmid-encoded en~ymes.3~ or by hydroxylation of the methyl group to form benzo-
Anaerobic degradation of toluene is postulated to ate.37Dentitrifying bacteria, including several Pseudom-
proceed by ring substitution of carbon dioxide to form onus species, have been reported to favor anaerobic
toluene degradation via methyl group hydroxylation
(toluene pathway C ) . ' TAs
~ ~ previously indicated, aero-
bic biodegradation is advantageous; therefore, toluene
2,4,6-Trinitrotoluene
bioremediation would likely be conducted under these
conditions. However, at least one anaerobic pathway is
needed for thermodynamic comparison. Thus, toluene
degradation via methyl group hydroxylation will serve
for this comparison. Under anaerobic conditions, ring
cleavage is facilitated by a hydration rea~tion.'~.~' Fur-
ther catabolism of the cleaved ring involves the incorpo-
Hydride-Meisenheimer complex
-.%: N02'
H
ration of three coenzyme A molecules.16
TNT Pathway C
Using indigenous microorganisms from TNT-contami-
nated soil inoculated with either dinoseb-degrading soil
bacteria or anaerobic TNT-degrading methanogenic
bacteria, Funk et al.I5recently reported the biodegrada-
2,4-Dinitrotoluene tion of TNT beyond triaminotoluene. The stepwise re-
duction of the TNT nitro groups yields triaminotoluene
NO2
as previously outlined in TNT pathway A (Fig. la).
Figure 2. Proposed mechanism for TNT pathway B nitro group The proposed anaerobic pathway for triaminotoluene
removal. The hydride-Meisenheimer complex is a key interme- biodegradation (Fig. lc) proceeds through methyl
diate.13.29,43 phloroglucinol and p-~resol.'~ p-Cresol is known to be
200 BIOTECHNOLOGY AND BIOENGINEERING. VOL. 51, NO. 2. JULY 20, 1996
catabolized under both aerobic and anaerobic condi- dard state is not known for most biochemical com-
tions by a variety of mi~roorganisms.’~ p o u n d ~ . ~This
~”~ is especially true for xenobiotic com-
pounds such as TNT and its metabolic products.
Therefore, a method to estimate the Gibbs free energy
pCresol Pathways A, B, and C of formation for TNT biodegradation intermediates is
Several microorganisms can transform p-cresol, the needed.
TNT pathway C metabolic product, into TCA cycle Group contribution methods have been widely used
intermediates under both aerobic and anaerobic condi- to estimate the ideal gas properites of pure compounds.
tions. As with toluene biodegradation pathways, aerobic These methods are based largely on the structures of
p-cresol catabolism occurs either through direct ring the compounds and to a lesser extent on state variables
attack by oxygen-dependent enzymes (p-cresol path- such as temperature and pre~sure.’~ Mavrovounioti~~~~~~
way A) or through hydroxylation of the methyl group recently developed a group contribution method that
(p-cresol pathway B).3,”723,25In the first approach, 4- accurately estimates the Gibbs free energy of formation
methyl catechol is formed by monooxygenase attack on for the biological standard state. Use of the group contri-
the aromatic ring.23 Ring cleavage and mineralization bution method requires that biochemical compounds
occur via the meta-cleavage pathway.’ The second deg- be decomposed into functional groups (e.g., carboxyl
radation approach involves methyl group hydroxylation group, hdyroxyl group, etc.). Each functional group is
to form 4 - h y d r o x y b e n ~ o a t eProtocatechuate
.~~~~~ is cre- assigned a value based on the group’s partial contribu-
ated after a subsequent oxygenase reaction and is miner- tion to the total thermodynamic property of the com-
alized via the 3-oxoadipate (ortho-cleavage) pathway.21 pound. Functional group values were obtained using a
Anaerobic degradation of p-cresol is postulated to pro- multiple linear regression routine based on biochemical
ceed through methyl group hydroxylation to form 4- compounds with known Gibbs free energies of forma-
hydroxybenzoate (p-cresol pathway C).8,’4 Reaction tion.33,34The biological standard Gibbs free energy
with coenzyme-A creates 4-hdyroxybenzoyl-CoA, change is affected only by the functional groups which
which is subsequently dehydroxylated to yield benzoyl- change during the reaction; therefore, only the product
CoA, a key intermediate.2n.42Further catabolism in- and reactant functional groups which change are in-
volves ring cleavage through a hydration reaction and cluded in the free energy calculation. The biological
the incorporation of three coenzyme A molecules. standard Gibbs free energy change is determined by
subtracting these reactant functional groups from the
product functional groups.
METHOD
Although the group contribution method could theo-
We postulate that the likely rate-controlling steps (or retically be used for any organic compound in aqueous
sets of steps) in TNT biodegradation and mineralization solution, the data used to develop the method were
pathways outlined above will have large negative Gibbs heavily biased toward biochemical compounds and reac-
free energy changes. The biological standard Gibbs free t i o n ~For
. ~ ~this reason, some of the functional groups
energy change is given by the following equation: that occur in xenobiotic compounds such as TNT were
not included in the published database. Functional
AGO1 = -RT In K (1) groups such as the nitro group, -NO2, simply are not
where the equilibrium constant, K, represents the activi- found in natural biological systems. Because the re-
ties of the products divided by the reactants with each moval or reduction of the nitro groups is a key step in
activity raised to the power of its stoichiometric coeffi- TNT biodegradation, the unavailability of a nitro group
~ i e n t The
. ~ ~biological standard state is defined as a contribution presented a major obstacle to using this
dilute aqueous solution at pH 7 and 25°C in which the method. A nitroaromatic Gibbs free energy of forma-
reacting species have molal concentrations equal to tion was needed to determine the contribution of the
unity.41 The biological standard Gibbs free energy nitro group. A nitroaromatic compound was preferable
change represents the useful energy liberated by such because its group contributions would better reflect pos-
chemical reactions, which the cell may then capture for sible nitro group and aromatic ring interactions.
other cell functions. The biological standard Gibbs free Nitrobenzene was selected by default, because it was
energy change, however, does not predict the kinetics the only nitroaromatic compound for which thermody-
or rate of the reaction. namic data were available.32From a Gibbs free energy
The Gibbs free energy of formation for a compound of hydration and a Gibbs free energy of formation in
is the free energy required to create the compound from the ideal gas state, the Gibbs free energy of formation
its elements. The difference in Gibbs free energies of of nitrobenzene in aqueous solution was
formation between the products and reactants of a par- The nitro group contribution was then obtained by back-
ticular reaction represents the standard Gibbs free en- calculating its value using known contributions for the
ergy change, AGO, for that reaction. Unfortunately, the aromatic ring. The calculated nitro group contribution
Gibbs free energy of formation at the biological stan- of +6.9 kcal/mol is similar to those for other nitrogen-
202 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 51, NO. 2, JULY 20, 1996
Table 111. TNT pathway B r e ~ u l t s . ' ~ An explosive compound such as TNT possesses an
enormous potential free energy; therefore, it is quite
possible that individual TNT biodegradation steps (or
sets of steps) would have relatively large negative Gibbs
free energy changes as compared to glycolytic and TCA
cycle steps. If intermediate steps with large -AGO/ exist,
these reactions represent a potentially rate-limiting pro-
cess through which the reaction must proceed. The use
of genetic engineering techniques to increase specific
enzyme levels or otherwise alter regulation will require
identification of the enzymes catalyzing these possible
intermediate reactions. In any case, the free energy cal-
culations afforded by the group contribution method
[using the bacterium, Desulfovibrio sp. (B strain)]," and may help identify those reaction steps which limit the
they "did not identify metabolites other than toluene overall rate of biodegradation and mineralization. This
even after 6 months of incubation." Therefore, the pres- analysis assumes that the transport of intermediate me-
ent work both predicts and provides supporting evi- tabolites across cell walls and between microorganisms
dence that the removal of the nitro groups on TNT in a consortium is not rate-limiting.
and the cleavage of the aromatic ring are major rate- The overall Gibbs free energy changes for the TNT
controlling steps in TNT biodegradation. biodegradation pathways are given in Table V. Because
The Gibbs free energy change is a state function; a consortium of microorganisms could be used to de-
therefore, the free energy change between two specified grade TNT and its intermediates, all possible pathway
TNT metabolic intermediates will be constant and inde- combinations were used to generate this table. For accu-
pendent of the reaction path. Several TNT biodegrada- rate pathway comparisons, the thermodynamic data
tion reactions involve intermediate steps that are not given in Table V are based on TNT as the starting
included in the pathways described here, because the substrate and citrate as the common metabolic product.
mechanisms of these reactions are not well known. The Although TNT pathways A, B, and C are initially anaer-
calculated free energy change for some steps is quite obic, these pathways coupled with their respective aero-
large; therefore, these reaction "steps" are probably bic toluene or p-cresol pathways possess the largest
composed of several intermediate steps which have not overall negative free energy changes. Pathway combina-
been fully determined. A close examination should be tions which are completely anaerobic have the smallest
conducted to determine whether these are indeed one- negative free energy changes. Aerobic reactions obvi-
step mechanisms with large -AGO' or whether they are ously release more free energy than their anaerobic
instead composed of several intermediate steps, one or counterparts. Therefore, the aerobic TNT degradation
more of which may have a large -AGO/. pathways would be expected to liberate more free en-
ergy than their anaerobic counterparts, and the results
summarized in Table V fulfill this expectation.
Table IV. TNT pathway C r e s ~ l t s . ' ~
Within each initial TNT degradation route, the path-
way combination possessing the largest overall negative
204 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 51, NO. 2, JULY 20, 1996
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