Beruflich Dokumente
Kultur Dokumente
Key words: aromatics biodegradation, energetics, intermediates, oxygenation, phenanthrene, stoichiometry, yield
Abstract
Oxygenation reactions significantly alter the energy and electron flows and, consequently, the overall stoichiometry
for the microbial utilization of aromatic compounds. Oxygenation reactions do not yield a net release of electrons,
but require an input of electrons to reduce oxygen molecules. The biodegradation pathway of phenanthrene as a
model compound was analyzed to determine the impact of oxygenation reactions on overall stoichiometry using the
half-reaction method. For individual oxygenation reactions, the half-reaction method for analyzing the electron and
energy flows must be modified, because the reactions do not release electrons for synthesis or energy generation.
Coupling the oxygenation reaction to subsequent reaction steps provides a net electron release for the coupled
reactions. Modeling results indicate that oxygenation reactions increase the oxygen requirement and reduce the
cell yield, compared to the conventional mineralization represented by hydroxylation reactions in place of oxy-
genations. The computed yields considering oxygenation reactions conform better to empirical yields reported
in the literature than do yields computed by the hydroxylation single-step methods. The coupled-reaction model
also is consistent with information about the ways in which micro-organisms that degrade aromatics accumulate
intermediates, regulate degradation genes, and organize enzyme clusters.
For the case of the oxygenation method, complete genations. This extra energy yield partially offsets the
mineralization of one mole phenanthrene incorpor- loss of 24 electrons shunted to O2 reductions.
ates six moles of O2 , and the standard reduction half Table 2 summarizes all the key parameters for the
reaction is estimation of the overall stoichiometry for one-step
mineralization of phenanthrene using Equations (2),
14 1 30 6
H2 CO3 +H+ +e = C14 H10 + H2 O+ O2 (g) (7), (10), and (11). In these calculations, pyruvate
42 42 42 42 is assumed to be a carbon source for both methods,
because pyruvate is always released as an interme-
(G0d = 85.52 kJ/e-eq). (16) diate in the biodegradation pathway. The fs0 value
(0.482) in the oxygenation method is 26.4% lower
This reaction indicates that only 42 electron equi- than fs0 (0.655) in the hydroxylation method. How-
valents per mole of phenanthrene are available for ever, this decrease is not as much as the electron
energy generation or biomass synthesis for the actual loss to oxygenation reactions (i.e., 24/66 or 36.4%).
degradation involving oxygenation, and the other 24 This result quantities the off-setting effect of the in-
electron equivalents are invested to reduce oxygen mo- creased free energy of the electron-donor reaction
lecules. The free energy for reaction 16 is G0d = when oxygenation reactions are considered.
85.52 kJ/e-eq. Thus, mineralization with oxygenations
is more energy yielding (per electron transferred from
the donor to the acceptor) than oxidation without oxy-
220
Table 2. Key parameters and calculated values for single-step
mineralization of phenanthrene by hydroxylation and oxy-
negative electron flows (T 0). Thus, an oxygenation
genation methods reaction fundamentally alters electron and energy bal-
ances (e.g., Equations (7) and (8)), which require that
Hydroxylation method Oxygenation method the donor reaction releases electrons, or T > 0. To
perform the electron and energy balances for reactions
G0d 25.87 85.52
with T 0, we first had to consider how the mi-
G0r 103.96 163.61
croorganisms supply electrons for situations in which
T 66/66 42/66
H 0/66 0/66
T 0.
O 0/66 24/66 When T = 0 (i.e., reactions C, H, and M), the
A 0.503 0.319 reaction releases no electrons and cannot supply any
fs0 0.665 0.482 electrons for energy generation and synthesis. In other
fe0 0.335 0.154 words, fs0 = fe0 = 0. However, these reactions
can release energy. Thus, reactions with T = 0 have
G0a = 78.06, G0cs = 35.72, G0syn = 31.35, G0pyr a mismatch between energy generation and electron
0
=35.72, Gcells = 31.35, m =1, K = 0.6. transfer. We reconcile this mismatch by coupling any
All free energies are in kJ/electron equivalent. oxygenation reaction having T = 0 with subsequent
reactions in the pathway so that a net electron release
Substituting T , fs0 , and fe0 values into Equa- results. For example, reaction C (T = 0) is combined
tion (14) predicts the following overall stoichiometric with reaction D, E, and F to yield a 12-electron release.
equations for each method: When T < 0, the oxygenation requires an input of
electrons from another source. Reaction A, G, and L
Hydroxylation method: have T < 0. In parallel to the situation for T = 0, we
couple subsequent reactions to the oxygenation to give
C14 H10 + 2.196NH+
4 + 5.520O2(g) + 2.412H2O
a positive T . The 3 coupled reactions having T > 0
are AF, GK, and LN. Electron-donor reactions and
= 3.020H2CO3 +2.196H+ + 2.196C5H7 O2 N (17) electron flows for the set of coupled reaction are sum-
marized in Table 3. The biodegradation pathway of
Oxygenation method: phenanthrene via the coupled-reaction model is shown
in Figure 3.
C14 H10 + 1.592NH+
4 + 8.541O2(g) + 4.225H2O
Coupling reactions make it possible to apply Equa-
tions (2) and (7) to compute fs0 and fe0 . The para-
= 6.041H2CO3 + 1.592H+ + 1.592C5H7 O2 N. (18) meters and fs0 values for each step are shown in
Table 4. Since pyruvate is released as an intermediate
The oxygenation method gives a higher oxygen re-
in all the coupled reactions, this compound is as-
quirement and a lower cell yield. Thus, conventional
sumed to be the carbon source. Each coupled reaction
mineralization, which is represented by the hydroxyla-
produces a positive electron transfer (T ) and, con-
tion method, should significantly overestimate cell
sequently, positive fs0 and fe0 values. Substituting the
yield or underestimate oxygen requirement for poly-
electron fraction values into Equation (14) provides
cyclic aromatic hydrocarbons, in which many oxy-
overall stoichiometric equation for each step degrad-
genation reactions are involved. The validity of this
ation reaction. Table 5 summarizes the mole-based
prediction is evaluated against experimental results in
stoichiometric coefficients for each step. The sum of
a later section.
the molar cell yield for the coupled reactions (1.589
Multi-step degradation involving oxygenations mole cells/mole substrate) is similar to that in the
single-step reaction (1.592 mole cells/mole substrate).
Complete phenanthrene degradation actually consists
of multiple enzyme reactions in series, and accu- Comparison of alternatives
mulation of intermediates sequesters electrons that
otherwise could be used for energy and synthesis. Table 6 compares normalized electron fractions used
Therefore, the energetics and stoichiometry for each for synthesis for each reaction. For each reaction step,
step should be analyzed. Phenanthrene degradation in- fs0 (m) values are fs0 for that step normalized to the
cludes several oxygenation reactions that have zero or number of electrons in phenanthrene.
221
Table 3. Electron donor reactions and electron flows for the coupled reaction for phenanthrene degradation
Rxn. H R T O G0d
AF GK LN
G0d 90.83 108.43 70.93
G0r 168.91 186.51 148.98
T 12/66 10/46 20/28
H 46/66 28/46 0/28
O 8/66 8/46 8/28
A 0.309 0.280 0.351
fs0 0.139 0.170 0.529
fe0 0.043 0.048 0.185
G0a = 78.06, G0pyr = 35.72, G0cs = 35.72, G0syn = 31.35,
0
Gcells = 31.35, m =1, K = 0.6.
All free energies are in kJ/electron equivalent.
AF GK LN sum 1 step
(eq. 18)
CA 1 0 0 1 1
CG 1 1 0 0 0
CL 0 1 1 0 0
O2 2.709 2.547 3.298 8.554 8.541
H+ 1.458 0.391 0.260 1.589 1.592
H2 O 0.625 1.828 1.779 4.232 4.225
Figure 3. Release of electrons during degradation of phenanthrene H2 CO3 0.709 2.047 3.298 6.054 6.041
via the coupled-reaction model. CA , phenanthrene; CG , 1-hy- NH+4 0.458 0.391 0.740 1.589 1.592
droxy-2-naphthoate; and CL , salicylate. Note that coupled reactions
GK and LN divide the naphthalene lower pathway into two C5 H7 O2 N 0.458 0.391 0.740 1.589 1.592
segments. Also note that e are held by intracellular carriers, such Ci represents the reactant in the reaction i.
as NADH+ 2. Negative and positive values represent a reactant and
product, respectively.
222
Table 6. Comparison of normalized electron fractions for cell
synthesis in each of the approaches
Using a molecular weight for cells of 113 g/mole
(for C5 H7 O2 N), the cell yields (Table 6) are 1.39,
Reaction T fs0 fs0 (m)a fs0 (s)b True 1.01, and 1.01 g cells/g phenanthrene for hydroxyla-
yieldc tion single-step, oxygenation single-step, and the sum
of coupled reactions, respectively. The yield is 0.89 g
Coupled AF 12/66 0.139 0.139 0.138
cells/g phenanthrene if the 6 oxygenation reactions
reaction GK 10/46 0.170 0.118 0.115
LN 20/28 0.529 0.224 0.229
and the 24 electrons involved in them are eliminated
Sum 0.481 0.482 1.01
from the computation of G0r . Table 7 summar-
izes experimentally estimated yield values for phen-
Single-step 42/66 0.482 0.482 0.482 1.01 anthrene. In some cases, lower yield values have
Oxygenation been reported (Aichinger et al. 1992; Bouchez et al.
Single-step 66/66 0.665 0.665 0.665 1.39 1996; Keuth and Rehm 1991; Stucki and Alexander
Hydroxylation 1987; Weissenfels et al. 1990; Wodzinski and John-
Single-step 42/66 0.423 0.423 0.423 0.89
son 1968). However, the experimental conditions in
Ignoring all these studies significantly affected the apparent yield
oxygenations values, making them incomparable to our model pre-
dictions. In many cases, maintenance or decay reduced
a f 0 in each step of multi-step degradation, normalized to the num-
s the apparent cell yield, in particular with low spe-
ber of electrons in an initial donor substrate, fs0 (m) = fs0 cific growth rates and long-term cultivation (e.g., 10
T /(T EQS).
b Fraction of original electrons going to synthesis if the f 0 of days in Bouchez et al. (1996) and Stucki and Al-
s
the total reaction is apportioned according to the number of exander (1987)). In other cases, accumulations of
electrons released in each step, fs0 (s) = fs0 (single-step) intermediates or dead-end products (e.g., Aichinger
T /[T (single-step) EQS]. et al. 1992; Bouchez et al. 1996; Keuth and Rehm
c g cells/g phenanthrene.
1991; Wodzinski and Johnson 1968) sequestered elec-
trons and carbon (H > 0), thereby reducing the
observed yields per phenanthrene consumed. Finally,
fs0 T non-optimal nutrient sources could bring about lower
fs0 (m) = (19)
T EQS cell yield values; e.g., nitrate, used in Bouchez et al.
(1996) and Weissenfels et al. (1990), or unknown ni-
in which EQS is the number of electron equivalents in
trogen sources, used in Guha and Jaffe (1996), incurs
the original substrate (66 e-eq/mole for phenanthrene).
additional synthesis costs (McCarty 1969) that reduce
These are compared to fs0 (s), which is the fraction of
yields. Only the study of Zhang et al. (1994) avoided
original electrons going to synthesis if the fs0 of the
these complications and provided yield values directly
total reaction is apportioned according to the number
comparable to our predictions. The yields from the
of electrons released in each step.
two oxygenation methods conform best to the compar-
fs0 (single-step) T able experimental yield reported in the literature, 1.0 g
fs0 (s) = . (20) cells/g phenanthrene (Zhang et al. 1997), than does the
T (single-step) EQS
hydroxylation method (1.39 g cells/g phenanthrene)
The comparison of fs0 (m) to fs0 (s) in Table 6 shows or ignoring all energy associated with oxygenation
that a step-by-step computation of fs0 does not yield reactions (0.89 g cells/g phenanthrene).
values that are weighted averages of the single-step We used the same methods to estimate yield values
oxygenation method. The difference between fs0 (m) for another PAH compound, naphthalene, for which
and fs0 (s) depends on the free energy for the energy- experimental yield values are more often reported in
generating redox couple (G0r ) alone, because the the literature. The coupled-reaction model has 2 steps
synthesis cost in all the coupled reactions is the same and the intermediate is salicylate. The predicted yield
as the single-step oxygenation reaction. For example, values are 1.411, 1.059, 1.058, and 0.94 g cells/g
the second coupled reaction (GK) gives a higher naphthalene for hydroxylation single-step, oxygena-
fs0 (m) value than an fs0 (s) value, because G0r for tion single-step, the sum of coupled reactions, and
this reaction (186.51 kJ/e-eq) is lower than that for ignoring all oxygenation reactions, respectively. Sim-
the single-step reaction (163.61 kJ/e-eq), while the ilar to the case of phenanthrene, the yields for the two
sum of each computational approach is very similar. oxygenation methods conform best to the two com-
223
Table 7. Cell yield values and cultivation conditions reported in the literature. Boldface type indicates that the experimental conditions did not
conform to our model conditions
parable yields reported in the literature: 0.93 0.12 g threne and anthracene. Yang et al. (1994) also demon-
cells/g naphthalene (Buitron and Capdeville 1993) and strated that a single gene cluster can encode multiple
1.2 g cells/g naphthalene (Volkering et al. 1993). PAH degradations. The biochemistry and genetics of
A physiological basis for the coupled-reaction the naphthalene degradation pathway contained on the
model is supported by other information reported in NAH7 plasmid has been well characterized (Yen and
the literature. The first supporting evidence is the Serdar 1988). Naphthalene-oxidation genes are organ-
typical pattern of intermediate accumulation. The in- ized in two operons. The first operon includes genes
termediate 1-hydroxy-2-naphthoate, which is the com- nahABCDEF, coding for the upper pathway, and the
pound produced from the first-step in the coupled- second operon includes genes nahGHIJK, coding for
reaction model, is the most frequently accumulated the lower pathway (salicylate oxidation via the ca-
intermediate during phenanthrene degradation (Evans techol meta-cleavage) (Yen and Gunsalus 1985). Thus,
et al. 1965; Guerin and Jones 1988; Kiyohara and the genes needed for the coupled reactions seem to be
Nagao 1978). Although not frequently occurring, regulated as gene clusters in each operon. This sug-
salicylate, the compound produced from the second gests that cells may regulate several sequential reac-
coupled step, also is accumulated in the media during tions in order to link electron-requiring and -producing
phenanthrene degradation via the naphthalene path- reactions. Thus, regulating the expressions of genes to
way or during naphthalene degradation (Evans et al. correspond to the coupled reactions seems to be an ef-
1965; Wodzinski and Johnson 1968). On the other ficient and economic strategy for ensuring an adequate
hand, other intermediate compounds are not found fre- supply of electron and energy for all the individual
quently in the media during phenanthrene degradation. reactions.
For example, 1-hydroxy-2-naphthoate, instead of 3,4- The third type of support is the organization of
dihydroxyphenanthrene (the product of reaction B), enzymes for the coupled reactions into multi-enzyme
was detected during the enzyme reactions of crude cell complexes having coupled catalytic functions. In some
extracts (Barnsley 1983b; Ensley et al. 1982; Evans et cases, enzymes are bound to the surface of a mem-
al. 1965). brane in adsorptive arrays or are more strongly in-
The second type of supporting evidence is the or- corporated into the lipid bilayer of the membrane as
ganization of gene regulation. The upper pathway of integral arrays (Friedrich 1984). Through structural
phenanthrene degradation to 1-hydroxy-2-naphthoate organization, the proximity of active sites may then
is very similar to the pathway for naphthalene degrad- render it possible for the product of the first enzyme to
ation to salicylate (Kiyohara et al. 1994). Sanseverino get rapid access to the second enzyme. This physical
et al. (1993) demonstrated that NAH7, a plasmid en- juxtaposition of enzymes in a sequence can markedly
coding naphthalene degradation, and other NAH7-like accelerate the overall reaction rate without the spilling
plasmids could also mediate metabolism of phenan- of intermediates (Gaertner 1978).
224
The location of enzymes involved in phenanthrene large as the diversion of electrons to reduce O2 , due
or naphthalene degradation has not been intensively to the off-setting effect of the increased free energy
studied. Several enzymes involved in degradation of release when O2 is a direct cosubstrate.
naphthalene, phenanthrene, or benzene were purified For individual oxygenation reactions, the half-
from the supernatant of cell extracts, indicating that reaction method for analyzing the electron and energy
these enzymes probably are not integrated into the flows is inadequate, because the reactions do not re-
lipid bilayer (Axcell and Geary 1975; Ensley et al. lease electrons for synthesis or energy generation. We
1982; Evans et al. 1965). Nevertheless, the enzymes overcame this complication by coupling the oxygen-
may be associated with the cell membrane in adsorpt- ation reaction to subsequent reaction steps so that
ive arrays, which may readily be separated from the the coupled reaction had a net electron release. We
membrane (Kurganov 1985). This hypothesis seems predicted the stoichiometry of each coupled reaction,
reasonable, since phenanthrene or naphthalene should as well as the sum of the coupled reactions. The
be strongly associated with cell membrane surfaces yield for the sum of coupled reactions was similar to
due to their hydrophobic properties (Stringfellow and a single-step mineralization involving oxygenations,
Alvarez-Cohen 1999); then, having the enzyme reac- much smaller than for single-step mineralization in
tions occur at the membrane is an economic strategy, which hydroxylations replace all oxygenations, and
as addressed by Robertson and Button (1987) for larger than ignoring all energy released from oxygen-
toluene degradation. Barnsley (1983b) observed that ation reactions
the maximum rate of oxidation of naphthalene meas- Oxygenation methods, single step or coupled, con-
ured with whole cells was 348 nmol/min-mg protein, formed better to empirical yields reported in the lit-
whereas with uncentrifuged disrupted cells it was 30 erature than did the hydroxylation single-step method
nmol/min-mg protein. For centrifuged extracts of the or ignoring energy released from oxygenation reac-
same organism, Ensley et al. (1982) reported a max- tions. Reported observations on what intermediates
imum rate of 4.6 nmol/min-mg protein. This indicates frequently accumulate, the regulation of the PAH-
that separation of enzymes from cell structure may degrading genes, and the location of enzyme clusters
significantly reduce the enzyme activity. Thus, the also support that the coupled reactions have a solid
enzymes in the coupled reactions may be associated physiological basis.
with the membrane to some extent to take advantage
of physiological proximity.
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