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Biodegradation 11: 213227, 2000.

2001 Kluwer Academic Publishers. Printed in the Netherlands.


213

Microbial energetics and stoichiometry for biodegradation of aromatic


compounds involving oxygenation reactions

Seung H. Woo & Bruce E. Rittmann


Department of Civil Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3109, USA
( author for correspondence: present address: School of Environmental Engineering, Pohang University of Science
and Technology, San 31, Hyoja-Dong, Pohang 790-784, Korea; e-mail: shwoo@ced.postech.ac.kr)

Accepted 20 June 2000

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.

Introduction of the compound of interest (VanBriesen and Rittmann


2000).
Aromatic hydrocarbons are ubiquitous contaminants Mathematical modeling of the biodegradation of
in the environment, and some are considered to be an electron-donor substrate typically includes cell
hazardous due to their toxic and carcinogenic prop- growth, substrate consumption, and cell decay. Each
erties (Keith and Telliard 1979). These compounds, reaction rate can be linked to the others by yield
which may consist of one or more benzene rings, can values, which can be obtained from experimental res-
be degraded by numerous microorganisms existing ults or from theoretical stoichiometric relationships.
in the natural environment (Gibson and Subramanian The stoichiometric relationships are usually created
1984; Smith 1990). The complete mineralization of by the method of regularities (Erickson 1979; Roels
the aromatic hydrocarbons produces cell mass and 1980; Stouthamer and van Verseveld 1985) or the
inorganic materials, such as CO2 and H2 O. How- method of half reactions (Heijnen et al. 1992; Mc-
ever, the biodegradation of the compounds involves Carty 1969, 1972; VanBriesen and Rittmann 2000).
multiple degradation steps, and metabolic intermedi- Heijnen and Van Dijken (1992) compared several
ates often accumulate in the media (Guerin and Jones regularity constants for the estimation of cell yield
1988; Kiyohara and Nagao 1978). Therefore, it can and suggested that the Gibbs energy-dissipation value
become necessary to analyze individual degradation provides the best tool for estimating of cell yield
steps in order to completely model the biodegradation among black-box models. However, the regularity or
214

Gibbs energy-dissipation method cannot predict cell


yield without experimental yield data, peculiarly for
hydrocarbon substrates with more than six carbons.
Furthermore, when intermediate formation is critical
during multi-step substrate degradation, a modified
half-reaction methodology is the best means for es-
timation of stoichiometric relationships (VanBriesen
and Rittmann 2000). The half-reaction method estim-
ates the cell yield based on a balancing of energy and
electrons flows for the catabolic and anabolic reactions
(McCarty 1969, 1972).
Microbial degradation of aromatic compounds in-
cludes dioxygenation or monooxygenation reactions,
which insert hydroxyl groups into the compound in
order to activate the molecule before ring cleavage.
In general, an oxygenation reaction involves direct in-
corporation of molecular oxygen from O2 rather than
from water (Gibson and Subramanian 1984; Hayaishi
et al. 1970). The aromatic substrate is oxidized dur-
ing an oxygenation, but reduction of two molecular
oxygens in O2 to the 2 oxidation state requires four
electrons. As a result, oxygenation reactions do not Figure 1. Electron and energy flows for biodegradation of an elec-
yield a net release of electrons, but require an input of tron-donor substrate. (a) traditional complete mineralization and (b)
electrons from an intracellular electron carrier such as intermediate formation.
NADH+ 2 . This characteristic of oxygenation reactions,
unlike more normal oxidation reactions, fundament- are evaluated based on experimental results reported in
ally alters the energy and electron flows when cells the literature.
are oxidizing aromatic hydrocarbons. In particular,
polycyclic aromatic hydrocarbons (PAHs) have two or
more condensed rings, and, therefore, many oxygen- Theoretical background
ation reactions are included for a full ring-cleavage
of the compounds. For example, the complete bio- Energy and electron balances without oxygenation
degradation pathway for naphthalene, which has 2 reactions
rings, entails 4 oxygenation reactions (Gibson and
Subramanian 1984), while the complete biodegrada- The stoichiometry of biological reactions relies upon
tion of the 3-ringed phenanthrene has 6 oxygenations relationships describing energy and electron balances.
(Gibson and Subramanian 1984). The multiple oxy- Generally, when an electron-donor substrate is ox-
genation reactions can have an important impact on idized to release available electrons, some electrons
the yield and stoichiometry. are transferred to an electron-acceptor substrate for
In this paper, we analyze the biodegradation path- energy generation, while the remainder of the elec-
way of phenanthrene, a 3-ring PAH compound, as trons are incorporated into newly generated cell mass.
a model compound to determine the impact of oxy- The proportioning of electrons between the synthesis
genation reactions on overall stoichiometry. For this and energy-generating reactions defines the yield, or
analysis, the half-reaction method following McCarty the ratio of bacteria produced to substrate consumed.
(1969) and VanBriesen and Rittmann (2000) forms These flows of electron and energy are shown schem-
the basis for analyzing multi-step degradations that atically in the top panel of Figure 1.
involve oxygenations. Because oxygenation steps do Considering only synthesis, the energy generated
not provide net electron gains, we must consider how by shuttling electrons from the donor to the acceptor
the cells link other electron-releasing reactions to drive must equal the energy invested in cell synthesis. Fol-
the oxygenation reactions. The feasibility of our hypo- lowing McCarty (1969, 1972), the general relationship
thesis and relationships among several enzymatic steps for the energy balance is
215

and Rittmann (2000) defined T as the fraction of elec-


 
fe0 KG0r = fs0 G0syn (1) trons from the donor transferred to either the energy-
generation or the biomass-synthesis pathways, while

in which G0r is the standard free energy of the H is the fraction of electrons held in the intermediates.

energy-generating redox couple, G0syn is the stand- This is shown in the bottom panel of Figure 1. Then,
ard free energy of the cell synthesis reactions, K is
the efficiency of energy capture in the energy gen- T +H =1 (5)
eration reaction, fe0 is the fraction of electron-donor and
electron equivalents sent to the acceptor to drive the
energy generating redox reaction, and fs0 is the frac-
fs0 + fe0 = T . (6)
tion of electron-donor electron equivalents invested in
biomass via the synthesis reaction. Equation (1) says VanBriesen and Rittmann (2000) showed that Equa-
that the energy captured from transfer of fe0 electron tions (2) and (6) could be solved simultaneously to
equivalents from the donor to the acceptor is inves- obtain
ted to synthesize fs0 electron equivalents of biomass.
The efficiency of energy capture is termed K and T TA
fs0 = and fe0 = . (7)
has been computed empirically for a variety of sys- 1+A 1+A
tems (McCarty 1969). The values ranged from about When intermediate formation does not occur, Equa-
0.40 to 0.80 for aerobic and anaerobic cultures and tion (6) reduces to Equation (3), since T = 1.0.
for heterotrophic and autotrophic microorganisms. An
average value, 0.60, has been used with a good suc- Energy and electron balances for oxygenation
cess (McCarty 1969, 1972; VanBriesen and Rittmann reactions
2000).
The ratio of fe0 /fs0 can be defined as A, and For oxygenation reactions, the electron balances must
rearranging Equation (1) gives: be modified, because not all the electrons released
from the electron-donor substrate are either transferred

f0 G0syn to cell synthesis processing or retained in the interme-
A = e0 = . (2) diates. Instead, some of electrons are transferred to
fs KG0r
molecular oxygen. Thus, electrons originally in the
A defines the relative electron flow between cell syn- primary electron-donor substrate follow one of four
thesis and energy generation for the available electrons paths: transferred to the electron acceptor to generate
transferred from the electron-donor substrate for the energy, utilized to build macromolecules in biomass
reaction of interest. synthesis, utilized to reduce O2 , or sequestered in the
The values fs0 and fe0 are coupled through the elec- intermediate. We define O as the fraction of elec-
tron balance. For a direct mineralization reaction, all trons from the donor invested to reduce O2 and R as
the electrons present in the electron donor substrate the fraction of electrons released or not sequestered
are sent either to the acceptor (fe0 ) or to biomass syn- in the intermediate. Then, the electron balances for
thesis (fs0 ), and the sum of fs0 and fe0 must equal 1 oxygenation reactions are given by:
(McCarty, 1969, 1972):
T +H +O =1 (8)
fe0 + fs0 = 1. (3)
Equations (2) and (3) can be solved simultaneously to R = T + O. (9)
compute fs0 and fe0 as T , H , R, and O values can be computed directly by
electron tracking for the substrate half reaction. This
1 A
fs0 = and fe0 = . (4) is illustrated below for phenanthrene.
1+A 1+A The sum of fs0 and fe0 still follows Equation
For reactions involving intermediate formation, (6), because only transferred electrons are utilized for
all the electrons in the electron-donor substrate are either energy generation or biomass synthesis. There-
not released, because some of the electrons are re- fore, simultaneously solving Equations (2) and (6)
tained in the intermediates. Previously, VanBriesen produces values of fs0 and fe0 that are the same as
216

Equation (7). Although Equation (7) is the same for


oxygenation and nonoxygenation reactions, the T val-  
ues differ, because R = T for a nonoxygenation,  G0pyr G0cs 
G0syn = + G0cells . (11)
while R > T for an oxygenation. Km

G0cs is standard free energy for the carbon-source re-

Calculating and formulating the overall stoichiometry duction half reaction (kJ/e-eq). G0pyr is standard free
energy of formation of pyruvate and is 35.72 kJ/e-eq.

Using the energy and electron balances described G0cells is the free energy to synthesize macromolec-
above, the electron fractions allocated to each flow ules and was estimated empirically as 31.35 kJ/e-eq
can be obtained from known thermodynamic values (McCarty 1969). As with the energy-generation reac-
for each half reaction. The free energy of the energy- tion, inefficiencies in energy transfer are included via
generation redox couple in Equation (1) is computed K. The exponent m accounts for the fact that the con-
as the difference between the free energy of the donor version from the carbon source to the oxidation state
and acceptor half reactions: of the common organic component (pyruvate is used
here) may be energy generating, giving an energy loss
   due to inefficiency (m is 1), or energy utilizing, giv-
G0r = G0a G0d (10)
ing an additional energy cost due to inefficiency (m is
 +1).
in which G0d is the standard free energy for the To complete the framework for the stoichiomet-

electron-donor reduction half reaction and G0a is the ric relationship, half reactions for the cell synthesis,
standard free energy for the electron-acceptor reduc- electron donor, and electron acceptor are needed. The
tion half reaction, where the prime ( ) indicates that cell synthesis half reaction can be modeled by pos-
the pH is fixed at 7.0 (McCarty 1969). Free energies tulating a chemical formula for the cell mass and
for many electron-donor and electron-acceptor half re- assuming synthesis is from simple, inorganic forms
actions are tabulated (McCarty 1969, 1972; Stumm of carbon and nitrogen. Hoover and Porges, (1952)

and Morgan 1996). For example, G0a for the oxy- suggest C5 H7 O2 N as a suitable formula for cells
gen half reaction is 78.06 kJ/electron equivalent. If based on elemental analysis of biomass. This formula
the half reaction is not tabulated, the calculation of has been widely used in biological modeling (Mc-

G0d is straightforward when values for the standard Carty 1972; Metcalf and Eddy 1991; VanBriesen and

free energy of formation (G0f ) are known for all the Rittmann 2000). VanBriesen and Rittmann (2000) pre-
species in the donor half reaction. If the standard free viously considered the differences caused by selection
energy of formation of the electron-donor substrate or of C5 H7 O2 N versus CH2 O0.6 N0.2 , which also is used
an intermediate is not known, it can be estimated us- in modeling. The difference of cell yields was less than
ing group-contribution theory (Mavrovouniotis 1990, 5%. Utilizing C5 H7 O2 N for cells and assuming the

1991). When intermediates are involved, G0d must cells are formed from carbonate and ammonium, a half
be computed with the actual intermediate included reaction (Rc ) for cell synthesis can be constructed (by

(VanBriesen and Rittmann 2000). When G0d is for convention) as a reduction reaction and in terms of one
an oxygenation reaction, O2 must be included. In equivalent of electrons (McCarty 1969; VanBriesen

short, G0d must be for the reduction half reaction and Rittmann 2000):
(Rd ) that includes exactly the reactants and products
involved. This is illustrated below for phenanthrene, 5 1 19 +
and Appendix A shows how group-contribution theory H2 CO3 + NH+
4 + 20 H + e

 20 20
is used to compute unlisted G0f values. (12)
1 13
When NH+ 4 is available as the N source, the syn- = C5 H7 O2 N + H2 O.
 20 20
thesis cost to create biomass (G0syn ) has two com-
ponents: the energy required to transform the carbon The free energy required to drive this reaction is
in the carbon source to the oxidation state of cellular supplied from the redox reaction between an electron
carbon and the energy required to create and assemble donor and acceptor. Bacteria prefer molecular oxygen
the cell macromolecules (McCarty 1969, 1972). The as the electron acceptor when aromatic hydrocarbons
two components are summed in Equation (11): are used for the electron donor. The half reaction (Ra )
217

for the utilization of molecular oxygen as the electron


acceptor is:
1 1
O2 (g) + H+ + e = H2 O. (13)
4 2
The full biodegradation stoichiometry (Rt ) can be
obtained by coupling the electron-donor half reaction
(Rd ) of interest with the half reactions for electron
acceptor and cell synthesis via fs0 , fe0 , and T values
(McCarty 1969; VanBriesen and Rittmann 2000).

Rt = T Rd + fe0 Ra + fs0 Rc . (14)

Electron-donor half reactions for phenanthrene


degradation

Each reaction step in a biodegradation pathway can


have its own electron-donor reaction (Rd ). To illus-
trate a pathway of electron-donor reactions that in-
clude oxygenations, phenanthrene, a 3-ring polycyclic
aromatic hydrocarbon, is selected. Phenanthrene is de-
graded by some soil bacteria through one of two differ-
ent routes (Cerniglia 1992; Gibson and Subramanian
1984; Smith 1990). Both routes have the same up-
per pathway for the degradation of phenanthrene to
1-hydroxy-2-naphthoate. However, the lower path-
way is divided into two routes (Kiyohara and Nagao
1978). In one route, 1-hydroxy-2-naphthoate is ox-
idized to 1,2-dihydroxynaphthalene, which is further
degraded via the naphthalene pathway to salicylate,
which can be further metabolized. In the other path- Figure 2. Biodegradation pathway of phenanthrene via the
naphthalene pathway. The left column is the upper pathway,
way, the ring of 1-hydroxy-2-naphthoate is cleaved while the right column is the naphthalene lower pathway. Capital
and further metabolized via the phthalate pathway letters indicate enzyme reactions or enzymes. A, phenanthrene
(Barnsley 1983a; Kiyohara and Nagao 1978; Rib- dioxygenase; B, cis-3,4-dihydroxy-3,4-dihydrophenanthrene
bons and Evans 1960). In this study, the naphthalene dehydrogenase; C, 3,4-dihydroxyphenanthrene dioxygenase;
D, 2-hydroxy-2H -benzo[h]chromene-2-carboxylate isomerase;
pathway was analyzed as a model system for the stoi- E, cis-4-(1 -hydroxynaphth-2 -yl)-2-.oxo-but-3-enoate hy-
chiometry calculations. Figure 2 shows the transform- dratase-aldolase; F, 1-hydroxy-2-naphthaldehyde dehydrogenase;
ation steps for phenanthrene biodegradation via the G, 1-hydroxy-2-naphthoate monooxygenase; H, 1,2-dihydroxyn-
commonly illustrated naphthalene pathway (Cerniglia aphthalene dioxygenase; I, 2-hydroxychromene-2-carboxylate
isomerase; J, trans-o-hydroxybenzylidenepyruvate hy-
1992; Eaton and Chapman 1992; Evans et al. 1965; dratase-aldolase; K, salicylaldehyde dehydrogenase; L,
Gibson and Subramanian 1984; Kiyohara et al. 1994; salicylate monooxygenase; M, catechol 2,3-dioxygenase; N,
Smith 1990). 2-hydroxymuconate semialdehyde hydrolase with subsequent
In the overall degradation of phenanthrene via the dehydrogenation and hydrogenation steps.
naphthalene pathway, 6 oxygenation reactions are in-
volved; 4 are dioxygenation reactions (reactions A,
C, H, and M), and 2 are monooxygenation reactions into a substrate molecule without producing a water
(reactions G and L). The monooxygenation reactions molecule (Hayaishi et al. 1970). In both cases, each
incorporate one oxygen atom from molecular oxygen oxygen atom is reduced by two electrons.
into a substrate molecule, and the other oxygen atom Table 1 summarizes the reduction half reactions
goes into a water molecule produced from the reaction. (i.e., Rd ) for each step. Table 1 also lists the electron-
The dioxygenations incorporate both oxygen atoms transfer parameters for one mole of substrate (H  , R  ,
218

T  , and O  ) and the standard free energy (G0d ) for only 42 electron equivalents are available for energy
one electron equivalent. In the phenanthrene pathway, generation or biomass synthesis, because 24 electron

G0f values for phenanthrene and all its intermedi- equivalents are utilized to reduce 6 oxygen molecules
ates were estimated using group-contribution theory in oxygenation reactions. Thus, oxygenation reactions
(Mavrovouniotis, 1990, 1991), which is described in consume a substantial fraction (36.4%) of the total
Appendix A. In two cases, three reactions were com- electrons present in phenanthrene. These activation
bined (reactions DF and IK), because they consist of reactions divert electrons from synthesis and energy
isomerase (D, I), or hydratase-aldolase (E, J) reactions generation and ought to affect the biomass yield for
that do not involve any transfer of electrons. These complete mineralization. Furthermore, individual re-
non-redox reactions were combined with a subsequent actions can have T values that are positive, zero, or
dehydrogenation reaction to allow an electron transfer negative. Only a positive T value supplies electrons

(i.e., R > 0). Appendix B illustrates how the G0d for energy generation and synthesis. Therefore, accu-
values are computed. mulation of intermediates that are further oxidized by
The first step, a dioxygenation reaction from phen- oxygenation reactions can profoundly affect whether
anthrene to cis-3,4-dihydroxy-3,4-dihydrophenanth- or not electrons become available to support biomass
rene, illustrates the profound impact of oxygenation synthesis.
reactions that require a reduced electron carrier as
a cosubstrate. Phenanthrene and cis-3,4-dihydroxy-
3,4-dihydrophenanthrene contain 66 and 64 electron Results and discussion
equivalents per mole, respectively. Thus, reaction A
releases 2 electron equivalents per mole from phenan- Single-step mineralization
threne (R  = 2), while 4 electron equivalents (O  = 4)
are incorporated to reduce molecular oxygen, which The effect of oxygenation reactions on the stoi-
is incorporated into phenanthrene as 2 OH substitu- chiometry of complete mineralization is explored by
ents. Two of the 4 electron equivalents come from comparing two scenarios: the pathway in Table 1,
phenanthrene, and the other 2 come from a reduced which involves six oxygenation steps, and an altern-
electron carrier, such as NADH+ 2 shown in Figure ate pathway in which each of the six oxygenations
2. As a result, the T  value is negative (2), which is replaced by one or two hydroxylation reactions so
represents that the reaction requires an input of 2 elec- that one hydroxylation is used for each oxygen atom
tron equivalents, rather than transferring electrons to inserted. In a hydroxylation reaction, the organic sub-
cell synthesis or energy generation. Similar negative strate is oxidized by two electrons, an OH substituent
electron transfers occur for reactions G and L, which is added, and H2 O is the source of oxygen in OH.
are monooxygenations. A comparison of the two methods is valuable, since
For one-step dioxygenation reactions (C, H, and the hydroxylation pathway corresponds to conven-
M), the T  values are zero, because the number of elec- tional mineralization reactions that have been used for
trons released from the reaction (R  = 4) is exactly the determination of overall stoichiometry in the past.
same with the number of electrons required for oxygen For the case of the hydroxylation method, the
reduction (O  = 4). This indicates that net electron standard half reaction for the electron donor, phen-
transfer does not occur (T  = 0). Thus, electrons re- anthrene (C14 H10 ), is written as a reduction for one
moved from the donor substrate are not available for electron equivalent:
either energy generation or biomass synthesis.
For reactions that are not oxygenation reactions, 14 1 42
H2 CO3 + H+ + e = C14 H10 + H2 O
the O  values are zero, because oxygen is not a cosub- 66 66 66
strate. As a result, the T  value is identical to the R 
value, which means that all electrons released from 
(G0d = 25.87 kJ/e-eq). (15)
the donor half reaction are transferred to either energy
generation or biomass synthesis. Quantitatively, all T  This reaction indicates that full mineralization of
values are positive, because R  values are necessarily phenanthrene yields 66 electron equivalents per mole
positive. of phenanthrene. The mineralization of phenanthrene

In total, complete oxidation of one mole of phen- produces 25.87 kJ per electron equivalent (G0d ) at
anthrene liberates 66 electron equivalents. However, pH = 7.
219
Table 1. Electron donor reactions and electron flows for each step of phenanthrene degradation via the naphthalene
pathway

Rxn. H R T O G0d

A C14 H10 + O2 + 2H+ + 2e = C14 H12 O2 64 2 2 4


1 C H + 1 O + H+ + e = 1 C H O (r) 126.82
2 14 10 2 2 2 14 12 2
B C14 H12 O2 = C14 H10 O2 + 2H+ + 2e 62 2 2 0
1 C H O + H+ + e = 1 C H O (r) 43.30
2 14 10 2 2 14 12 2
C C14 H10 O2 + O2 = C14 H9 O 4 +H
+ 58 4 0 4
+
C14 H9 O4 + H = C14 H10 O2 + O2 (r)
DF C14 H9 O +
4 + 8H2 O = C11 H7 O3 + 3H2 CO3 + 12H + 12e
46 12 12 0
1 C H O + 3 H CO + H+ + e = 1 C H O + 8 H O (r) 34.40
12 11 7 3 12 2 3 12 14 9 4 12 2
G C11 H7 O +
3 + O2 + 3H + 2e = C10 H8 O2 + H2 CO3 44 2 2 4
1 1 3 + 1 1
12 C11 H7 O3 + 2 O2 + 2 H + e = 2 C10 H8 O2 + 2 H2 CO3 (r) 167.24
H C10 H8 O2 + O2 = C10 H7 O 4 + H + 40 4 0 4
C10 H7 O +
4 + H = C10 H8 O2 + O2 (r) --
IK C10 H7 O +
4 + 8H2 O = C7 H5 O3 + 3H2 CO3 + 12H + 12e
28 12 12 0
1 C H O + 3 H CO + H+ + e = 1 C H O + 8 H O (r) 35.32
12 7 5 3 12 2 3 12 10 7 4 12 2
L C7 H5 O +
3 + O2 + 3H +2e = C6 H6 O2 + H2 CO3 26 2 2 4
1 C H O + 1 O + 3 H+ + e = 1 C H O + 1 H CO (r) 167.24
2 7 5 3 2 2 2 2 6 6 2 2 2 3
M C6 H6 O2 + O2 = C6 H5 O 4 +H
+ 22 4 0 4
C6 H5 O +
4 + H = C6 H6 O2 + O2 (r)

N C6 H5 O4 +14H2 O = 6H2 CO3 + 21H+ + 22e 0 22 22 0
6 21 + 1 14
22 H2 CO3 + 22 H + e = 22 C6 H5 O4 + 22 H2 O (r) 36.70

1 step C14 H10 +30H2 O + 6O2 = 14H2 CO3 + 42H+ + 42e 0 66 42 24


14 H CO + H+ + e = 1 C H + 30 H O + 6 O (r) 85.52
42 2 3 42 14 10 42 2 42 2
1. All free energies are in kJ/electron equivalent.
2. (r) represents a standard half reaction as a reduction form with one electron equivalent.
3. H  , R  , T  , and O  represent the total number of electrons held in intermediates, not held in intermediates, transferred
to energy or synthesis reactions, and used to reduce O2 , respectively. H , R, T , and O are computed by dividing H  , R  ,
T  , and O  by the number of electrons originally in the donor substrate.
4. Electrons are associated with an intracellular electron carrier, such as NADH+ 2 , as shown in Figure 2.

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 C14 H10 +2O2 + 8H2 O = C11 H7 O +


3 + 3H2 CO3 + 13H + 12e
46 20 12 8
1 C H O + 3 H CO + 13 H+ + e = 1 C H + 2 O + 8 H O (r) 90.83
12 11 7 3 12 2 3 12 12 14 10 12 2 12 2
GK C11 H7 O3 + 2O 2 + 8H 2 O = C 7 H 5 O
3 + 4H 2 CO 3 + 10H + + 10e 28 18 10 8
1 4 + 1 2 8
10 C7 H5 O3 + 10 H2 CO3 + H + e = 10 C11 H7 O3 + 10 O2 + 10 H2 O (r) 108.43
+
LN C7 H5 O3 +2O2 + 14H2 O = 7H2 CO3 + 19H + 20e 0 28 20 8
7 19 + 1 2 14
20 H2 CO3 + 20 H + e = 20 C7 H5 O3 + 20 O2 + 20 H2 O (r) 70.93

1 step C14 H10 + 30H2 O + 6O2 = 14H2 CO3 +42H+ + 42e 0 66 42 24


14 H CO + H+ + e = 1 C H + 30 H O + 6 O (r) 85.52
42 2 3 42 14 10 42 2 42 2
All free energies are in kJ/electron equivalent.
(r) represents a standard half reaction as a reduction form with one electron equivalent.
O2 refers to gas-phase oxygen, or O2 (g).

Table 4. Key parameters and calculated values for the coupled


reaction for phenanthrene degradation

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.

Table 5. Mole-based stoichiometric coefficient for steps in


coupled reaction for phenanthrene degradation

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

Reference Reported Estimated Cultivation Intermediate N source


yield value yield value time production

Wodzinski & Johnson (1968) 0.4a NIg Yes NH+4


Stucki & Alexander (1987) 3.5b 0.55a 10 d NIg NH+4
Weissenfels et al. (1990) 0.24c 0.45a 2d NDh NH4 NO3
Keuth & Rehm (1991) 74.4d 0.84a 30 h Yes NH+4
Aichinger et al. (1992) 0.33e 0.69a NIg 0.08e NIg
Stringfellow & Aitken (1994) 1.3a NIg NDh NH+4
Guha & Jaffe (1996) 0.392a 24 h NIg NIg
Bouchez et al. (1996) 35f 0.63a 10 d 14% C NH4 NO3
Zhang et al. (1997) 1.0a 120 h NIg NH+4
Jahan et al. (1999) 1.02a 120 h NIg NO3
a g cells/g phenanthrene. b g protein/mol C. c mg protein/mg C. d mg protein/mmol phenanthrene. e mg COD/mg COD. f % carbon. g NI, not
indicated. h ND, not detected.

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.
Acknowledgments

Conclusions We would like to thank Dr. J. M. VanBriesen for


discussion on microbial thermodynamics. This work
was supported by a grant from the Korea Science and
We evaluated the impact of oxygenation reactions
Engineering Foundation.
on thermodynamic relationships and overall stoi-
chiometry for biodegradation of phenanthrene as a
model polynuclear aromatic compound. In particu- Appendix A
lar, we used the half-reaction method for analyzing
multi-step degradations that involved oxygenations. Estimating standard free energy of formation using
Since oxygenation reactions do not yield a net release group contribution theory
of electrons, but require an input of electrons to re-
duce oxygen molecules, we extended the conventional In order to complete stoichiometric relationships for
energetic relationships to consider how electrons are biological degradation reactions using half-reaction
supplied to these reactions. method, it is necessary to know the standard free
Oxygenation reactions significantly altered the en- energy of the donor, acceptor, and cell synthesis re-
ergy and electron flows, and this changed the stoi- actions. For relevant electron-acceptors and common

chiometry for the complete mineralization of phenan- organic electrondonors, G0d values are tabulated
threne. Oxygenation reactions increased the oxygen (McCarty 1969, 1971; Stumm and Morgan 1996). If

requirement and reduced the cell yield, compared to the half reaction is not tabulated, the G0d value can
the conventional mineralization represented by the hy- be calculated from the standard free energy of forma-

droxylation method. The decrease in yield was not as tion (G0f ) for all species in the half reaction. For the
225

phenanthrene pathway, since the G0f values for the ample, for coupled reaction AF, the electron-donor
electron-donor substrate and intermediates were not half reaction is:
found in the literature, the values were estimated us- 1 3 13
ing group contribution theory (Mavrovouniotis 1990, C11 H7 O3 + H2 CO3 + H+ + e
12 12 12
1991).
1 2 8
The calculations of the standard free energies = C14 H10 + O2 (g) + H2 O.
of formation for phenanthrene (C14 H10 ), 1-hydroxy- 12 12 12

2-naphthoate (C11 H7 O
3 ), and salicylate (C7 H5 O3 ), For this reaction, the G0f values tabulated in the lit-
which are relevant to coupled-reaction model, are erature are 237.0, 0, 39.96, 0, and 622.4 kJ/mole
shown in Table A1. The chemical structure for each for H2 O, O2 (g), e , H+ (with pH = 7.0), and H2 CO3 ,
compound is shown in Figure 3. respectively (Stumm and Morgan 1996), and the es-
timated values are 310.99 and 288.42 for C14 H10
Table A1. Estimation of the free energy of formation for the and C11 H7 O 3 , respectively (see Appendix A). The
compounds involved in the coupled-reactions in the phenanthrene
pathway from contributions of groups free energy for this reaction is calculated as:

Compound Group or Number of Contribution Total
G0d (reaction)
correction occurrences (kJ/mol) contribution  
(kJ/mol) = G0f (products)
 
C14 H10 Origina 1 98.65 98.65 G0f (reactants)
Correction 1 16.72 16.72  
8(237)
for hydrocarbon = 310.99
12 + 12 +
20
12
CH=b 10 35.11 351.1  
>C=c 4 10.45 41.8 288.42
12 + 3(622.4)
12 + 13(39.96)
12 +0
Total 310.99

C11 H7 O Origina 1 98.65 98.65


= 90.83 kJ/e-eq.
3
CH=b 6 35.11 210.67
>C=c 2 10.45 20.9
>C=d 2 6.27 12.54 Nomenclature
OHe 1 132.92 132.92
COO 1 300.96 300.96 A electron equivalents of substrate converted
Total 288.42 for energy per electron equivalent of cells
C 7 H5 O
3 Origina 1 98.65 98.65 synthesized.
CH=b 4 35.11 140.45
EQS electron equivalents in one mole of substrate,
>C=d 2 6.27 12.54
OHe 1 132.92 132.92 e-eq/mole substrate.
COO 1 300.96 300.96 fe0 fraction of electron-donor electron equival-
Total 379.54
ents used for energy generation.
a A contribution that must be added to every compound. fs0 fraction of electron-donor electron equival-
b Participating in a benzene ring.
ents used for cell synthesis.
c Participating in two fused benzene rings.
d The formal double bond and a formal single bond participating fs (m) fs0 in each step of multi-step degradation,
0
in a benzene ring. normalized to the number of electrons in an
e Attached to benzene ring.
initial donor substrate.
fs0 (s) fraction of original electrons going to syn-
thesis if the fs0 of the total reaction is appor-
tioned according to the number of electrons
Appendix B released in each step.
0 
Ga standard free energy for the electron acceptor
Calculation of the free energy for the electron-donor reduction half reaction, kJ/e-eq.
half reaction 0
Gcells standard free energy to synthesize macro-
molecules, kJ/e-eq.
The overall free energy of the donor half reaction can 
G0cs standard free energy for the carbon source
be computed using the free energy of formations of
reduction half reaction, kJ/e-eq.
all reactants and products in the half reaction. For ex-
226

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