International Biodeterioration & Biodegradation 65 (2011) 883e889

Contents lists available at ScienceDirect

International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Characterization of hydrophobic organic contaminant biodegradation

by COD analysis
Mikhail Baboshin, Ludmila Golovleva*
G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, Prospekt Nauki 5, Pushchino, Moscow Region, Russia

a r t i c l e i n f o a b s t r a c t

Article history: Biodegradation of hydrophobic organic contaminants is often accompanied by the accumulation of
Received 2 March 2011 hydrophobic intermediates. This paper is devoted to the experimental evaluation of electron balance
Received in revised form between the hydrophobic intermediates and the other biodegradation products. The ratio of the fraction
25 April 2011
of electrons that are not held in the hydrophobic intermediates to the total fraction of electrons released
Accepted 26 April 2011
from the substrate during its conversion is called a degradation level (DL). Under certain conditions, the
Available online 23 July 2011
DL value is equal to the angular coefficient of the plot in the coordinates ð1  ðS=S0 Þ; 1  ðCOD=COD0 ÞÞ,
where S and COD are current values of substrate concentration and chemical oxygen demand of the
Keywords:
PAH
ethylacetate extract from the culture; S0 and COD0 are the respective initial values. This approach has
HOC been used for analysis of the conversion of PAHs by Sphingomonas sp. VKM B-2434. The DL values
COD obtained correspond well to most published data on PAH metabolism by this strain. The DL values
Intermediates significantly increased as a result of addition of the strains consuming the products of conversion of PAHs
Hydrophobic by Sphingomonas sp. VKM B-2434. The proposed method seems useful for characterizing biodegradation
Sphingomonas sp. VKM B-2434 of hydrophobic pollutants by both pure and mixed microbial cultures.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction characterizing the products as a whole and allowing verification of

Pollution of soils and water ecosystems by hydrophobic organic
contaminants such as polycyclic aromatic hydrocarbons (PAHs) is
a problem of great concern. Biodegradation of the contaminants is
one of the possible ways of remediation (Allard and Neilson 1997;
Bamforth and Singleton 2005). Organic substrate oxidation by
microorganisms results in either its complete mineralization or
accumulation of the products of incomplete oxidation. Accumula-
tion of hydrophobic intermediates is of particular interest, because
these products inhibit biodegradation of pollutants (Casellas et al.
1998; Kazunga and Aitken 2000; Kazunga et al. 2001) and may
be extremely recalcitrant (Schmidt et al. 2010) and hazardous for
the environment (Allard and Neilson 1997; Lundstedt et al. 2007).
The measurement of concentrations of all intermediates is a labor-
consuming task complicated by the absence of the respective
analytical standards (Lundstedt et al. 2007) and a universal tech-
nique applicable to the analysis of all intermediates simulta-
neously; therefore some of the intermediates may not be seen
during analysis. Consequently, there is a need for integral criteria
the data of chemical analysis of intermediates. One of such criterion
is the electron balance between different fractions of products
(VanBriesen and Rittmann 2000). Electrons released from the
substrate may be invested in biomass, transferred to an electron
acceptor, or sequestered in the intermediates. The ratio of the
fraction of electrons that are not held in the hydrophobic inter-
mediates to the total fraction of electrons released from the
substrate during its conversion will be called here a degradation
level (DL). The value of DL ¼ 1 when metabolites are not accumu-
lated, and DL ¼ 0 when the substrate conversion is not accompa-
nied by oxidation.
In this paper, the DL values are evaluated for conversion of
selected PAHs by Sphingomonas sp. VKM B-2434, the chemical
oxygen demand (COD) of ethylacetate extracts of tested culture, is
used for the evaluation.
2. Materials and methods
2.1. Reagents

The PAHs were of high purity (>98%; SigmaeAldrich). Other

* Corresponding author. Tel.: þ7 495 6257448; fax: þ7 495 9563370. reagents were produced in Russia and qualified as no less than
E-mail address: golovleva@ibpm.pushchino.ru (L. Golovleva). analytical reagent grade. Solvents were distilled before use.

0964-8305/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2011.04.008
884 M. Baboshin, L. Golovleva / International Biodeterioration & Biodegradation 65 (2011) 883e889
2.2. Incubation medium
The mineral medium contained (g l1): NH4NO3, 1.0; KH2PO4,
1.0; K2HPO4, 1.0; MgSO4$7H2O, 0.2; CaCl2, 0.02; FeCl3, 2 drops of
saturated solution; KOH, up to pH 7.5. The mineral medium was
filtered, 100 or 200 ml were poured into flasks (750 ml), and ster-
ilized. After sterilization, the medium was supplemented with
vitamin B12 (1 mg l1) and a set quantity of PAH as a solution in
acetone in a concentration of 5 g l1 for anthracene and 50 mg l1
for other substrates.
2.3. Culture
Sphingomonas sp. VKM B-2434, capable of conversion of a broad
range of PAHs, has been characterized previously (Baboshin et al.
2008; Baboshin and Golovleva 2010). Additional strains FON-9,
HNA-21, and HNA-32, growing on 9-fluorenone, 1-hydroxy-2-
naphthoic acid, and 3-hydroxy-2-naphthoic acid, respectively, but
incapable of converting PAHs, were isolated from polluted bottom
sediments by the method of enrichment culture.
2.4. Conversion of PAHs utilized by Sphingomonas sp. VKM B-2434
as a sole carbon and energy source
One ml of the respective culture (or two cultures) was added to
a flask (750 ml) containing 100 ml of the medium. Cultures of VKM
B-2434, FON-9, HNA-21, and HNA-32, grown for 3 days in the media
with acenaphthene (0.5 g l1), 9-fluorenone (0.1 g l1), 1-hydroxy-
2-naphthoic acid (0.1 g l1), and 3-hydroxy-2-naphthoic acid
(0.1 g l1), respectively, were used for inoculation. The flasks were
cultivated on a shaker (30  C, 120 rpm). After incubation, the
contents of a flask was acidified by 0.5 ml of 25% H2SO4 and twice
extracted with ethylacetate (30 þ 10 ml). The volume of the
extracts was brought up to 40 ml with ethylacetate; a 1-ml aliquot
was taken from each extract for PAH concentration measurement
by gas chromatography and an aliquot of 1e20 ml (depending on
the initial PAH concentration in the experiment) was taken for COD
measurement.
2.5. Co-metabolic conversion of PAHs
A flask with 200 ml of the incubation medium was inoculated as
described above and incubated at 30  C under stirring and bubbling
with the air saturated with acenaphthene vapor; for this purpose,
the air (0.1 l min1) was passed through a column (10 cm,
Ø 25 mm) with crystalline acenaphthene and fed into the flask with
the culture; acenaphthene was supplied to the culture at a rate of
about 115 mg h1. In a set period of time, the incubation was
stopped and 1 ml of 25% H2SO4 was added into the flask, followed
by twofold extraction with ethylacetate (40 þ 10 ml) and
measurement of COD and PAH concentration in the extract. The
quantity of the substrate subjected to bioconversion was calculated,
taking into account the losses of PAH with the output airflow in
sterile control. It was assumed that the losses of conversion prod-
ucts could be neglected, because the presence of oxygen atoms in
their molecules contributed to formation of hydrogen bonds in
solution. The absence of 9-fluorenone losses was shown
experimentally.
Each PAH biodegradation experiment was run twice.
2.6. Gas chromatographic analysis of the culture extract
Polycyclic aromatic hydrocarbon concentrations in the extracts
were determined by gas chromatography in a Crystal 2000 M chro-
matograph (Chromatec, Russia) with a flame-ionization detector and
an HP-5 column (30 m  0.32 mm  0.25 mm). The injector and
detector temperatures were 250 and 290  C, respectively. The column
temperature in the course of analysis increased from 150 to 250  C at
a rate of 10  C min1. Samples (1 ml) were injected by an autosampler.
2.7. COD measurement in the culture extract
The standard method of COD detection in water (ISO 1990) was
modified for our purposes. An aliquot of culture extract with the
content of dissolved substances equivalent to no more than 3 mg of
oxygen was placed into a round-bottom flask (50 ml). Ethylacetate
was evaporated in a rotor evaporator at 38  C under vacuum (w100
mbar) until dryness; 5 ml of acetone was added to the dry residue
and then evaporated as well. The flask was kept open overnight for
volatilization of residual acetone; then, 10 ml of 0.25 N K2Cr2O7
solution, 0.1 g of Ag2SO4, and 10 ml of sulfuric acid were added into
the flask and boiled for 2 h with reflux condenser. Then the solution
was quantitatively transferred into a glass, supplemented with 2
drops of 0.4% diphenylamine solution in 80% sulfuric acid and 3 ml
of phosphoric acid, and titrated with 0.25 N Mohr’s salt solution
until the blue color disappeared. The COD of the culture extract, i.e.,
the amount of oxygen (in milligrams) needed for complete oxida-
tion of substances extracted from 1 L of the culture, was calculated
by the formula
10 1
h ¼ ðVX  VÞ 0:25  8  ; (1)
VX Vcult
where VX and V e Mohr’s salt volumes spent for titration of the
blank and tested samples, respectively; 10/VX e correction factor for
bringing Mohr’s salt concentration to exactly 0.25 N; 0.25 e Mohr’s
salt concentration; 8 e oxygen equivalent; and Vcult e culture
volume corresponding to the quantity of extract in the assay.
2.8. Calculation of indices of electron balance
Oxidation of a substance containing carbon, hydrogen, and
oxygen atoms to carbon dioxide and water can be described by the
equation
y
Cx Hy Oz þ n½O/xCO2 þ H O: (2a)
2 2
Stoichiometric coefficient
y
n ¼ 2x þ  z; (2b)
2
showing the number of oxygen atoms needed for oxidation of one
Cx Hy Oz molecule will be called molar COD. Molar COD is equal to
the double quantity of available electrons in the Cx Hy Oz molecule,
i.e., electrons passing to the oxygen atoms under oxidation of the
substance (Minkevich and Eroshin 1973).
Let us assume that microbial culture utilizes hydrophobic
substrate as a sole carbon and energy source, and it is possible to
measure substrate concentration, to extract hydrophobic
substances from the culture with ethylacetate, and to estimate total
COD of this extract.
In accordance with the definition that DL is a ratio of the elec-
tron fraction transferred into the products other than hydrophobic
intermediates to the total fraction of electrons released from the
substrate, the DL is equal to the ratio
dCOD
DL ¼ ; (3)
nS  dS
where dS is the change in substrate concentration in the culture as
a result of microbial conversion (expressed in mol l1); nS is the
 M. Baboshin, L. Golovleva / International Biodeterioration & Biodegradation 65 (2011) 883e889 885

molar COD of the substrate; the nS  dS value evaluates the electron 2.9. Statistical treatment of results
fraction released from the substrate; dCOD is the change in the COD
value of ethylacetate extract of the culture (expressed in moles of Coefficients a and b of the linear regression y ¼ a þ bx were
atomic oxygen per litre of the culture), and the dCOD value evalu- determined by the least-squares method. If coefficient a showed no
ates the electron fraction transferred into the non-hydrophobic significant difference from zero (by t-criterion), the function was
products. considered as a direct proportionality, y ¼ bx. The confidence
If the initial biomass concentration is low and the culture interval for angular coefficient b of the function y ¼ bx was esti-
medium contains no hydrophobic substances except for the mated by t-distribution by the formula (Doerffel 1990):
substrate, the COD value of the extract in the beginning of vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
uPm
incubation is fully determined by the initial substrate concen- u ðy  Yi Þ2
tration (S0):
Db ¼ tðP; f Þt i ¼ 1Pmi ; (9)
f  i ¼ 1 x2i

COD0 ¼ nS  S0 ; (4) where m is the number of points (excluding zero points); xi is

argument values; yi is experimental values of the function; Yi ¼ bxi
and the formula (3) can be written as is calculated values of the function; and tðP; f Þ is the quantile of t-


 distribution at a confidence probability P and a number of degrees
COD S of freedom f ¼ m  1.
d ¼ DL  d ; (5)
COD0 S0
where S0 and COD0 are the initial values of the substrate and COD 3. Results
concentrations, respectively.
Integration of Eq. (5) at a constant DL leads to the direct pro- 3.1. Experimental determination of molar COD for PAHs
portionality between the COD conversion level and the substrate and their metabolic products
conversion level with a coefficient of proportionality equal to DL:


 Chemical oxygen demand values of the samples (0.5e3.0 mg) of
COD S PAHs and a number of compounds known as the intermediates of
1 ¼ DL  1  : (6)
COD0 S0 bacterial metabolism of PAHs have been measured. The molar COD
value calculated by these experimental data is in good agreement
In accordance with the formula (Eq. (6)), the DL is equal to the
with the theoretical molar COD value, i.e., stoichiometric coefficient
angular coefficient of the plot in the coordinates
n in Eq. (2a), for each of the 13 tested substances (Fig. 1).


S COD
1 ; 1 ; (7)
S0 COD0 3.2. Estimation of the DL values for PAH conversion
by Sphingomonas sp. VKM B-2434
in the case of variable DL the plot will be non-linear.
The coordinates (Eq. (7)) are non-dimensional; therefore the
The curves of acenaphthene, fluorene, phenanthrene, anthracene,
substrate and COD concentrations may be expressed in any units,
fluoranthene, and pyrene conversion by Sphingomonas sp. VKM
although the above expressions are deduced for molar concentra-
B-2434 and the respective curves of COD dynamics of the culture
tions. In this work we use mass concentrations (milligrams per litre
extract are presented in Fig. 2. The data in Fig. 3 are given in the
of the culture) for both substrate and COD.
The hydrophobic products of substrate conversion can be
divided into two groups:

1. Hydrophobic intermediates of the degradation pathway; and

2. Hydrophobic products of anabolism (i.e., biomass lipids).

The contribution of the second group of products to the change

in COD of the ethylacetate extract can be neglected. Indeed, the
biomass yield during the growth on aromatic substrates is usually
much less than 1 (Bouchez et al. 1996; VanBriesen 2001) and the
content of lipids in biomass is usually less than 10% of dry weight
(O’Leary 1962). Consequently, the yield of lipids from the substrate
does not exceed several percent, which is comparable with the
error of COD measurement.
Thus, the experimentally determined DL value is almost fully
determined by hydrophobic intermediates.
The averaged molar COD of hydrophobic intermediates is
equal to

nP ¼ ð1  DLÞnS ;
where nS is the molar COD of the substrate estimated by the
equation of oxidation of the substrate (2a); DL is estimated by the
graph slope in the coordinates (Eq. (7)).
The value nP allows for the assumption of just which products
are formed and for checking the correctness of the metabolic
scheme proposed on the basis of chemical analysis of the products.
(8)
Fig. 1. Correspondence between theoretical (nt) and experimental (ne) values of molar
COD for acenaphthene (1), fluorene (2), phenanthrene (3), anthracene (4), fluo-
ranthene (5), pyrene (6), 1,2-acenaphthene quinone (7), 9-fluorenone (8), 9,10-
phenanthrene quinone (9), 1-hydroxy-2-naphthoic acid (10), 3-hydroxy-2-naphthoic
acid (11), o-phthalic acid (12), and salycilic acid (13). The dotted line shows straight
line ne ¼ nt where the points would lie under the equality of experimental molar COD
values to theoretically predicted values. Averages are based on five independent
measurements. Confidence intervals correspond to a confidence probability of 0.95.
886 M. Baboshin, L. Golovleva / International Biodeterioration & Biodegradation 65 (2011) 883e889

500 1500 50 160

Fluoranthene, mg l-1
400 1200 40
Acenaphthene, mg l-1 120

COD, mg l-1
C OD , mg l-1
300 900 30
80
200 600 20

40
100 300 10

0 0 0 0
0 10 20 30 40 50 0 20 40 60 80

time, h time, h
120 350 70 200

100 300 60
160
Phenanthrene, mg l-1

A nthracene, mg l-1
250 50
80
COD, mg l-1

COD, mg l-1
200 120
40
60
150 30 80
40
100 20
20 40
50 10

0 0 0 0
0 20 40 60 0 100 200 300
time, h time, h
60 180
25 80
160
50
140 20
60
Fluorene, mg l-1

P yren e, m g l-1

40 120
COD, mg l-1

C O D , m g l-1
100 15
30 40
80
10
20 60

40 20
10 5
20

0 0 0 0
0 20 40 60 80 100 0 50 100
time, h time, h
Fig. 2. The dynamics of substrate concentration () and COD of the culture extract (þ) under conversion of PAHs by Sphingomonas sp. VKM B-2434. The dynamics of substrate
concentration in the sterile control (Δ) and COD in the control without the substrate (,) are also shown for co-metabolic conversion of fluorene and pyrene. Data from two
completely independent experiments are shown in each graph.

coordinates of dependence of the COD conversion level on the sp. VKM B-2434 (Fig. 4). On addition of the strain HNA-32 utilizing
substrate conversion level. These plots were used for calculation of DL 3-hydroxy-2-naphthoic acid, the DL of anthracene conversion
and the mean molar COD of products for each substrate (Table 1). In increased from 0.28 to 0.92. Addition of the strain HNA-12 utilizing
the experiment with fluorene, the culture was extracted by two 1-hydroxy-2-naphthoic acid resulted in significant increase in the
methods: the common one and the one without preliminary acidifi- DL of phenanthrene conversion. In this case, the DL value varied in
cation by sulfuric acid; it was shown that most of the products (about the course of incubation due to the lag phase of the strain HNA-12
70% by COD) extracted in acidic medium were not extracted at pH 7.5. (which corresponded to accumulation and subsequent utilization
of 1-hydroxy-2-naphthoic acid); the mean DL value of phenan-
3.3. Estimation of the DL values for PAH conversion threne conversion by the binary culture was close to 1. Addition of
by mixed cultures the strain FON-9 utilizing 9-fluorenone increased the DL of fluorene
conversion from 0.40 to 0.61; at the same time, the strain FON-9
Values for DL significantly increase as a result of addition of the almost completely degrades 9-fluorenone, DL ¼ 0.92 under the
strains utilizing the products of PAH conversion by Sphingomonas growth of the strain FON-9 on 9-fluorenone.
 M. Baboshin, L. Golovleva / International Biodeterioration & Biodegradation 65 (2011) 883e889 887

Fig. 3. Dependence of the COD conversion level on the substrate conversion level for
the conversion of acenaphthene (1), fluorene (2), phenanthrene (3), anthracene (4),
fluoranthene (5), and pyrene (6) by Sphingomonas sp. VKM B-2434. The angular
coefficients of approximating straight lines are equal to DL values given in Table 1.
Fig. 4. Dependence of the COD conversion level on the substrate conversion level for
the conversion of phenanthrene (1), anthracene (2), and fluorene (3) by the binary
cultures including Sphingomonas sp. VKM B-2434 and a second strain consuming 1-
hydroxy-2-naphthoic acid, 3-hydroxy-2-naphthoic acid, and 9-fluorenone, respec-
tively. The curves corresponding to the monoculture of Sphingomonas sp. VKM B-2434
are shown by the dotted lines and designated by the respective apostrophized
numbers.

4. Discussion
Sphingomonas sp. VKM B-2434 was shown to be quantitative
The COD analysis applied in this work to study the biodegrada- (Baboshin et al. 2008). Experimentally measured molar COD values
tion of PAHs is extensively used in environmental chemistry to
of the products of phenanthrene and anthracene conversion by
assess organic matter concentrations in water (ISO 1990). The Sphingomonas sp. VKM B-2434 coincided with the theoretically
technique provides accurate measurement of molar COD values of
calculated molar COD of hydroxynaphthoic acids (Table 1).
different PAHs and their metabolic products, i.e., the number of There are grounds for believing that the analysis used gave
oxygen atoms needed for complete oxidation of one molecule of the
correct results for other substrates as well. VKM B-2434 performs
compound under consideration. Application of this technique to substantial degradation of acenaphthene and fluoranthene, and
biodegradation studies is based on the assumption that during the
this process is accompanied by considerable growth of biomass
conversion of hydrophobic substrate by microbial culture, the (Baboshin et al. 2008). The DL values of acenaphthene and fluo-
change in ethylacetate extract COD per unit of converted substrate
ranthene conversion that we have measured are high (0.99 and 0.83,
concentration is almost completely determined by the nature of respectively). They correspond to low values of the mean molar COD
formed hydrophobic intermediates and makes it possible to calcu-
of products (0.3 and 6.3, respectively), much lower than the molar
late the mean molar COD value of these intermediates. This thesis COD of 3-hydroxyphthalic acid, which is the final product in the
has been confirmed experimentally. Previously, phenanthrene and previously proposed scheme of acenaphthene and fluoranthene
anthracene oxidation into the respective hydroxynaphthoic acids by degradation by Sphingomonas sp. VKM B-2434 (Baboshin et al.
2008). In the cited work it was proposed that 3-hydroxyphthalic
acid was not a terminal product. Here, we have obtained more
Table 1 direct evidence of this hypothesis. Previously, we noticed that VKM
Degradation level (DL) and mean molar COD of hydrophobic intermediates (np) for B-2324 metabolism of acenaphthene differed substantial from that
PAH degradation by the strain Sphingomonas sp. VKM B-2434. of fluoranthene (in spite of the common metabolic pathway
Substrate DL np Identified intermediatesa beginning from naphthalene-1,8-dicarboxylic acid). In particular,
(theoretical values of molar the culture can convert a much higher amount of acenaphthene
COD are given in brackets) (>1 g l1) compared to fluoranthene (<0.1 g l1). These data are
Acenaphthene 0.991  0.014 0.261  0.406 Naphthalene-1,8-dicarboxylic indirect evidence of toxic product accumulation at the initial stage
acid (24) of fluoranthene metabolism. In the present work it has been shown
3-Hydroxyphthalic acid (14)
Fluorene 0.396  0.032 18.724  0.992 9-Fluorenone (29)
that the DL of fluoranthene conversion is much less than for ace-
Hydroxyfluorenone (28) naphthene; it indicates accumulation of a fluoranthene conversion
Phenanthrene 0.297  0.015 23.199  0.495 1-Hydroxy-2-naphthoic acid (23) product absent in acenaphthene metabolism. According to data in
Anthracene 0.281  0.026 23.727  0.858 3-Hydroxy-2-naphthoic acid (23) the literature, accumulation of substance in the fraction of degra-
Fluoranthene 0.831  0.025 6.253  0.925 2-Hydroxy-1-acenaphthoic
dation pathway metabolites in some cases may be negligibly small
acid (28)
Naphthalene-1,8-dicarboxylic (Solano-Serena et al. 1999; Annweiler et al. 2000), whereas in other
acid (24) cases it is significant (Lobos et al. 1992; Bouchez et al. 1996). It may
Pyrene 0.161  0.043 31.043  1.591 Pyrene-4,5-dihydrodiol (36) be supposed that the product inhibition of microbial growth is
Hydroxypyrene (36) highly probable when accumulation of intermediates occurs. This
a
According to the data of the work (Baboshin et al. 2008). phenomenon seems to take place during the conversion of PAHs by
888 M. Baboshin, L. Golovleva / International Biodeterioration & Biodegradation 65 (2011) 883e889
Sphingomonas sp. VKM B-2434, with the exception of acenaph-
thene: The DL value of its conversion is close to 1. Indeed, the limit of
initial concentration (0.08e0.4 g l1 depending on the substrate),
above which complete substrate conversion is not observed, has
been obtained for each PAH utilized by Sphingomonas sp. VKM B-
2434 as a growth substrate, with the exception of acenaphthene
only (Baboshin et al. 2008).
The lowest DL among the tested PAHs was observed for pyrene.
It seems that Sphingomonas sp. VKM B-2434 performs minimal
transformation of the pyrene molecule. This observation corrobo-
rates our previous data (Baboshin et al. 2008) and other data in the
literature on pyrene metabolism by sphingomonads (Ho et al.
2000).
The molar COD of the products of fluorene conversion by
Sphingomonas sp. VKM B-2434 is much less than the theoretical
value for previously identified metabolites: 9-fluorenone and
hydroxyfluorenones; the most part of COD is concentrated in acid
extract. This fact demonstrates the incompleteness of the previ-
ously proposed scheme of fluorene metabolism and deeper
degradation of molecule of this substrate accompanied by forma-
tion of acid metabolites as a result of cleavage of aromatic rings.
Analysis of COD can be used for estimation of the DL of PAH
conversion by mixed cultures. Results of the experiments with
binary cultures including strain-degraders of hydroxynaphthoic
acids show that the addition of strains utilizing the products of
conversion of a given substrate by a given microbial culture may
enhance DL value to 1, i.e., result in complete degradation of
molecule of the given substrate. At the same time, the addition of 9-
fluorenone degrader considerably increased the DL of fluorene
conversion but did not lead to complete fluorene degradation
ðDL < 1Þ. This is probably due to the fact that 9-fluorenone is a by-
product of fluorene degradation by Sphingomonas sp. VKM B-2434,
as has been shown previously for Arthrobacter sp. F101 (Casellas
et al. 1997, 1998).
The value of DL is equal to the part of electrons coming from
substrate to oxygen or non-hydrophobic products; i.e., it partially
characterizes the balance of electrons during substrate biodegra-
dation. It approximately corresponds to the value
T ¼ fe0 þ fs0 ; (10)
which has been introduced in the theoretical studies on stoichi-
ometry of microbial growth (McCarty 1975; VanBriesen and
Rittmann 2000) and characterizes the part of electrons sent from
the donor either to the acceptor (fe0 ) or to biomass synthesis (fs0 ). If
the transfer of electrons to the lipids of biomass and non-
hydrophobic products not related to biomass can be neglected,
then DL and T are equivalent.
In more general terms, the task of electron balance consists of
the analysis of distribution of electrons between three fractions of
the products: intermediates of the degradation pathway, biomass,
and final metabolic products. In the absence of intermediate frac-
tion, electron balance can be found from oxygen uptake by the
culture (Aichinger et al. 1992; Smets et al. 1996; Chandran and
Smets 2001). In the presence of intermediate fraction, respiro-
metric data will show the total electrons of two fractions: biomass
and intermediates. Experimental estimation of the electron fraction
of intermediates based on respirometric data only is obviously
impossible. Additional measurement of COD of the intermediate
fraction by the proposed method will give a more correct value of
biomass yield.
Carbon balance is another method of describing biodegradation
stoichiometry (Lobos et al. 1992; Bouchez et al. 1996; Solano-Serena
et al. 1999; Annweiler et al. 2000). Carbon balance is often estimated
by using 14C-radiolabelled substrates (Heitkamp and Cerniglia 1988;
Boonchan et al. 2000; Schneider et al. 2000). For many substrates
(particularly PAHs), only a limited set of partially labeled radiomers
is available commercially, which hampers interpretation of such
experiments from the standpoint of stoichiometry.
The method applied in this work is intended for the analysis of
biodegradation of hydrophobic substrates, which are co-extracted
with metabolites of the degradation pathway. The method presup-
poses evaporation of a sample to dryness and, as a consequence, is
unfit for the analysis of conversion of volatile substrates; the losses
of substance during evaporation become significant for substrates
more volatile than acenaphthene (data not shown), which has
a vapor pressure of 4.02 Pa (Alexander 1999). At the same time, the
advantage of the method described is a simple and direct
measurement of the electron fraction of metabolites of the degra-
dation pathway. Such analysis makes it possible to: (1) assess
biodegradation efficiency; (2) assess the possibility of product
inhibition of microbial growth; (3) suppose exactly which conver-
sion products are formed; and (4) verify the correctness of metabolic
scheme proposed on the basis of chemical analysis of the interme-
diates. The proposed method seems useful for characterization of
biodegradation of a broad range of organic pollutants by both pure
and mixed microbial cultures.
References
Aichinger, G., Grady Jr., C.P.L., Tabak, H.H., 1992. Application of respirometric
biodegradability testing protocol to slightly soluble organic compounds. Water
Environment Research 64, 890e900.
Alexander, M., 1999. Biodegradation and bioremediation. Academic Press, San
Diego.
Allard, A.S., Neilson, A.H., 1997. Bioremediation of organic waste sites: a critical
review of microbiological aspects. International Biodeterioration & Biodegra-
dation 39, 253e285.
Annweiler, E., Richnow, H.H., Antranikian, G., Hebenbrock, S., Garms, C., Franke, S.,
Franke, W., Michaelis, W., 2000. Naphthalene degradation and incorporation of
naphthalene-derived carbon into biomass by the thermophile Bacillus ther-
moleovorans. Applied and Environmental Microbiology 66, 518e523.
Baboshin, M.A., Akimov, V.N., Baskunov, B.P., Born, T.L., Khan, S.U., Golovleva, L.A.,
2008. Conversion of polycyclic aromatic hydrocarbons by Sphingomonas sp.
VKM B-2434. Biodegradation 19, 567e576.
Baboshin, M.A., Golovleva, L.A., 2010. The strategy of strain selection for a mixed
culture performing rapid conversion of a mixture of polyaromatic compounds.
Microbiology (Moscow) 79, 73e82.
Bamforth, S.M., Singleton, I., 2005. Bioremediation of polycyclic aromatic hydro-
carbons: current knowledge and future directions. Journal of Chemical Tech-
nology and Biotechnology 80, 723e736 (Review).
Boonchan, S., Britz, M.L., Stanley, G.A., 2000. Degradation and mineralization of
high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal-
bacterial cocultures. Applied and Environmental Microbiology 66, 1007e1019.
Bouchez, M., Blanchet, D., Vandecasteele, J.P., 1996. The microbiological fate of
polycyclic aromatic hydrocarbons: carbon and oxygen balances for bacterial
degradation of model compounds. Applied Microbiology and Biotechnology 45,
556e561.
Casellas, M., Grifoll, M., Sebate, J., Solanas, A.M., 1997. New metabolites in the
degradation of fluorene by Arthrobacter sp. strain F101. Applied and Environ-
mental Microbiology 63, 819e826.
Casellas, M., Grifoll, M., Sebate, J., Solanas, A.M., 1998. Isolation and characterization
of a fluorenone-degrading bacterial strain and its role in synergistic degrada-
tion of fluorene by a consortium. Canadian Journal of Microbiology 44,
734e742.
Chandran, K., Smets, B.F., 2001. Estimating biomass yield coefficients for autotrophic
ammonia and nitrite oxidation from batch respirograms. Water Research 35,
3153e3156.
Doerffel, K., 1990. Statistic in der analytischen Chemie. Deutscher Verlag für
Grundstoffindustrie GmbH, Leipzig.
Heitkamp, M.A., Cerniglia, C.E., 1988. Mineralization of polycyclic aromatic hydro-
carbons by a bacterium isolated from sediment below an oil field. Applied and
Environmental Microbiology 54, 1612e1614.
Ho, Y., Jackson, M., Yang, Y., Mueller, J.G., Pritchard, P.H., 2000. Characterization of
fluoranthene- and pyrene-degrading bacteria isolated from PAH-contaminated
soils and sediments. Journal of Industrial Microbiology and Biotechnology 24,
100e112.
ISO, 1990. Water quality e determination of chemical oxygen demand International
Standard ISO 6060. International Organization for Standardization, Geneva.
Kazunga, C., Aitken, M.D., 2000. Products of incomplete metabolism of pyrene by
polycyclic aromatic hydrocarbon-degrading bacteria. Applied and Environ-
mental Microbiology 66, 1917e1922.
 M. Baboshin, L. Golovleva / International Biodeterioration & Biodegradation 65 (2011) 883e889
Kazunga, C., Aitken, M.D., Gold, A., Sangaiah, R., 2001. Fluoranthene-2,3- and -1,5-
diones are novel products from the bacterial transformation of fluoranthene.
Environmental Science and Technology 35, 917e922.
Lobos, J.H., Leib, T.K., Su, T.M., 1992. Biodegradation of bisphenol A and other
bisphenols by a Gram-negative aerobic bacterium. Applied and Environmental
Microbiology 58, 1823e1831.
}
Lundstedt, S., White, P.A., Lemieux, C.L., Lynes, K.D., Lambert, I.B., Oberg, L.,
Haglund, P., Tysklind, M., 2007. Sources, fate, and toxic hazards of oxygenated
polycyclic aromatic hydrocarbons (PAHs) at PAH-contaminated sites. Ambio 36,
475e485.
McCarty, P.L., 1975. Stoichiometry of biological reactions. Progress in Water Tech-
nology 7, 157e172.
Minkevich, I.G., Eroshin, V.K., 1973. Productivity and heat generation of fermenta-
tion under oxygen limitation. Folia Microbiologica 18, 376e385.
O’Leary, W.M., 1962. The fatty acids of bacteria. Microbiology and Molecular Biology
Reviews 26, 421e447.
889
Schneider, J., Grosser, R.J., Jayasimhulu, K., Xue, W., Kinkle, B., Warshawsky, D., 2000.
Biodegradation of carbazole by Ralstonia sp. RJGII.123 isolated from a hydro-
carbon contaminated soil. Canadian Journal of Microbiology 46, 269e277.
Schmidt, S.N., Christensen, J.H., Johnsen, A.R., 2010. Fungal PAH-metabolites resist
mineralization by soil microorganisms. Environmental Science and Technology
44, 1677e1682.
Smets, B.F., Jobbagy, A., Cowan, R.M., Grady Jr., C.P.L., 1996. Evaluation of respiro-
metric data: identification of features that preclude data fitting with existing
kinetic expressions. Ecotoxicology and Environmental Safety 33, 88e99.
Solano-Serena, F., Marchal, R., Ropars, M., Lebeault, J.M., Vandecasteele, J.P., 1999.
Biodegradation of gasoline: kinetics, mass balance and fate of individual
hydrocarbons. Journal of Applied Microbiology 86, 1008e1016.
VanBriesen, J.M., Rittmann, B.E., 2000. Mathematical description of microbiological
reactions involving intermediates. Biotechnology and Bioengineering 67, 35e52.
VanBriesen, J.M., 2001. Thermodynamic yield predictions for biodegradation
through oxygenase activation reactions. Biodegradation 12, 265e281.