231

Hydrocarbon biodegradation in oxygen-limited

sequential batch reactors by consortium from
weathered, oil-contaminated soil
S.A. Medina-Moreno, S. Huerta-Ochoa, and M. Gutiérrez-Rojas

Abstract: We studied the use of sequential batch reactors under oxygen limitation to improve and maintain consortium
ability to biodegrade hydrocarbons. Air-agitated tubular reactors (2.5 L) were operated for 20 sequential 21-day cycles.
Maya crude oil – paraffin mixture (13 000 mg/L) was used as the sole carbon source. The reactors were inoculated
with a consortium from the rhizosphere of Cyperus laxus, a native plant that grows naturally in weathered, contami-
nated soil. Oxygen limitation was induced in the tubular reactor by maintaining low oxygen transfer coefficients (kLa <
20.6 h–1). The extent and biodegradation rates increased significantly up to the fourth cycle, maintaining values of
about 66.33% and 460 mg·L–1·d–1, respectively. Thereafter, sequential batch reactor operation exhibited a pattern with a
constant general trend of biodegradation. The effect of oxygen limitation on consortium activity led to a low biomass
yield and non-soluble metabolite (0.45 g SS/g hydrocarbons consumed). The average number of hydrocarbon-degrading
microorganisms increased from 6.5 × 107 (cycles 1–3) to 2.2 × 108 (cycles 4–20). Five bacterial strains were identi-
fied: Achromobacter (Alcaligenes) xylosoxidans, Bacillus cereus, Bacillus subtilis, Brevibacterium luteum, and
Pseudomonas pseudoalcaligenes. Asphaltene-free total petroleum hydrocarbons, extracted from a weathered, con-
taminated soil, were also biodegraded (97.1 mg·L–1·d–1) and mineralized (210.48 mg CO2·L–1·d–1) by the enriched
consortium without inhibition. Our results indicate that sequential batch reactors under oxygen limitation can be
used to produce consortia with high and constant biodegradation ability for industrial applications of
bioremediation.

239batch reactors, oxygen limitation, consortium, hydrocarbon biodegradation.

Key words: sequential

Résumé : Nous avons réalisé une étude sur des réacteurs séquentiels discontinus, dans des conditions limitées en oxy-
gène, afin d’améliorer et de conserver la capacité des consortiums à biodégrader les hydrocarbures. Les réacteurs tubu-
laires en agitation aérienne (2,5 L) ont fonctionné pendant 20 cycles séquentiels de 21 jours. Un mélange de pétrole
brut Maya et de paraffine (13 000 mg/L) a été utilisé comme seul source de carbone. Les réacteurs ont été inoculés
avec un consortium issu de la rhizosphère de la plante indigène Cyperus laxus qui pousse à l’état sauvage dans des
sols altérés contaminés. Une limitation en oxygène a été produite dans le réacteur tubulaire en modérant les coeffi-
cients de transfert d’oxygène (kLa < 20,6 h–1). L’ampleur et les taux de biodégradation ont augmenté significativement
jusqu’au quatrième cycle, en conservant des valeurs d’environ 66,33 % et de 460 mg·L–1·jour–1 respectivement. Par la
suite, les réacteurs séquentiels discontinus ont démontré une tendance générale de biodégradation. L’impact de la limi-
tation d’oxygène sur l’activité des consortiums a mené à un faible rendement de biomasse et de métabolites insolubles
(0,45 g de solides en suspension par g d’hydrocarbures consommés). Le nombre moyen de micro-organismes dégradant
les hydrocarbures a augmenté de 6,5 × 107 (cycles 1–3) à 2,2 × 108 (cycles 4–20). Cinq souches bactériennes ont été
identifiées comme étant Achromobacter (Alcaligenes) xylosoxidans, Bacillus cereus, Bacillus subtilis, Brevibacterium luteum
et Pseudomonas pseudoalcaligenes. Les hydrocarbures de pétrole totaux sans asphaltène, extraits d’un sol altéré conta-
miné, ont également été biodégradés (97,1 mg·L–1·jour–1) et minéralisés (210,48 mg CO2·L–1·jour–1) par le consortium
enrichi sans inhibition. Nos résultats indiquent que les réacteurs séquentiels discontinus fonctionnant avec une quantité
limitée d’oxygène peuvent être utilisés pour produire des consortiums pourvus d’une capacité élevée et constante de
biodégradation pour des applications industrielles de bioremédiation.

Received 3 August 2004. Revision received 19 November 2004. Accepted 20 November 2004. Published on the NRC Research
Press Web site at http://cjm.nrc.ca on 20 May 2005.
Abbreviations: AF-TPH, asphaltene-free total petroleum hydrocarbons (g); [O2]sat, maximum dissolved oxygen concentration
(saturation) at any time (mg O2/L); [O2], dissolved oxygen concentration at time t (mg O2/L); kLa, volumetric oxygen transfer
coefficient (h–1); MCP, 1:1 mixture (w/w) of Maya crude oil and n-paraffin (g); MPN, most probable number; QO2, oxygen specific
consumption rate (mg O2·(g SS)–1@h–1); SOC, soluble organic carbon (g/L); and SS, suspended solids (g/L).
S.A. Medina-Moreno, S. Huerta-Ochoa, and M. Gutiérrez-Rojas.1 Departamento de Biotecnología, UAM-Iztapalapa. Av. San
Rafael Atlixco No. 186 Col. Vicentina, C.P. 09340, México, D.F.
1
Corresponding author (e-mail: mgr@xanum.uam.mx).

Can. J. Microbiol. 51: 231–239 (2005) doi: 10.1139/W04-130 © 2005 NRC Canada
232
Mots clés : réacteurs séquentiels discontinus, limitation d’oxygène, consortiums, biodégradation d’hydrocarbures.
[Traduit par la Rédaction] Medina-Moreno et al.
Introduction
Due to the improper handling and transport of petroleum,
oil spills have been a major cause of environmental pollution
at different sites around the world. In particular, operations
in the oil industry have caused serious pollution problems in
the tropical regions of Mexico (Adams et al. 1999). The
company responsible for hydrocarbon exploitation and dis-
tribution in Mexico (Petroleos Mexicanos) is now interested
in restoring polluted sites, mainly soils and sediments.
Bioremediation processes are considered decontamination
options because they are basic technologies that can be de-
veloped at the local level and they have a low cost in com-
parison with other technologies (Boopathy 2000). The
effectiveness of bioremediation often depends on the nature
of the microorganisms present, and on how their activity can
be maintained and improved under controlled conditions.
Since bacterial consortia have greater metabolic versatility
over pure cultures, these groups are being used in the treat-
ment of crude oil wastes at extreme locations such as the
Arctic (Braddock et al. 1999) and tropical regions
(Palittapongarnpim et al. 1998). In several studies, different
strategies have been used to improve the hydrocarbon
biodegradation capability of consortia in liquid culture.
Venkateswaran and Harayama (1995) found that for liquid
cultures (5000 mg Arabian light crude oil/L), 6 sequential
transfers of a crude oil-degrading mixed culture improved
the ratio of hydrocarbon biodegradation rate to the amount
of degrading microorganisms with only 1 batch. Márquéz et
al. (2001) developed a 2 step sequential procedure in liquid
culture (60 000 mg diesel/L) to improve the ability of a mi-
crobial consortium to degrade diesel from recently contami-
nated soil. The maintenance of consortia has also been
carried out in liquid cyclone fermentors (Van Hamme and
Ward 1999) supplemented with 23 000 mg/L of refinery
sludge using mixed microbial cultures from hydrocarbon-
polluted soil. Oxygen limitation in aerobic hydrocarbon deg-
radation by bacterial consortia in liquid cultures has been
proven to modify degradation rates (Ebenhöh and Berthe-
Corti 2001) and consortium composition (Eriksson et al.
2003), which results in bioreactors with decreased perfor-
mance. On the other hand, the addition of an immiscible or-
ganic liquid in stirred-aerated, multiphase bioreactors has
been shown to decrease the volumetric oxygen transfer coef-
ficient (kLa) (Nielsen et al. 2003). Since there is little infor-
mation regarding sequential reactors used in maintaining and
improving the ability of consortia to biodegrade crude oil
under oxygen limitation, study of these systems is necessary
so as to develop hydrocarbon biodegradation consortia for
industrial applications.
The aim of this work was to study biodegradation of
Maya crude oil – n-paraffin (MCP) mixtures by a consor-
tium from weathered soil, in air-agitated sequential batch re-
actors. The main objective was to determine whether oxygen
limitation and sequential batch operation allow for the main-
tenance and improvement of the biodegradation ability of
the consortium. We also evaluated the ability of the consor-
Can. J. Microbiol. Vol. 51, 2005
239
tium to biodegrade a more complex mixture such as total
petroleum hydrocarbons extracted from weathered soil.
Materials and methods
Consortium, medium, and hydrocarbons
The consortium was isolated from the rhizosphere of
Cyperus laxus (Díaz-Ramírez et al. 2003), a native plant that
grows naturally in weathered, oil-contaminated sites
(Gallegos-Martínez et al. 2000). The mineral medium con-
tained the following (in g/L): 6.75 NaNO3, 2.15 K2HPO3,
1.13 KCl, and 0.54 MgSO4. The pH was adjusted to 6.5 with
1 mol/L HCl. The hydrocarbons used were MCP, which is
Maya crude oil (Mexican Institute of Petroleum) mixed in a
1:1 ratio (w/w) with n-paraffins (C10 to C28; Biocode-
Hycel, Mexico),and asphaltene-free total petroleum hydro-
carbons (AF-TPH). TPH was extracted from the same
weathered soil according to the EPA (1994) 3540 technique.
Asphaltenes were removed by cold precipitation with n-
pentane (J.T. Baker, EDOMEX Mexico) and vacuum filtra-
tion (BÜCHI V-800, BÜCHI, Flawil, Switzerland) (ASTM-
D2007-91, 1991). AF-TPH composition was determined by
chromatography column according to Díaz-Ramírez et al.
(2003). Initial AF-TPH composition was 48.5% aliphatic
fraction; 29.0% aromatic fraction; and 22.5% polar fraction.
Reactor set-up and operation
Two glass columns (2.5 L) were used without mechanical
agitation and operated in sequential 21-day cycles at 30 °C
and a specific air flow rate of 2 L·(L medium)–1@min–1
(Fig. 1). Each reactor containing 1500 mL of mineral me-
dium and 13 000 mg MCP/L was initially inoculated with a
consortium culture (500 mL) that was previously grown with
MCP as the sole carbon source. At the end of each cycle,
1500 mL of culture was removed. The remainder of the cul-
ture was used as the inoculum for the next cycle, and initial
conditions were restored. Each reactor was sampled (10 mL)
4 times at 0, 3, 7, 12, 16, and 21 d. The abiotically lost MCP
was determined by operating a sterile reactor for 1 cycle un-
der the same conditions without inoculum.
Oxygen transfer and oxygen-specific consumption rate
Volumetric oxygen transfer coefficients (kLa) and oxygen-
specific consumption rates (QO2) were estimated according
to Fujio et al. (1973). A dissolved-oxygen analyzer (model
DO-40; New Brunswick Scientific, Edison, New Jersey) was
used to measure oxygen concentration.
Soluble organic carbon and suspended solid analyses
Reactor samples were centrifuged (Solbat Centrifuge
Equipment Co., Pueblo, Mexico) at 4000g in Corex tubes for
30 min. Three phases were formed: organic, aqueous, and
solid. Soluble organic carbon (SOC) was determined from
the aqueous phase using a total organic carbon analyzer
(Shimadzu model 5000A, Australia). The amount of sus-
pended solids (SS) from the solid phase and the fraction that
© 2005 NRC Canada
Medina-Moreno et al.
Fig. 1. Reactor set-up used. (1) glass columns with water jacket (8.5 cm i.d., and 45 cm length), (2) glass ring air distributors, (3) air
flow meter, (4) sampling port, (5) bath temperature for the jackets (pump and heater), (6) traps, (7) thermometers, (8) air distributor to
reactors, (9) pressure regulator, (10) air filter, (11) general air line, (12) dissolved oxygen probe electrode, (13) dissolved oxygen ana-
lyzer.
remained trapped in the organic phase were determined. The
SS fraction trapped in the organic phase was recovered by
three 1:5 liquid–liquid (v/v, i.e., organic phase/solvent) ex-
tractions with a 1:3 hexane–isotonic solution (v/v). The or-
ganic phases were pooled and stored. All SS (hexane–
isotonic extracts and solid phase) were re-centrifuged 3
times with 1:20 acetone–isotonic solution (v/v). The SS were
determined with a spectrophotometer at 620 nm (LKB
Biochrom Ultraspec II, Cambridge, UK). The SS and MCP
carbon fractions were determined with a total organic carbon
solid peripheral apparatus.
Microbial analysis
The total number of heterotrophs, determined by CFU,
was assessed for several cycles (0, 7, 16, and 21 d) by the
spread-plate counting technique on trypticase soy agar
(TSA; Bioxon, EDOMEX, Mexico) according to Lorch et al.
(1995). Colonies with different characteristics were isolated
and their identification was carried out by Microbial ID Inc.
(Newark, Delaware) based on cellular fatty acid analyses.
The criterion used for bacterial strain identification was a
similarity index higher than 0.7. The number of
hydrocarbon-degrading microorganisms was determined by
the MPN technique according to Wrenn and Venosa (1996).
Tetrazolium blue (Sigma, Steinheim, Germany) was used as
microbial growth indicator.
AF-TPH biodegradation and mineralization assays
Baffled 125-mL Erlenmeyer flasks containing 50 mL of
sterile mineral medium and sealed with Teflon Mininert
valves were used. The flasks were inoculated with 1 mL of
233
reactor culture broth at 4 different AF-TPH concentrations
(3000, 6000, 9000, and 12 000 mg/L). For AF-TPH determi-
nation, the flasks were sampled at 90, 186, 282, 382, and
478 h. Headspace CO2 was measured every 12 h by gas
chromatography (GOW-MAC 550, TCD detector, Las Ve-
gas, Nevada). Uninoculated and AF-TPH-free inoculated
controls were used to determine abiotic loss and endogenous
respiration, respectively. C/N and C/P ratios for every AF-
TPH concentration were adjusted to 10 and 20, respectively.
All flasks were shaken (200 r·min–1, 30 °C) and aerated un-
der sterile conditions after headspace sampling.
Hydrocarbon analysis
Residual MCP was extracted and determined according to
Van Hamme and Ward (1999). Extracts were vacuum con-
centrated (Rotary Evaporator R-205, BÜCHI) and dried to a
constant mass in a fume hood. Residual MCP was
gravimetrically measured. Extraction was 95% efficient. Re-
sidual MCP was also analyzed by gas chromatography (SRI
8610) with a FID detector (30 m × 0.25 mm (i.d.) capillary
column DB-5, J&W Scientific, California) and helium as the
carrier gas. The rate of temperature change (from 50 °C to
300 °C) was 5 °C/min. Residual AF-TPH was recovered in the
same manner as MCP and quantified with an infrared spec-
trometer (Perkin Elmer 200 FT-IR; PerkinElmer Instruments,
Shelton, Connecticut) according to García-Rivero et al. (2002).
Results and discussion
Oxygen transfer and specific consumption rate
Since aeration brings about the abiotic loss of hydrocar-
© 2005 NRC Canada
234 Can. J. Microbiol. Vol. 51, 2005

Fig. 2. Dissolved oxygen kinetics (% oxygen saturation) for different cycles and days according to Numerical method (Fujio et al.
1973). (a) Reactor 1, cycle 2, day 21; (b) reactor 2, cycle 8, day 14; (c) reactor 1, cycle 10, day 5; and (d) reactor 2, cycle 13, day 8.

Table 1. Volumetric oxygen transfer coefficients (kLa) and specific oxygen consumption rates (QO2) of both reactors
for various cycles and days.
Cycle Suspended Total O2 consumption
Reactor No. No. day kLa (h–1) solids (g/L) QO2 (mg O2·(g SS)–1@h–1)* rate/O2 transfer rate
1 2 21 17.1 3.9 7.5 3.9
2 2 21 16.4 4.2 6.7 3.0
1 4 10 18.4 3.2 18.5 4.5
2 4 10 19.6 3.3 16.6 2.9
1 6 7 16.4 2.1 14.4 2.9
2 6 7 18.6 2.2 13.2 3.2
1 8 14 17.4 4.0 22.5 5.7
2 8 14 20.6 4.1 14.4 3.4
1 10 6 18.3 2.0 17.6 2.0
2 10 6 19.8 2.3 15.3 2.8
1 13 8 17.5 2.8 16.8 3.9
2 13 8 19.4 3.2 13.5 3.6
*Specific oxygen consumption rate was determined with a saturation value of 5 mg O2/L corresponding to Mexico City conditions
(585 mmHg and 25 °C).

bons and foam formation, low air flow rates (2 L air·(L
medium)–1@min–1) were maintained to minimize loss as well
as problems with reactor operation. Twelve dissolved oxy-
gen analyses for different cycles were conducted. Figure 2
shows examples of results from experimental dissolved oxy-
gen. The volumetric oxygen transfer coefficients (kLa) and
specific consumption rate (QO2) for both reactors are shown
in Table 1. No significant differences were observed in kLa
values for the 2 reactors and the different cycles. The kLa
values remained stable within a range of 16.4 to 20.6 h–1
and, as expected, were very low despite the presence of the
organic phase (Nielsen et al. 2003). On the other hand, QO2
values were high, also as expected, because hydrocarbons
are highly reduced compounds. The QO2 values increased 2-
fold after cycle No. 4, revealing greater metabolic activity.
Since suspended solids (biomass included) did not change
significantly for the same cycle, total oxygen consumption
rates (QO2@SS) were always greater than oxygen transfer
rates (kLa·([O2]sat – [O2])) demonstrating that microbial cul-
tures were oxygen limited (Table 1, last column). Eriksson
et al. (2003) reported that during polycyclic aromatic hydro-
carbon degradation at low temperature and limited aerobic

© 2005 NRC Canada

Medina-Moreno et al. 235

Fig. 3. Suspended solids (SS) and soluble organic carbon (SOC)
production rates during twenty 21-day cycles. SS production
rates (䊏) and SOC production rates (ⵧ) were estimated by lin-
ear regression. Error bars correspond to SD for quadruplicate
samples.
Fig. 5. GC-FID chromatograms for MCP mixture on days 0 and
21 in the control reactor (abiotic conditions) and standard mix-
ture of n-alkanes (C9 to C28).

Fig. 4. Yields of SS/MCP (Maya crude oil – n-paraffin) mixtures

degraded (g/g) during twenty 21-day cycles. Error bars corre-
spond to SD for quadruplicate samples.

Fig. 6. MCP mixture biodegradation in the reactors during 21-

day cycles. The MCP biodegradation rates (䉫) were estimated
by linear regression from residual MCP values vs. time; they are
expressed as mg of MCP degraded·bioreactor L–1@day–1. The ex-
tent of MCP biodegradation (bars 䊏) was estimated using the
initial (day 0) and final (day 21) values of every cycle. Error
bars correspond to SD of quadruplicate samples.

conditions, the composition and activity of microbial com-

munities appeared to be more affected by the presence of
oxygen than by temperature or inoculum source.

SS and SOC production

The MCP biodegradation process during operation of the
oxygen-limited sequential batch reactor yielded SS and
SOC. SS formation during aerobic hydrocarbon bio-
degradation can be related to biomass (Solano et al. 1999)
and non-soluble metabolite formation (Coty and Leavitt
1999). Furthermore, SOC formation can be associated with
oxidized hydrocarbons such as aliphatic organic acids,
diacids, and aromatic ketones (Langbehn and Steinhart
1995). SS and SOC production rates are shown in Fig. 3. SS
production rates, which were approximately 198 mg·L–1@d–1,

© 2005 NRC Canada

236 Can. J. Microbiol. Vol. 51, 2005

Fig. 7. GC-FID chromatograms of residual MCP mixture of 1 reactor on days 0, 7, 16, and 21 for 2 different cycles: (a) cycle 6 and
(b) cycle 20.

Table 2. Growth of heterotrophs and hydrocarbon-degrading microorganisms for several 21-day cycles.
Cycle 1 Cycle 4 Cycle 7 Cycle 15 Cycle 20
7 7 7 7 7 7 7 7
Day Het × 10 Deg × 10 Het × 10 Deg × 10 Het × 10 Deg × 10 Het × 10 Deg × 10 Het × 107 Deg × 107
0 3.6±0.10 0.20±0.05 2.6±0.15 0.14±0.03 2.5±0.20 1.1±0.06 3.8±0.05 1.4±0.04 4.4±0.10 1.7±0.12
7 3.3±0.20 0.25±0.02 200±8.00 19±1.00 370±23 10±0.12 286±17 18±1.50 360±25 13±1.00
16 89.0±6.00 6.5±0.15 980±8.00 48±2.00 960±43 68±6.00 770±40 89±4.10 890±65 78±4.80
21 16±0.60 0.51±0.03 9.7±0.70 1.56±0.20 7.1±0.70 2.7±0.20 12±0.70 3.6±0.20 11±0.75 4.4±0.10
Note: Het, heterotrophic microorganisms determined by serial dilutions and growth in trypticase soy agar (TSA) plates (30 °C, 3 d) according to Lorch
et al. (1995). Deg, hydrocarbon-degrading microorganisms determined by most probable number (MPN) with tetrazolium salt according to Wrenn and
Venosa (1996). Het and Deg are both expressed in CFU/mL.

did not change significantly throughout the 20 cycles. How-
ever, SOC production rates increased significantly up to ap-
proximately 131 mg·L–1@d–1 from cycle No. 4 on up,
probably because of soluble metabolite accumulation. To
understand why the SS production rate was constant, SS
yield with respect to degraded MCP was estimated. Yield
decreased steadily until the fourth cycle, reaching constant
values of about 0.45 g SS / g MCP (Fig. 4). Raymond
(1999) studied the aerobic biodegradation of n-octadecane
by Nocardia sp. in aerated, 2-L, mechanically agitated reac-
tors, and obtained final values for a biomass-hydrocarbon
yield of 0.81 to 0.84 g/g. In our work, consortium activity
was affected by oxygen limitation (Table 1, last column),
which resulted in the high transformation of initial MCP into
more soluble forms (SOC) and into low biomass and non-
soluble metabolite (SS) production. Nevertheless, oxygen
limitation could work as an effective mechanism in improv-
ing the ability of the consortium to biodegrade MCP. Fur-
thermore, sequential batch operation, more precisely
discharge-charge manipulation, offers the advantage of re-
storing initial conditions that avoid problems such as prod-
uct inhibition.
Abiotic loss of hydrocarbons
An abiotic reactor was run and sampled under the same
conditions during one 21-day cycle. During the first hours, a

© 2005 NRC Canada

Medina-Moreno et al.
Fig. 8. CO2 (mg/L medium) evolved during the biodegradation
assay with asphaltene-free total petroleum hydrocarbons (AF-
TPH). The curves correspond to each of the following initial AF-
TPH concentrations: 3000 mg/L (∆); 6000 mg/L (*); 9000 mg/L
(X); 12 000 mg/L (䊊); and the control (endogenous respiration;
ⵧ).
complete dispersion-homogenization of MCP was observed
owing to aeration. The rate of MCP loss was up to
548 mg·L–1@d–1 on the third day, followed by a rapid de-
crease. The total MCP loss at the end of the cycle was
26.9% (data not shown). Figure 5 shows chromatograms of
MCP as unresolved complex material, similar to the
chromatograms (bitumen degradation) reported by Potter
and Duval (2001), and presents standard n-alkanes. The
abiotic loss of hydrocarbons was mainly due to the exhaust
of volatile and semivolatile compounds with less than 12
carbon atoms. To ensure that reliable hydrocarbon
biodegradation data are obtained, the MCP residual values
for the reactors were corrected taking into account both
abiotic loss rates and extraction efficiency.
Hydrocarbon biodegradation
The extent and volumetric rates of MCP biodegradation
during operation of the sequential batch reactor increased
significantly (Fig. 6) up to the fourth cycle, reaching values
of about 66.33% and 460 mg·L–1@d–1, respectively. Con-
sidering that abiotic loss was approximately 27%, only 7%
of the initial MCP remained as a non-biodegradable fraction,
which was constant for all cycles. The results show an im-
provement in the capability of the consortium to biodegrade
MCP under assay conditions. Extensive emulsification and
foam were notably present in each cycle (probably because
of the biosurfactant produced) throughout the entire experi-
ment. To qualitatively determine the hydrocarbons
biodegraded by consortium, the residual MCP was also ana-
lyzed by gas chromatography. After cycle 3, the unresolved
complex mixture of residual MCP exhibited a constant de-
pletion of C13 to C28 compounds at day 7, 16, and 21 from
reactors, as shown in cycles 6 and 20 (Fig. 7). The sum of
areas under the C13 to C28 peaks account for 60%–70% of
the nonvolatilized MCP fraction (Fig. 5). Since the results
exhibit an overall depletion of compounds different from
those that occurred under abiotic conditions (see Fig. 5), it
can be concluded that the consortium was able to use com-
237
plex hydrocarbon molecules (C13 to C28 compounds) as
sources of carbon and energy. The remaining fractions were
not analyzed; however, when the same consortium was used
to biodegrade asphaltene-free Maya crude oil, it was re-
vealed that the final fraction was found to consist predomi-
nantly of polar compounds (Díaz-Ramírez et al. 2003). In
our work, asphaltenes were not removed from MCP. Thus,
hydrocarbons remaining at the end of the cycles were ex-
pected to be rich in polar and asphaltene fractions.
The extent of biodegradation in our study was acceptable
and similar to that obtained during liquid culture
biodegradation of Tapis light crude oil (Palittapongarnpim et
al. 1998) and twice the degree reported by Sugiura et al.
(1997) with Maya crude oil; native consortia were used in
both studies. Furthermore, laboratory sequential batch opera-
tions with liquid cultures have also been reported to improve
the hydrocarbon biodegradation ability of consortia
(Venkateswaran and Harayama 1995; Márquéz et al. 2001).
However, no reports are available regarding the stability of
responses of consortia to biodegraded hydrocarbons, includ-
ing studies of prolonged periods in sequential batch reactors
along with oxygen limitations. Our results confirm that se-
quential batch operations under oxygen limitation allow the
consortium to improve and to maintain a MCP-degradation
capability by reaching stable general trend responses, which
results in a pattern of constant biodegradation.
Microbial growth
The MPN method was used to enumerate the hydrocarbon-
degrading microorganisms and to justify the use of TSA
plates for total counts of the consortium populations. Van
Hamme et al. (2000) used similar techniques (TSA plate
count and 5-tube MPN method) to enumerate consortium
populations that were able to degrade Bow River crude oil in
liquid culture. Table 2 shows the consortium growth in reac-
tors for both total heterotrophs and hydrocarbon-degrading
microorganisms during various cycles. At the beginning of
every cycle, the total number of initial heterotrophs was high
(2.0–4.4 × 107 CFU/mL). The total number of heterotrophs
and hydrocarbon-degrading microorganisms always reached
a maximum at day 16, which was around 2 orders of magni-
tude higher than the initial count. After day 16, the viable
population decreased, probably because of an insufficient
amount of hydrocarbons to sustain growth. The average
number of hydrocarbon-degrading microorganisms increased
from 6.5 × 107 (cycles 1–3) to 2.2 × 108 (cycles 4–10). Fur-
thermore, the ratio of the hydrocarbon-degrading population
to the heterotroph population clearly increased with cycle
number, suggesting that sequential batch operations im-
proved consortium biodegradation capability. Similar results
(Venkateswaran and Harayama 1995) have been obtained
with liquid cultures sequentially enriched with bacterial con-
sortium during pretreated Arabian crude oil biodegradation.
Several isolates from TSA plates were identified accord-
ing to the Microbial Identification System so as to determine
the bacterial species in the consortium. Five isolates were
identified as Achromobacter (Alcaligenes) xylosoxidans,
Bacillus cereus, Bacillus subtilis, Brevibacterium luteum,
and Pseudomonas pseudoalcaligenes. Several of these gen-
era have been reported as being capable of degrading hydro-
© 2005 NRC Canada
238
Table 3. Asphaltene-free total petroleum hydrocarbon (AF-TPH) biodegradation and mineralization.
Initial AF-TPH Biodegradation*
concentration (mg/L) (%)
12 000 15.9
9 000 21.1
6 000 30.8
3 000 47.6
*Estimated using final and initial AF-TPH concentration.
†
Estimated by linear regression.
‡
Estimated with CO2 values after 200 h (corrected for endogenous respiration).
carbons (Komancová et al. 2003; Kumamaru et al. 1998;
Lee and Cutright 1997).
AF-TPH biodegradation
AF-TPH biodegradation assays were performed at differ-
ent initial substrate concentrations to determine whether
consortium degradation ability can also be effective with a
more recalcitrant, complex hydrocarbon mixture without
substrate inhibition. The assay was developed without soil to
avoid the interference of sorption phenomena and the nega-
tive effect of hydrocarbon bioavailability. The inoculum was
taken from reactors at day 7 of cycle 15. Since AF-TPH
were extracted from weathered soil, abiotic loss was not ob-
served in control experiments (data not shown). The extent
and rates of biodegradation are shown in Table 3. The con-
sortium was able to biodegrade AF-TPH at all assayed con-
centrations. Since the culture aqueous phase was always
saturated, statistically (Tukey, α = 0.01) constant
biodegradation rates averaging 88.4 mg·L–1@d–1 were ob-
served. These values were 5 times lower than those obtained
from MCP-biodegradation rates, probably because of the re-
calcitrant nature of AF-TPH.
AF-TPH mineralization by the consortium was also deter-
mined by the amount of CO2 evolved (Fig. 8). The CO2 pro-
duced at all concentrations was higher than the control, and
clearly none of these reached a maximum, suggesting that
the time allowed for AF-TPH biodegradation and mineral-
ization was not sufficient. During CO2 production, 2 stages
were distinguished. In the first stage (up to 200 h), CO2 pro-
duction rates were lower than in the second. This can be
seen as consortium acclimation to a new carbon source.
Later, a significant increase was observed probably due to a
new population distribution as in the case of all MCP cycles,
in which hydrocarbon-degrading microorganisms became
predominant on day 21. CO2 production rates (mineraliza-
tion) and CO2 yields were affected by initial AF-TPH con-
centration, unlike the biodegradation rate, indicating
metabolite accumulation. García-Rivero et al. (2002) found
that in batch-agitated slurry soil, both mineralization and
yield decreased when initial TPH-toluene concentrations in-
creased. These authors put forward the idea that substrate in-
hibition was due to the higher amount of TPH released by
toluene into the aqueous phase. In our study, AF-TPH were
slowly released from the organic to the aqueous phase, re-
sulting in a non-inhibited mineralization and yield pattern.
Therefore, biodegradation assays confirmed that the consor-
tium was able to biodegrade and mineralize, without inhibi-
tion, a more complex and recalcitrant hydrocarbon-mixture
such as AF-TPH.
Can. J. Microbiol. Vol. 51, 2005
Biodegradation CO2 production rate‡ Yield‡ (mg
rate† (mg·L–1·d–1) (mg·L–1·d–1) CO2/mg AF-TPH)
97.1 210.48 1.86
93.8 179.28 1.61
85.2 165.36 1.36
77.5 60.24 0.61
Conclusions
The ability of a bacterial consortium to biodegrade a MCP
mixture in liquid culture was improved and maintained dur-
ing prolonged time periods in air-agitated, sequential batch
reactors under oxygen limitation. Although oxygen limita-
tion leads to low biomass and non-soluble metabolite pro-
duction and to soluble metabolite accumulation, reactor
operation performance shows a constant general trend in
biodegradation. The extent of hydrocarbon biodegradation
was similar to or higher than that obtained in other studies in
liquid cultures. Furthermore, sequential batch operation al-
lowed consortium enrichment by increasing the number of
hydrocarbon-degrading microorganisms. Furthermore, the
enriched consortium was proven to be capable of
biodegrading and mineralizing, without inhibition, a more
complex and recalcitrant hydrocarbon mixture such as AF-
TPH extracted from a weathered, contaminated soil.
Acknowledgements
The authors acknowledge the financial support received
from CONACyT (The National Council for Science and
Technology) and PEMEX-Refinación (the State oil indus-
try).
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