Les antibiotiques sont produits sous des formes pharmaceutiques telles que des pilules acheter du zithromax
elles permettent d'injecter la quantité de préparation strictement nécessaire.
Application of bioaugmentation to improve the activated sludge system into the contact oxidation system treating petrochemical wastewater
Bioresource Technology 100 (2009) 597–602
Contents lists available at
Application of bioaugmentation to improve the activated sludge systeminto the contact oxidation system treating petrochemical wastewater
Fang Ma a,b,*, Jing-bo Guo a,b, Li-jun Zhao c, Chein-chi Chang d, Di Cui a,b
a School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, PR Chinab State Key Lab of Urban Water Resources and Environment, Harbin Institute of Technology, Harbin 150090, PR Chinac School of Chemical Engineering, China University of Petroleum, Beijing 102249, PR Chinad Department of Civil and Environmental Engineering, University of Maryland, Baltimore, MD 21250, USA
In this paper, bioaugmentation was applied to upgrade a full-scale activated sludge system (S2) into a
Received 1 March 2008
contact oxidation system (S1). Results showed that when chemical oxygen demand (COD) and ammonia
Received in revised form 29 June 2008
nitrogen (NHþ-N) concentration of the petrochemical wastewater were 320–530 mg/L and 8–25 mg/L,
Accepted 30 June 2008
respectively, the bioaugmented process (S1) took only 20 days when they were below 80 mg/L and
Available online 2 September 2008
10 mg/L, respectively. However, the unbioaugmented conventional activated sludge process (S2) spent30 days to reach the similar efﬂuent quality. As the organic loading rate (OLR) increased from 0.6 to
0.9 and ﬁnally up to 1.10 kg COD/m3 d, S1 showed strong resistance to shock loadings and restored after
three days compared to the seven days required by S2. Based on the results of this paper, it shows that
Contact oxidation processActivated sludge process
bioaugementation application is feasible and efﬁcient for the process upgrade due to the availability of
the bioaugmented specialized consortia.
Ó 2008 Elsevier Ltd. All rights reserved.
the chemical property and concentration of the pollutants, andthe activity of the bioaugmented bacteria. Therefore, process per-
Bioaugmentation is the application of indigenous or allochtho-
formances were unpredictable and the full-scale applications of
nous or genetically modiﬁed organisms to polluted hazardous
bioaugmentation to the existing industrial wastewater treatment
waste sites or bioreactors in order to accelerate the removal of
facilities were rarely reported.
undesired pollutants (
The petrochemical wastewater treatment plant (WWTP) studied
in this research was located in northeast China. Its inﬂuent was a
ulating strains which are efﬁcient in degrading target pollutants,
mixed waste stream from an oil reﬁnery factory and various
bioaugmentation could effectively remove the refractory organics
petrochemical industries producing dyestuff, chemical fertilizers,
involved in wastewater. Previous studies indicated that bioaug-
calcium carbide, glycol, oxirene, acrylon, synthetic resin and pesti-
mentation was feasible for the treatment of waste streams pro-
cides. The wastewater contains numerous refractory organics such
duced from pharmaceutical factories (),
as petroleum hydrocarbons, benzene hydrocarbons, aniline, nitro-
coke plants (), pulp mills
benzene, phenols as well as their derivatives. These organics are
dye (and other industries.
highly toxic and inhibitory to microbial activity and would lead to
However, those researches on bioaugmentation were limited to
a series of problems, such as poor efﬂuent quality and unstable
lab-scale reactors or target organic substances such as 2-chloro-
operation. Therefore, as both the amount and type of petrochemical
phenol, 2,4-dichorophenol, EDTA and dichloroethene
products increased, the existing anoxic–oxic (A/O) activated sludge
process can not meet the demands of the increasingly complicated
petrochemical wastewater. It is urgent to develop and apply inno-
The efﬁciency of
vative technologies for the proper treatment of petrochemical
the bioaugmentation depends on many factors, which include
Based on the achievements acquired in the pilot study
bioaugmentation was applied in the full-scale petro-
* Corresponding author. Address: School of Municipal and Environmental Engi-
chemical WWTP to improve the existing activated sludge process
neering, Harbin Institute of Technology, Harbin 150090, PR China. Tel.: +86 451
by upgrading it to a contact oxidation process. Successful bioaug-
E-mail address: (F. Ma).
mentation depends mainly on the behavior of the inoculated
0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
F. Ma et al. / Bioresource Technology 100 (2009) 597–602
strains in the environment where they are introduced. Therefore,
the growth rate of the organisms must be higher than decreasingrate of washout and predation (
2.1. Full-scale A/O contact oxidation process
). To avoid this, repeated inoculation of highlycompetent pollutant-degrading specialized bacteria was applied
The parallel biological systems in the petrochemical WWTP were
investigated in the present study. The layouts of S1 (the bioaug-
Although periodic addition could
mented contact oxidation upgrading system) and S2 (the conven-
provide the system with sufﬁcient biomass, it could not justify
tional activated sludge system without bioaugmentation) were
the high cost and complex operation. For this reason, the present
shown in . During our study, another conventional activated
application of bioaugmentation was combined with immobiliza-
sludge system (S3) was shut down for the maintenance and repair
tion technology through the contact oxidation process. It proved
purpose. This S3 system was used to study the comparison of
to be a good solution towards the prevention of the microorgan-
start-up time between the bioaugmented contact oxidation system
isms from being washed out or grazed by other microorganisms
and the conventional activated sludge system without bioaugmen-
such as protozoa (
tation. The schematic diagram of the A/O tank for S1 was presented
Meanwhile, it was
in . The difference of S1 to S2 was the polyurethane foams
proved that the immobilized cells were more efﬁcient than free-
packed within S1. The A/O tank had a size of 60 m 40 m 8 m
living cells. Therefore, the immobilized bacteria required a less
(L W H) and the effective depth of water was 7.2 m. The tank
lag period before the biodegradation could take place (
was made up of ﬁve compartments. The ﬁrst and the fourth com-
). In addition, immobilized
partments (A1 and A2) without aeration facilities acted as anoxic
microorganisms can withstand pH, temperatures and high concen-
tanks. The other three aeration compartments (O1, O2, and O3)
trations of pollutants, which are lethal to free-living cells
packed with polyurethane foams as the carriers were contact oxida-
tion tanks. Agitators and vertical bafﬂes were installed in anoxic
Compared to the previous applications of bioaugmentation
tanks for the adequate mixture of the wastewater and to avoid the
which mainly involved lab-scale systems
accumulation of suspended solids in the biological system. Thus, un-
the present study was unique for
der the same efﬂuent and environmental conditions, the existing
its full-scale biological treatment system with genuine process var-
activated sludge system without bioaugmentation was operated in
iability. The main objectives of this research were: (1) to evaluate
parallel with the bioaugmented contact oxidation upgraded process
the feasibility of bioaugmentation application for the rapid upgrade
with the purpose to investigate their different performances.
of the activated sludge process to the contact oxidation process, (2)to verify the performances of the bioaugmented system, (3) to
2.2. Characteristics of petrochemical wastewater
investigate the differences of the bacterial community structure be-tween the upgrade system and the original system, (4) to explore
Before entering the biological systems, the petrochemical
feasible and reliable strategies for successful bioaugmentaion.
wastewater mentioned above was pretreated by neutralizationand primary sedimentation. The temperature of the wastewaterduring the upgrading phase was 27–32 °C. Characteristics of thepetrochemical wastewater entering the biological system were
2.3. Upgrading procedures
After carriers were installed in the contact oxidation tank, bio-
logical system S1 was bioaugmented with mixed cultures of spe-cialized bacteria targeting to various refractory organics. These
Table 1The characteristics of petrochemical wastewater
Fig. 1. The layout of S1 (A/O contact oxidation process with bioaugmentation and
a Note: Integrated wastewater discharge standard of China
S2 (A/O conventional activated sludge process without bioaugmentaion).
); parameters except for pH are in mg/L.
Fig. 2. The schematic diagram of S1.
F. Ma et al. / Bioresource Technology 100 (2009) 597–602
bacteria, mainly consisting of Pseudomonas, Bacillus, Acinetobacter,
collected directly in the activated sludge form. All these sampling
Flavobacterium and Micrococcus, were enriched from the activated
were performed in a steady operational state. One milliliter of sus-
sludge of various petrochemical WWTP through isolation and
pended samples was washed with 500 lL sodium phosphate and
acclimation. Details for the isolation and acclimation process of
then the mixture was centrifuged at 12,000 rpm for 10 min. Geno-
the specialized bacteria were presented elsewhere (
mic DNA was extracted from the above supernatant by a bacterial
). Meanwhile, certain necessary organic substrates and inor-
Genomic DNA Extraction Kit (TaKaRa, Dalian, China) according to
ganic trace elements were added to stimulate the growth of these
the supplier instructions.
microorganisms. Batch cultivation was adopted in a way that thepartial wastewater in the tank was discharged and fresh petro-
2.5.2. PCR ampliﬁcation
chemical wastewater was introduced. Through this, suspended
The V3 region of 16S rDNA genes were ampliﬁed by using uni-
biomass was washed out to avoid competing with the ﬁxed micro-
versal primers F338GC (5'-CGCCCGCCGCGCGCGGCGGGCGGGGC-
organisms for substrates (
The organic loading rate (OLR) was increased stepwise from 0.04
(5'-ATTACCGCGGCTGCTGG-3'). The ﬁnal PCR mixture (50 lL)
to 0.5 kg COD/m3 d at the end of the upgrading period as the ﬂow
contained 100 ng DNA extract, 2 lL of each primer, 4 lL deoxynu-
rate reached the design value of 700 m3/d. The preliminary cultiva-
cleoside triphosphates, 5 lL 10 PCR buffer (Mg2+ plus), 0.5 lL Taq
tion and acclimation were ﬁnished twelve days later.
polymerase, and 0.5 lL BSA. The touchdown PCR protocol included
Metabolic rate is the amount of energy expended in a given per-
8 min of initial denaturation at 94 °C, 30 cycles of 94 °C for 40 s
iod. Oxygen serves as an electron acceptor in the metabolism of the
(denaturation), 55 °C for 40 s (annealing) and 72 °C for 30 s (exten-
aerobic bacteria. Thus, the metabolic rate of microorganisms in
sion). PCR products were stored at 4 °C and detected by electropho-
each compartment of S1 could be limited through the adjustment
resis on a 2% agarose gel stained with ethidium bromide. All
of DO concentration. Then, unique bacterial community structure
biochemical reagents were purchased from TaKaRa, Dalian, China.
would form in different locations of the biological system (The average DO concentrations in three oxic
2.5.3. DGGE analysis
tanks were 1.45, 2.40, and 6.0 mg/L, respectively.
DGGE was performed on a D-Code Universal Mutation Detec-
tion System (Bio-Rad, Hercules, CA, USA). Five microliter of PCR
2.4. Shock loading experiments
products and 10 lL of 10 loading buffer were loaded onto 8%(w/v) polyacrylamide gels using a denaturing gradient ranging
After continuous ﬂow and steady-state were realized, shock
from 35% denaturant at the top of the gel to 60% denaturant at
loading experiments were carried out to investigate the perfor-
the bottom (100% denaturant contains 7 M urea and 40% (v/v)
mances of bioaugmented system under perturbation conditions.
formamide). Electrophoresis was performed at 60 °C, initially at
The shock loadings were generated by increasing the inﬂow rate
20 V for 30 min and then at 150 V for 9 h. Finally, gels were stained
of the biological system. The corresponding OLR for the system
with SYBR Green 1 and visualized and photographed by a transil-
was elevated and the hydraulic retention time (HRT) of the petro-
lumination scanner. Bacterial community structures were ana-
chemical wastewater was reduced. This suggested that the biolog-
lyzed by visually identifying DNA bands that migrated at
ical system should remove more pollutants during less time.
different distance in each lane on the denaturing gels.
Otherwise, the efﬂuent quality would deteriorate. The experimen-tal design conditions were described in
2.6. Analytical methods
2.5. Bacterial community structure analysis
Efﬂuent from each compartment and inﬂuent from the distribu-
Polymerase chain reaction-denaturing gradient gel electropho-
tion tank were regularly collected for the off-line testing of ammo-
resis (PCR-DGGE) had been developed to analyze bacterial commu-
nia nitrogen (NHþ-N) and chemical oxygen demand (COD)
nity structures without the inherent biases of cultivation (
according to standard methods (
). Thus it becomes one of the most efﬁcient molecular bio-
The DO concentration and temper-
technologies in monitoring the microbial communities of the envi-
ature of the wastewater were measured by a DO sensor. Organic
ronmental samples (Biomass samples were
pollutants contained in the inﬂuent and efﬂuent of S1 and S2 were
collected from each compartment of S1 and S2. The gene fragments
detected by gas chromatography-mass spectrometry (GC–MS) ma-
of mixed bacteria were ﬁrst extracted from the above biomass
chine (GC-6890N/MS-5973N, Agilent, USA). The chromatography
samples and then the V3 region of 16S rRNA was ampliﬁed by
conditions were described elsewhere
polymerase chain reaction (PCR). The PCR products were then ana-lyzed by denaturing gradient gel electrophoresis (DGGE). The spe-
3. Results and discussion
ciﬁc steps were as follows.
3.1. COD and NHþ-N removal efﬁciency at steady-state
2.5.1. Extraction of genomic DNA
Biomass samples of the full-scale contact oxidation process
It took the bioaugmented A/O contact oxidation system (S1) 20
were collected by washing the bioﬁlm attached on the carrier with
days to meet the national discharge standards. For the unbioaug-
sterilized water. The biomass of the activated sludge process was
mented activated sludge system (S3), it required 30 days to reachthe same efﬂuent quality as S1. This demonstrated that bioaug-mentation was a powerful tool to shorten the adaptation time ofthe biological system. As shown in when the COD of the
inﬂuent varied between 320–530 mg/L, the average efﬂuent COD
Shock loading experiments schedule
concentrations were 70 mg/L for S1 and 79 mg/L for S2. Though
Inﬂow rate (m3/h)
OLR (kg COD/m3 d)
the difference was small, it was still quite encouraging considering
the low biodegradability and great quantity of the petrochemical
wastewater. Although the NHþ-N contained in the inﬂuent was
lower than 25 mg/L, the average concentration of NHþ-N in the
F. Ma et al. / Bioresource Technology 100 (2009) 597–602
Fig. 5. PCR-DGGE ﬁngerprints in each stage of S1 and S2.
Fig. 3. Efﬂuent COD and NHþ-N concentration of the bioaugmentd contact
oxidation system (S1) and the activated sludge (S2) without bioaugmentation.
restored only 3 days later when the OLR increased to 0.6 and thento 0.9 kg COD/m3 d. The efﬂuent NHþ-N of S1 was even undetect-
efﬂuent of S2 was 12.4 mg/L. As for S1, despite the generation of
able in the later phase of Test 2. When short-time shock loading oc-
NHþ-N by nitrogen-containing organics, its efﬂuent NHþ-N con-
curred with OLR rising to 1.1 kg COD/m3 d, the efﬂuent quality of
centration was 4.1 mg/L and the average removal efﬁciency was
S1 was only slightly inﬂuenced and still conformed to discharge
72%. Thus, under the same working conditions, the bioaugmented
standards. S1's efﬂuent quality began to improve 24 h later rather
system performed better than the unbioaugmented system, espe-
than ﬁve days later for S2. The average COD removal efﬁciencies of
cially for nitriﬁcation. This may be the action of the bioaugmented
S2 when OLR stayed at 0.6, 0.9 and 1.1 kg COD/m3 d were 76.8%,
specialized bacteria and the formation of the bioﬁlm in the contact
77.8% and 75.6%, respectively, while those of S1 were 80.9%,
oxidation process. Bioﬁlm could retain sufﬁcient slow-growing
81.0% and 77.8%. As for NHþ-N, the conversion efﬁciencies of S1
bacteria with special metabolic capabilities.
were 67.2%, 94.9%, and 69.2% in the three serial tests, which wereobviously higher than S2 with 8.6%, 27.2% and 17.3% conversion
3.2. Shock loading resistant ability
efﬁciencies. Thus, under normal working conditions, the bioaug-menented S1 performed just slightly better than the unbioaug-
As described in the performances of the bioaugmented
mented S2. However, S1 showed better resistance to shock
contact oxidation process (S1) and the activated sludge process
loadings than S2. Thus, S1 was much potential when wastewater
without bioaugmentation (S2) with shock loadings were shown
volume and organic contents increased followed the enhancement
in . Along with the shock loadings, both S1 and S2 suffered
of production or the inevitable accidental wastewater discharge
efﬂuent quality perturbations, whereas the variation of S1 was
much smaller than that of S2, especially for nitriﬁcation efﬁciency.
It took S2 about one week to return to the normal states, while S1
3.3. Degradation and removal to refractory organics
The GC/MS results of the inﬂuent and the efﬂuent from the bio-
augmented contact oxidation process (S1) and the activated sludge
process without bioaugmentation (S2) were presented in .
The number of organics was reduced to 21 in the bioaugmented
system compared to 46 when bioaugmentation was not adopted.
Certain refractory hydrocarbons (including alkanes, alkenes, al-
kynes and aromatic hydrocarbons), ketones, phenols, heterocyclic
compounds, amines were removed in the bioaugmented system.
Organics numbers comparison of inﬂuent and efﬂuent
Fig. 4. Performance of the S1 and S2 during shock loading period.
Note: ‘‘ND" not detected.
F. Ma et al. / Bioresource Technology 100 (2009) 597–602
Contribution of each reactor to the pollutants removal
The results of this work lead to the following conclusions:
(1) Bioaugmentation with specialized bacteria targeted to vari-
ous refractory organics was successful in the full-scale
upgrade to a ﬁve-stage A/O oxidation contact process. For
Note: ‘‘–" more NHþ-N was observed in the efﬂuent compared to the inﬂuent.
the start-up time, the upgraded process spent only 20 days
when its efﬂuent COD and NHþ-N were below 80 mg/L and
10 mg/L, respectively, compared to 30 days for the activated
Although the organics were only a small portion of the total organ-
sludge system. Besides, the rapid upgrade period, the bio-
ic pollutants, they are hazardous if discharged to the environment.
augmented system also proved to be a powerful tool inimproving the degradation efﬁciency of recalcitrant com-
3.4. Contribution of each stage to pollutants removal and bacterial
pounds and the resistance to shock loadings.
(2) Organic pollutants were removed gradually in the bioaug-
mented system, which was un-isochronous with the nitriﬁ-
As petrochemical wastewater passed through each stage of the
cation process due to the diverse bacterial community and
A/O process, pollutants were removed through the combined func-
unique predominant bacteria presented in each stage of
tions of each compartment. By monitoring the steady-state COD
the bioaugmented system. Thus, real temporal and spatial
and NHþ-N concentration of wastewater sampled at each end of
multiple stages were accomplished by the collaborate func-
the stage, the role of each compartment in pollutants removal
tions of the unique bacterial communities formed in each
was investigated. As presented in , for COD removal, the
O1 stage of the bioaugmented system (S1) performed much better
(3) Successful bioaugmentation relies on various factors. Among
than that of the unbioaugmented system (S2), while the O2 stage
these factors, the survival of consortia inoculated into the
of S2 was slightly better than that of S1. However, the overall
system was the most signiﬁcant factor. Possible strategies,
COD removal efﬁciency of S1 was 84.2%, which was higher than
such as the adjustment of DO concentration in the biological
S2 with 74.4%. As for NHþ-N, 19.6% nitriﬁcation efﬁciency was
tank, should be considered to create the optimum opera-
achieved mainly in the O3 stage of S1. In S2, more NHþ-N was con-
tional conditions for the growth and reproduction of the bac-
verted by nitrogen-containing organics. As nitriﬁers failed to per-
teria inoculated. Thus, bioaugementation application is
form their functions, the NHþ-N was accumulated in the former
successful due to the availability of the bioaugmented spe-
four stages of S2. For S1, NHþ-N accumulation appeared in the ﬁrst
two stages, and then it began to decrease in the O2 stage. Most ofNHþ-N was converted in the O3 stage with a 49.1% conversion
From the data presented in , it could be inferred that the
pollutants in S1 were decomposed gradually through cooperative
We gratefully acknowledge the National Basic Research Program
action of each stage, rather than the random behavior of each stage
of China (973 Program) (Granted No. 2004CB418505), the National
contained in S2. It was hypothesized that the specialized bacteria
Natural Science Foundation of China (Granted No. 50778052) and
inoculated in S1 may lead to its different performances from S2.
the Heilongjiang Provincial Science and Technology Development
Therefore, bacterial community analysis was conducted through
Program (Granted No. CC05S301) for their ﬁnancial support.
PCR-DGGE technology to provide evidence for this hypothesis.
The PCR-DGGE ﬁngerprints were presented in It was obviousthat the lanes of samples collected from different locations in S2
appeared in almost the same bands. Thus, no detectable shift ofthe bacterial community was observed in different stages of the
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Código Orgánico Tributario LA ASAMBLEA NACIONAL DE LA REPÚBLICA BOLIVARIANA DE VENEZUELA CÓDIGO ORGÁNICO TRIBUTARIO DISPOSICIONES PRELIMINARES Artículo 1: Las disposiciones de este Código Orgánico son aplicables a los tributos nacionales y a las relaciones jurídicas derivadas de ellos. Para los tributos aduaneros se aplicará en lo atinente a los medios de extinción de las obligaciones, para los recursos administrativos y judiciales, la determinación de intereses y lo referente a las normas para la administración de tales tributos que se indican en este Código; para los demás efectos se aplicará con carácter supletorio.
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