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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 Bioresource Technology 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.
Petrochemical wastewater 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.
doi:10.1016/j.biortech.2008.06.066 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 Heterocyclic compounds 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- NHþ-N conversion 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.
community analysis (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 cialized consortia.
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.
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