bioaugmented remediation of high concentration btex-contaminated groundwater by permeable reactive...
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Journal of Hazardous Materials 244– 245 (2013) 765– 772
Contents lists available at SciVerse ScienceDirect
Journal of Hazardous Materials
jou rn al h om epage: www.elsev ier .com/ loc ate / jhazmat
ioaugmented remediation of high concentration BTEX-contaminatedroundwater by permeable reactive barrier with immobilized bead
ao-Ping Xina, Chih-Hung Wub, Cheng-Han Wuc, Chi-Wen Linc,∗
Department of Environmental Engineering and Energy, School of Chemical Engineering and Environment, Beijing Institute of Technology, Beijing 100081, PR ChinaGraduate School of Engineering Science and Technology, National Yunlin University of Science and Technology, 123 University Rd., Sec. 3, Douliou, Yunlin 64002, Taiwan, ROCDepartment of Safety, Health and Environmental Engineering, National Yunlin University of Science and Technology, 123 University Rd., Sec. 3, Douliou, Yunlin 64002, Taiwan, ROC
i g h l i g h t s
High concentration BTEX-polluted groundwater was remediated using bioaugmentation process.The actual mineralization rate was 5.8–11.4% depending on the BTEX compound.The low mineralization rate indicated that BTEX bioremediation requires a low oxygen supply.Bioaugmentation-PRB yielded much higher remediation efficiency compared to biostimulation.The CHXY119 & YATO411 immobilized bead has great potential for practical applications.
r t i c l e i n f o
rticle history:eceived 26 August 2012eceived in revised form 2 November 2012ccepted 3 November 2012vailable online 12 November 2012
eywords:TEXigh-concentration contaminated
a b s t r a c t
Ineffective biostimulation requires immediate development of new technologies for remediation ofhigh concentration BTEX-contaminated (benzene, toluene, ethylbenzene and xylene) groundwater. Inthis study, bioaugmentation with Mycobacterium sp. CHXY119 and Pseudomonas sp. YATO411 immo-bilized bead was used to remediate BTEX-contaminated groundwater with about 100 mg l−1 in totalconcentration. The batch test results showed that the CHXY119 and YATO411 immobilized bead com-pletely biodegraded each BTEX compound, and the maximum biodegradation rates were 0.790 mg l−1 h−1
for benzene, 1.113 mg l−1 h−1 for toluene, 0.992 mg l−1 h−1 for ethylbenzene and 0.231 mg l−1 h−1 for p-xylene. The actual mineralization rates were 10.8% for benzene, 10.5% for toluene, 5.8% for ethylbenzene
roundwaterioremediationioaugmentationermeable reactive barriers (PRBs)
and 11.4% for p-xylene, which indicated that the bioremediation of BTEX by the immobilized bead requiresa rather small oxygen supply. Degradation rates achieved by the bioaugmented permeable reactive bar-rier (Bio-PRB) system of the immobilized bead were 97.8% for benzene, 94.2% for toluene, 84.7% forethylbenzene and 87.4% for p-xylene; and the toxicity of the groundwater fell by 91.2% after bioremedi-ation by the bioaugmented PRB, which confirmed its great potential for remediating groundwater withhigh concentrations of contaminants.
. Introduction
The BTEX compounds, namely, benzene (B), toluene (T),thylbenzene (E) and xylenes (X), are often found in soil androundwater [1]. The toxicity effect and water solubility of BTEXndowed these compounds with great environmental hazard [2,3].n many countries, the groundwater was always used as drinking
ater source in which the allowable amounts of the BTEX was veryow due to their serious adverse impact on human health, espe-ially benzene being 0.05 mg l−1 for potable water [4]. Therefore,
∗ Corresponding author. Tel.: +886 5 534 2601x4425;ax: +886 5 531 2069.
E-mail address: [email protected] (C.-W. Lin).
304-3894/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jhazmat.2012.11.007
© 2012 Elsevier B.V. All rights reserved.
remediation of BTEX-contaminated groundwater and soil is a majorconcern worldwide [5].
In recent years, permeable reactive barriers(PRBs) have gradu-ally replaced the conventional pump-and-treat processes used toremediate contaminated groundwater [6]. A major advantage ofPRBs is the cost savings achieved by using the natural flow to bringthe contaminants in contact with the reactive materials withoutinstalling any above-ground facilities or energy inputs [7,8]. Thevarious filling materials proposed for use in groundwater remedi-ation, including zero-valent iron, activated carbon, fly ash, zeolite,waste green sand, and peat depending on the properties of contam-
inants (e.g., organic or inorganic) and reaction mechanisms (e.g.,degradation or adsorption) [9–14].The bioaugmented permeable reactive barriers (Bio-PRBs), inwhich biodegradation by microorganisms is used as a remediation
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echanism, have proven efficient, economical, environmentallyriendly when used for removing xenobiotics or monoaromaticompounds from the contaminated groundwater [5]. Biodegra-ation is also proposed to be the best method for BTEX removal1,5,15]. Although anaerobic biodegradation of organic pollutantss relatively simpler because it does not require an oxygen supply,erobic bioremediation of BTEX contaminated groundwater is stilln attractive option because of its fastness and its complete min-ralization, no generating toxic compounds such as H2S, NO2
– andrganic intermediates [16]. In aerobic Bio-PRBs, oxygen-releasingompounds (ORCs), which mainly consist of CaO2 or MgO2 andemain effective for six months up to one year, continuously andlowly produce O2 in the presence of H2O [17,18].
In situ bioremediation mainly divided into biostimulation andioaugmentation. The former is performed by stimulating activityf the intrinsic microorganisms to biodegrade the organic con-aminants through addition of exogenous oxygen and inorganicutrients [19]; the latter is performed by injecting competentiodegrading microorganisms to accelerate biodegradation ofollutants, in addition to the exogenous oxygen and inorganicutrients [5]. The biostimulation can effectively remediate the
ow concentration BTEX-contaminated groundwater (no more than mg l−1 for each pollutant) to meet the standard level of potableater [9,20,21]; however, when the BTEX compounds present in
roundwater are at much high concentrations such as 25 mg l−1
or each pollutant or above, the biostimulation approach harvestsuch low removal efficiencies that the remaining concentrationsf BTEX do not meet potable water standards [16]. Facing theow performance of biostimulation in treating high concentrationTEX-contaminated groundwater, bioaugmentation process posesotential due to higher bioremediation efficiency [5]. However,here were few reports about bioaugmentation remediation of highoncentration BTEX-contaminated groundwater.
This study applied bioaugmentation technology for in situ reme-iation of the high concentration BTEX-contaminated groundwaterith approximately 100 mg l−1 in total concentration for the first
ime. For evaluating the potential of bioaugmentation in reme-iation of high concentration BTEX-contaminated groundwater,ve aspects studies were conducted, (1) efficiency of immobilizedycobacterium sp. CHXY119 beads for biodegrading high con-
entration BTEX-contaminated groundwater contained in shakingasks; (2) performance improvement in biodegrading high con-entration BTEX-contaminated groundwater by the Mycobacteriump. CHXY119 and Pseudomonas sp. YATO411 immobilized bead inhaking flask; (3) relationship between biodegradation efficiencyf BTEX and rate of oxygen consumed by mixed cultures in immo-ilized beads in batch experiment; (4) performance of the mixedultures immobilized bead for bioremediation of high concentra-ion BTEX-contaminated groundwater in a PRB using ORCs as slowelease source for oxygen supply; (5) toxicity decrease evaluation ofhe BTEX-contaminated groundwater in the PRB using luminescentacteria toxicity test.
. Materials and methods
.1. Chemicals and strains
Benzene was purchased from Echo Chemical (Germany),oluene was obtained from Fisher Chemical (USA), and boththylbenzene and p-xylene were obtained from Tedia Com-any (USA). The BTEX compounds were of HPLC purity, the
emaining compounds were of analytical purity. The two com-etent degrading strains used to bioremediate the contaminatedroundwater, Mycobacterium sp. CHXY119 and Pseudomonasp. YATO411, worked in the form of immobilized beads.aterials 244– 245 (2013) 765– 772
The Pseudomonas sp. YATO411 [22] and Mycobacterium sp.CHXY119 were isolated from sludge after long term acclimationin the laboratory and then identified based on 16S rDNA.
2.2. Strain growth and immobilized bead preparation
Both Mycobacterium sp. CHXY119 and Pseudomonas sp.YATO411 were grown in nutrient broth liquid media in a shaker at25 ◦C at 150 rpm. The biomass was collected by centrifugation whenthe OD600 of media approached 1.0 and then washed three timeswith sterile water to remove the nutrients. The washed biomasswas then dissolved again with the same volume of sterile water toobtain cell suspension with OD600 1.0.
The immobilized bead was prepared with polyvinyl alcohol(PVA) and sodium alginate. Firstly, a colloidal solution was obtainedby dissolving the 9% PVA and 0.9% sodium alginate (w/v) in deion-ized water in a sterilizer at 120 ◦C. Secondly, the colloidal solutioncooled to about 25 ◦C of room temperature was mixed uniformlyeither with the Mycobacterium sp. CHXY119 cell suspension ofOD600 1.0 at a rate of 10% (v/v) to prepare the sole Mycobacteriumsp. CHXY119 immobilized bead or with both Mycobacterium sp.CHXY119 and Pseudomonas sp. YATO411 cell suspensions at a rateof 5% to prepare the Mycobacterium sp. CHXY119 and Pseudomonassp. YATO411 immobilized bead. Thirdly, the cell-contained col-loidal solution was slowly injected through a tip driven by aperistaltic pump into a curing solution containing 2.5% CaCl2 and 5%H3BO3 in w/v. The white cell-immobilized bead then formed after1–2 h of immersion. Finally, the formed cell-immobilized bead waswashed three times with sterile water, followed by immersion of6 h in 5% (w/v) KH2PO4 to promote the mechanical strength.
2.3. Simulated BTEX-contaminated groundwater
The simulated BTEX-contaminated groundwater was obtainedby adding required volume of benzene, toluene, ethylbenzeneand p-xylene into the inorganic salts solution contains: K2HPO4,1750 mg; KH2PO4, 2145 mg; MgSO4·7H2O, 100 mg; (NH4)2SO4,10 mg; FeSO4·7H2O, 1 mg; CaCl2·2H2O, 45 mg; CuCl2·2H2O,0.25 mg; CoCl2·6H2O, 0.25 mg; ZnSO4·7H2O, 1 mg; MnCl2·4H2O,1 mg; Na2MoO4·2H2O, 0.1 mg; NiCl2·6H2O, 0.02 mg; deionizedwater, 1000 ml. The total concentration of added BTEX was about100 mg l−1, in approximately equal weights of each compound.
2.4. Biodegradation of BTEX by immobilized bead in batch tests
Different amounts of cell-immobilized bead (1, 5, 10, 15and 20 g) were placed in 250 ml sealed brown flasks contain-ing 100 ml of oxygen-saturated (about 7–8 mg l−1 of dissolvedO2) simulated BTEX-contaminated groundwater with total dose of95 mg l−1 as sole carbon sources, in which 24.68 mg l−1 for ben-zene, 23.67 mg l−1 for toluene, 21.97 mg l−1 for ethylbenzene and24.68 mg l−1 for p-xylene. The immobilized bead-inoculated flaskswere incubated in a shaker at 25 ◦C at 150 rpm. In the course ofbiodegradation of BTEX, the headspace gas of flasks was sampledperiodically for gas chromatography with flame ionization analysis(GC-FID, GC-14B, Shimadzu, Japan) to measure the remaining con-centration of BTEX in the contaminated groundwater. The controlsoccurred without any inoculation.
2.5. Relationship between oxygen uptake and biodegradation ofBTEX
Fifty grams of cell-immobilized beads containing both Mycobac-terium sp. CHXY119 and Pseudomonas sp. YATO411 was inoculatedinto spherical bottles containing simulated contaminated ground-water consisting of 1000 ml of oxygen-saturated (about 7–8 mg l−1
B.-P. Xin et al. / Journal of Hazardous Materials 244– 245 (2013) 765– 772 767
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Fig. 1. Schematic diagram of an ox
f dissolved oxygen (DO)) water containing 20 mg l−1 of benzene,oluene, ethylbenzene or p-xylene. The inoculated bottles werencubated at 25 ◦C for reaction. During biodegradation, the DO ofhe groundwater was monitored with a portable DO metre every
min, and the remaining dose of pollutants was measured every h by GC-FID analysis of the headspace gas until DO value almosttabilized. The oxygen uptake rate for BTEX biodegradation wasalculated as follows:
xygen-uptake rate (OUR, mg · O2 l−1 h−1) = DO1 − DO2
t1 − t2(1)
here “DO1” is the DO concentration at time t1 (mg l−1), “DO2” ishe DO concentration at time t2 (mg l−1), and t (min) is measuringime.
.6. Peformance of immobilized mixed cultures in remediatingTEX-contaminated groundwater when using ORCs as releaseource for oxygen supply in a PRB
The PRB system used in the present studies was a slight mod-fication of the previous one [16]. The schematic diagram of the
odified PRB was shown in Fig. 1. Two rows of bioremediationells (#2, #3) as modification for storing the cell-immobilized beadere located behind the oxygen-releasing barrier. The total volume
f the PRB was 15.6 litter (52 cm × 15 cm × 20 cm, L × W × H); that ofhe oxygen-releasing barrier was 1.2 litter (4 cm × 15 cm × 20 cm,
× W × H) caging 800 g of ORCs consisting of 40% (w/w) CaO2 asell as cement, sand, KH2PO4, K2HPO4, and so on; that of biore-ediation wells was 1.133 litter (4 cm × 15 cm × 6, ϕ×H) holding
50 g of cell-immobilized beads. The rest of the tank was packedith 25 kg Ottawa standard sand, which resulted in a porosity value
f 0.388. The PRB tank was maintained at 25 ◦C by a water cir-ulation system outside the tank. A syringe pump was used for
ontinuous injection of the simulated BTEX-contaminated ground-ater containing 28.89 mg l−1 B, 29.05 mg l−1 T, 28.33 mg l−1 End 28.89 mg l−1 X into the PRB system. The inflow was set at.45 ml min−1; flow velocity was 50 cm d−1; hydraulic residence
releasing reactive barrier system.
time was 1.415 d; hydraulic conductivity was 2.9 × 10−2 cm s−1.The ORCs were replaced as needed. During bioremediation, both theinlet (#1) and outlet (#4) were sampled periodically to measure thechange in BTEX concentrations and to calculate the bioremediationefficiencies of the PRB system.
2.7. SEM analysis of cell-immobilized bead before and afterbioremediation
The scanning electron microscope (SEM) samples were pre-pared as described by Sastry et al. [23] and analyzed with a HitachiSU1510 Natural Scanning Electron Microscope.
2.8. PCR-DGGE analysis of the microbial community duringbioremediation
Two millilitre of outlet samples (#4) of the PRB were takenperiodically, and then the DNA extraction, PCR (polymerase chainreaction) amplication and DGGE (denaturing gradient gel elec-trophoresis) assay were performed as described in the previouspaper [16].
2.9. Toxicity analysis of the groundwater in the process ofbioremediation
At day 35 of the stable duration of the second stage, 2 ml ofgroundwater was sampled from #1 (inlet), #2, #3 and #4 (outlet)sites of the PRB, respectively. The solutions were analyzed usingthe Freshwater Luminescent Bacterium Vibrio fischeri sp. (Photo-
bacterium phosphoreum, NRRL number B-11177) for toxicity testbased on the method described by Ma et al. [24]. The dilution fac-tor for 50% of luminescent inhibition as EC50 value was obtainedfor reflecting the toxicity of liquid sample.
768 B.-P. Xin et al. / Journal of Hazardous Materials 244– 245 (2013) 765– 772
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F rium s5
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ig. 2. Biodegradation of BTEX serving as the sole carbon source by the Mycobacte g (©); 10 g (�); 15 g (�); 20 g (�); Blank (�).
. Results and discussion
.1. Biodegradation of BTEX by CHXY119 immobilized bead inatch test
Fig. 2 presents the changing concentrations of benzene, toluene,thylbenzene and p-xylene under different doses of immobi-ized Mycobacterium sp. CHXY119 as a function of incubationime. It was clear that benzene, toluene, ethylbenzene and-xylene exhibited different biodegradation efficiencies by immo-ilized Mycobacterium sp. CHXY119. Ethylbenzene was completelyemoved after 120 h of incubation, and the average biodegradationate was 0.239 mg l−1 h−1; p-xylene reached 100% biodegradationfter 210 h of contact, and the average biodegradation rate was.119 mg l−1 h−1. Moreover, there was no lag of biodegradation foroth ethylbenzene and p-xylene, i.e., biodegradation started as soons the pollutants came in contact with the cells. In contrast, toluenexhibited a biodegradation lag of about 80 h, followed by completeemoval after 400 h of contact with the immobilized Mycobacteriump. CHXY119 at an average biodegradation rate of 0.087 mg l−1 h−1.mmobilized Mycobacterium sp. CHXY119 had the worst efficiencyor removing benzene, which is the most serious threat to humanealth among the BTEX compounds and has a minimum permit-ed dose of 0.005 mg l−1 in drinking water. Only 5% of benzene wasemoved after 400 h of incubation.
Although different BTEX pollutants showed different removalynamics, the doses of the immobilized Mycobacterium sp.HXY119 had almost no effect on removal efficiency (Fig. 2),uggesting the treatment characteristic of the immobilized bead.he immobilized bead removed BTEX in two separate steps, i.e.,ransport from the outside of the bead to the inside and theniodegradation by the immobilized cells. In general, biodegrada-ion processes were relatively fast ones due to the high activityf the immobilized cells [25]; whereas the pollutant transporta-
ion processes relying on the thickness and structure of the beadsere slower ones as the rate-limiting step. Therefore, even loweroses of immobilized cells were effective for bioremediation ofTEX-contaminated groundwater.p. CHXY119 immobilized bead under different doses of bead at batch test. 1 g (�);
3.2. Improving biodegradation of BTEX by Mycobacterium sp.CHXY119 and Pseudomonas sp. YATO411 immobilized bead inshaking flasks
Although the Mycobacterium sp. CHXY119 immobilized beadcompletely removed ethylbenzene, p-xylene and toluene, thebiodegradation rates were rather low, leading to longer time forbioremediation. An even worse problem was the failure of immobi-lized Mycobacterium sp. CHXY119 to remove benzene, which is themost dangerous BTEX pollutant. To improve removal performance,Pseudomonas sp. YATO411, which is effective for biodegradingtoluene [22], was combined with Mycobacterium sp. CHXY119 in aMycobacterium sp. CHXY119 and Pseudomonas sp. YATO411 immo-bilization bead for biodegrading BTEX. Fig. 3 shows the variation inthe remaining doses of B, T, E and X under different concentrationsof the immobilized Mycobacterium sp. CHXY119 and Pseudomonassp. YATO411 as a function of incubation time. The combination ofPseudomonas sp. YATO411 and Mycobacterium sp. CHXY119 greatlypromoted BTEX biodegradation under limited O2 supply (Fig. 4).Notably, benzene, the most dangerous BTEX pollutant, was com-pletely removed by the mixed culture after 66 h of contact withoutany lag at bead dose of 15 g or higher; the biodegradation rate ofbenzene reached 0.790 mg l−1 h−1, being 3949.5 times as high asthe sole Mycobacterium sp. CHXY119 which yielded only about 5%removal efficiency after 400 h of incubation. As for toluene, the useof the mixed culture bead eliminated the lag phase of ca. 80 h thatoccurs when Mycobacterium sp. CHXY119 is used alone. Toluenealso shortened the overall biodegradation time from 400 to 24 h;meanwhile, the average biodegradation rate of toluene increased12.8-fold from 0.087 to 1.113 mg l−1 h−1. Similarly, the mixed cul-ture bead also decreased the time needed to achieve 100% removalof ethylbenzene from 120 to 24 h, and the average biodegrada-tion rate of 0.992 mg l−1 h−1 was 3.2 times higher than that ofMycobacterium sp. CHXY119 alone. In contrast with toluene and
ethylbenzene, a low bead dose of 10 g or lower required more than200 h for complete removal of p-xylene whereas a high bead dose of15 g or higher reduced the time needed for 100% biodegradation to120–150 h. The average biodegradation rate was 0.95 times faster
B.-P. Xin et al. / Journal of Hazardous Materials 244– 245 (2013) 765– 772 769
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Fig. 3. Biodegradation of BTEX serving as the sole carbon source by the Mycobacterium sp. CHXY119 and Pseudomonas sp. YATO411 immobilized beads under different dosesof bead at batch test. 1 g (�); 5 g (©); 10 g (�); 15 g (�); 20 g (�); Blank (�).
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ig. 4. Comparison of biodegradation rate between the sole Mycobacterium sp. CHp. YATO411 beads under different doses of bead at batch test. (a) the sole Mycobaseudomonas sp. YATO411 beads. Benzene ( ); toluene ( ); ethylben
han Mycobacterium sp. CHXY119 alone. These experimental resultshowed that the Mycobacterium sp. CHXY119 and Pseudomonas sp.ATO411 immobilized bead had much higher efficiency in biore-ediation of BTEX compared to Mycobacterium sp. CHXY119 alone,
nd the lower dose of mixed culture bead was still competentor BTEX removal despite the longer time required for completeiodegradation.
.3. Relationship between biodegradation efficiency of BTEX andxygen consumption rate by the immobilized mixed cultures inatch experiment
Because supply of oxygen was crucial for the aerobic bioreme-iation of contaminated groundwater [26], further investigationsere performed to elucidate the detailed relationships between
iodegradation efficiencies and oxygen consumption rates. Fig. 5
resents the time courses of BTEX removal and oxygen consump-ion rates. It was observed that for each pollutant of BTEX, thereas almost no oxygen utilization in the early stage of severalours, followed by growing oxygen consumption until that oxygen9 immobilized bead and combined Mycobacterium sp. CHXY119 and Pseudomonasm sp. CHXY119 immobilized bead; (b) combined Mycobacterium sp. CHXY119 and
); p-xylene ( ).
consumption rate reached the maximum, and then it sharplydeclined to zero again, even though that dissolved oxygen wasnot depleted. Accordingly, the BTEX concentration also remainedunchanged for early several hours, followed by active biodegra-dation up to zero. The experimental results confirmed that BTEXbiodegradation is highly dependent on oxygen consumption.
The maximum oxygen uptake rate was 0.9 mg-O2 l−1 h−1
for benzene, 1.38 mg-O2 l−1 h−1 for toluene, 0.62 mg-O2 l−1 h−1
for ethylbenzene and 0.96 mg-O2 l−1 h−1 for p-xylene. The totalamount of oxygen consumption was 6.65 mg l−1 for ben-zene, 6.58 mg l−1 for toluene, 3.66 mg l−1 for ethylbenzene and7.22 mg l−1 for p-xylene. The theoretical oxygen consumptionrequired for complete oxidation of the BTEX at 20 mg l−1 was61.6 mg l−1 for benzene, 62.6 mg l−1 for toluene, 63.4 mg l−1 forethylbenzene and 63.4 mg l−1 for p-xylene based on the equationsas follows [27].
C6H6(benzene) + 7.5O2 → 6CO2 + 3H2O (2)
C7H8(toluene) + 9O2 → 7CO2 + 4H2O (3)
770 B.-P. Xin et al. / Journal of Hazardous Materials 244– 245 (2013) 765– 772
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Fig. 6. The biodegradation of BTEX and change in DO with working time by theMycobacterium sp. CHXY119 and Pseudomonas sp. YATO411 immobilized bead whenusing ORCs as oxygen source in a PRB system with continuous flow at 3.45 ml min−1.Part I, without both cell beads and ORCs; Part II, with both cell beads and ORCs; Part
ig. 5. The relationship between the biodegradation of BTEX and the oxygen uptake read at batch test. (�) BTEX remaining concentration (mg l−1); (©) oxygen uptake
8H10(ethylbenzene) + 10.5O2 → 8CO2 + 5H2O (4)
8H10(xylene) + 10.5O2 → 8CO2 + 5H2O (5)
By comparing the actual oxygen consumption amounts with theheoretical ones for complete oxidation, the actual oxidation ratef the BTEX was only 10.8% for benzene, 10.5% for toluene, 5.8% forthylbenzene and 11.4% for p-xylene, respectively. Because BTEXas used as the sole carbon source in the mixed culture immo-
ilized bead, a certain proportion of pollutants, approximately aswice as much as the mineralized percent, were assimilated to formiomass [28]; so, the consumption rate of the BTEX stemming fromxidation and assimilation was about 32.4% for benzene, 31.5% foroluene, 17.4% for ethylbenzene and 34.2% for p-xylene, respec-ively. It was speculated that the ring cleavage of the BTEX occurredo meet so high consumption rates; however, the remaining whichept in the solution as metabolism products was needed to beurther identified. The very low oxidation rate meant that a veryimited oxygen supply could meet the biodegradation of much
ore amount of BTEX, with the BTEX transforming into harmlessr low toxic metabolism products by ring cleavage under aerobicondition. The biodegradation characteristic of the mixed culturemmobilized bead was advantageous for bioremediation of theTEX-contaminated groundwater due to the low required amountf oxygen.
.4. Bioremediation performance of high concentrationontaminated groundwater by the immobilized mixed culturessing ORCs as oxygen source in a PRB
Performance tests of the Mycobacterium sp. CHXY119 andseudomonas sp. YATO411 immobilized beads to determineheir efficiency for in situ bioremediation of BTEX-contaminatedroundwater in a PRB were needed to evaluate their potentialpplications in the field. Fig. 6 shows the time course of BTEXemoval and DO change in a PRB using ORCs as slow release source
or oxygen supply. It was found that during the first stage fromay 0 to day 7, no degradation of BTEX occurred in the absencef the Mycobacterium sp. CHXY119 and Pseudomonas sp. YATO411mmobilized bead. In the second stage from day 8 to day 47, whichIII, replacement of the spent ORCs with the fresh one; Part IV, removal of the cellbeads. Benzene (�), toluene (�), ethylbenzene (�) and p-xylene (�); DO of site #2(©) and #4 (�).
ous Materials 244– 245 (2013) 765– 772 771
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Table 1Remaining BTEX concentration and toxicity of groundwater at different bioremedi-ation degree from inlet to outlet at day 35 of the stable duration of the second stageinitiated by addition of both the immobilized bead and ORC.
Item #1 (inlet) #2 #3 #4 (outlet)
Benzene (mg l−1) 26.44 3.07 1.25 0.71Toluene (mg −1) 26.59 3.57 1.88 1.14Ethylbenzene (mg l−1) 25.93 4.05 2.27 1.44Xylene (mg l−1) 26.44 4.12 2.69 2.07
B.-P. Xin et al. / Journal of Hazard
as initiated by addition of both the immobilized bead and ORCs;or each pollutant of the BTEX, biodegradation efficiency graduallyncreased up to the maximum because of continuous growthf Mycobacterium sp. CHXY119 and Pseudomonas sp. YATO411,ollowed by a rather stable duration of bioremediation efficiencywing to the maximum biomass of Mycobacterium sp. CHXY119nd Pseudomonas sp. YATO411, and then a continuous decline iniodegradation efficiency occurred due to the shortage of oxygenupply and slow accumulation of intrinsic microorganisms. Inhe third stage from days 48 to 72, which was initiated by aeplacement of spent ORCs with the fresh ones, a similar pattern ofTEX biodegradation with the second stage was observed excepthat biodegradation efficiency was lower. Although the fresh ORCsupplied enough oxygen, the strong accumulation of intrinsicicroorganisms adversely affected the growth and activity of theycobacterium sp. CHXY119 and Pseudomonas sp. YATO411 immo-
ilized bead, leading to the lower remediation efficiency. In theourth stage from days 73 to 86, which was initiated by a removalf the immobilized bead, biodegradation efficiencies were lowerhan those in the third stage. The results showed that the Mycobac-erium sp. CHXY119 and Pseudomonas sp. YATO411 was requiredor the higher remediation efficiency, although the intrinsic
icroorganisms also had remediation ability to some degree.In the batch experiments, the Mycobacterium sp. CHXY119 and
seudomonas sp. YATO411 immobilized bead possessed very highioremediation ability towards BTEX and almost 100% of removalas attained for each pollutant of the BTEX. In contrast, the same
mmobilized bead in the PRB system removed 97.8% of benzene,4.2% of toluene, 84.7% of ethylbenzene and 87.4% of p-xylene
n the stable duration of the second stage from days 29 to 39. partial explanation could be the continuous treatment by PRBrocess. However, further study is needed. As for the difference
n biodegradation efficiency with different pollutants in the sametage and with different stages of the PRB system, gradually accu-ulated intrinsic microorganisms might play an important role
or it, although the detailed mechanism was still unknown. How-ver, despite the lower biodegradation efficiency of the BTEX inhe PRB system compared with the batch experiments, the bioaug-
entation process based on the Mycobacterium sp. CHXY119 andseudomonas sp. YATO411 immobilized bead indeed exhibiteduch higher bioremediation capacity compared to biostimulationhen facing the high-concentration contaminated groundwater.y addition of exogenous oxygen and inorganic nutrients to stim-late the activity of the intrinsic microorganisms, biostimulationchieved only 60% of biodegradation efficiency for benzene, 80%or toluene, 86% for ethylbenzene, and 78% for p-xylene [16]. Anfficiency increase of 37.8% for benzene, 14.2% for toluene and 9.4%or p-xylene was achieved using the bioaugmentation PRB pro-ess except for a slight decline of 1.3% for ethylbenzene. The highoncentration BTEX-contaminated groundwater around the worldighlighted the importance of the present studies, although thereere still a lot of works to do for further improving the bioremedi-
tion efficiency of the PRB system based on the Mycobacterium sp.HXY119 and Pseudomonas sp. YATO411 immobilized bead.
.5. SEM analysis of cell-immobilized bead before and afterioremediation
The immobilized bead collected from the end of the third stagen PRB was analyzed by SEM and compared with the raw beadFig. S1 in the Supplementary Material). After a long time of biore-
ediation, the cells inside the bead remained in a normal form,
uggesting that the mixed cells might still possessed the biodegra-ation ability towards the BTEX and played an important rolen bioremediation of groundwater even in the end of the thirdtage. However, the number of cells inside the bead was almost
Dilution factor for EC50 ofluminescent inhibition
38.6 ± 1.3 28.6 ± 3.2 25.4 ± 1.8 3.4 ± 1.5
unchanged compared with the raw one, indicating that the accu-mulation of the intrinsic microorganisms adversely affected thegrowth of the Mycobacterium sp. CHXY119 and Pseudomonas sp.YATO411 and thereby resulted in an evident decline in bioremedi-ation efficiency with increase of working time.
3.6. PCR-DGGE analysis of the microbial community duringbioremediation
To understand further whether the Mycobacterium sp. CHXY119and Pseudomonas sp. YATO411 existed or not in the PRB system atthe third stage from days 48 to 72 initiated by a replacement ofspent ORCs with the fresh ones, the variation of microbial commu-nities over the whole third stage was examined using PCR-DGGE(Fig. S2 in the Supplementary Material). The band d representingMycobacterium sp. CHXY119 and band e representing Pseudomonassp. YATO411 were found through the third stage, demonstratingthat Mycobacterium sp. CHXY119 and Pseudomonas sp. YATO411still played important role in bioremediation of the groundwatereven at the later stage. Meanwhile, the intrinsic microorganismsband a, b, c, f, g, h, i and j accumulated during remediation, resultingin decreasing in BTEX removal efficiency.
3.7. Toxicity analysis of the groundwater in the process ofbioremediation
With the decrease in BTEX concentration from inlet (#1) to out-let (#4) of the PRB because of biodegradation by Mycobacterium sp.CHXY119 and Pseudomonas sp. YATO411 immobilized beads, thedilution factor for 50% of luminescence inhibition decreased from38.6 to 3.4, toxicity fell by 91.2% (Table 1). Although complete tox-icity removal was not achieved after bioremediation, the 91.2% oftoxicity removal exhibited the great potential of the bioaugmenta-tion process by Mycobacterium sp. CHXY119 and Pseudomonas sp.YATO411 immobilized bead for remediation of the high concentra-tion BTEX-contaminated groundwater.
4. Conclusion
The Mycobacterium sp. CHXY119 and Pseudomonas sp. YATO411immobilized bead harvested almost 100% of removal for the BTEXin batch test. The actual oxidation rate was 10.8% for benzene,10.5% for toluene, 5.8% for ethylbenzene and 11.4% for p-xylene.The very low mineralization rate meant that a limited oxygen sup-ply could meet the biodegradation of much more amount of BTEX.The bioaugmentation PRB process achieved an efficiency increaseof 37.8% for benzene, 14.2% for toluene and 9.4% for p-xylene com-pared with biostimulation. The toxicity of the groundwater fell by91.2% after remediation by the bioaugmentation. The bioaugmen-
tation PRB of the Mycobacterium sp. CHXY119 and Pseudomonas sp.YATO411 immobilized bead exhibited great potential for remedi-ating high-concentration contaminated groundwater.
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72 B.-P. Xin et al. / Journal of Hazard
cknowledgements
The support from the National Science Council, Taiwan, ROCNSC 96-2221-E-224-093-MY3) is gratefully acknowledged. Theuthors wish to thank Ms. Mei-Shan Wang of our laboratoryGraduate student at National Yunlin University of Science andechnology) for data collection. Ted Knoy is also appreciated foris editorial assistance.
ppendix A. Supplementary data
Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.jhazmat.012.11.007.
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