e> Pergamon . War. Sci. Tech. Vol. 34,.No. S~, pp. 335-344,1996.Copynghl Ci 1996 IAWQ. Published byElsevier Science lId

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ANAEROBIC DEGRADATION OFPENTACHLOROPHENOL (PCP) INBIOLOGICAL EXPANDED-BED REACTOR

Hiroshi Tsuno, Masasumi Kawamura and Isao Somiya

D~paTfmenJofEnvironm~ntaJ Engineering. KyotoUniversity. Yoshida-Honmacht,Sakyo-ku; Kyoto 606-01, Japan

ABSTRACT

In this study, anaerobicdegradationof PCP was discussed in an expanded-bed GAC anaerobic reactor whichwas applied to treatment of wastewatercontaining high concentration of PCP-Na as well as acetate. Total­COD concentration in effluent was kept less than 100 mgIL except for the start-up duration and PCP-Naconcentration was kept less than 0.50 mgIL under the experimentalconditions (HRT of 5 days, influent PCP­Na concentrationof 100and 400 mgIL,and influentTotal-CODconcentration of 480 and 1600mgIL).1t wasindicated by methaneproduction rate and material balanceof PCP-Na that about 60% of PCP-Na loaded intothe reactor was transformed 10 methane and CO2 for 375 days. GClMS analysis of extracts of the GACmedium and effluent indicated that reductive dechlorination of pcp occurred and dechlorinatedphenols wereadsorbed on the GAC in the reactor. Microorganisms collected from the reactor degraded PCP-Na and 2-CPanaerobically and addition of acetate in culture accelerated anaerobic degradation of PCP-Na in batchexperiments.Copyrighte 1996IAWQ. Publishedby ElsevierScience Ltd.

KEyWORDS

Anaerobic treatment; activated carbon ; biodegradation; biological inhibition; chlorinated organics;expanded-bed reactor; pentachlorophenol.

INTRODucnON

Pentachlorophenol (PCP) is widely used as wood preserving-agent. biocide for agricultural purposes and in aVariety of other applications. Large quantiti,es of P~P are also p~od,uced from bleaching process withchlorine gas in pulp and paper industry, PCP IS an ~nv1ronmentally sl,8ID,fi~ant chemical because of its toxicproperties. The toxicity acts on a variety of organisms as a po!ent ,mhibltor ?f oxidative phosphorylation(Mikesell and Boyd. 1986). Though PCP has tx:n shown to resist biodegradation, several pathways for themicrobial degradation of PCP have been [dentifled. These pa~~~ys are pcp methylation, reductive orOxidative dehalogenation and ring cleavage (Kudo, .1989). Possibility ?f anaerobic biodegradation of PCPWas shown by the observation that an accumulatton o~ lesser c~lonnated phenols occurred with PCPdisappearance in anaerobic soil (Guthrie et al., 1984) and to anaer~blc sewage sl.udge (Larssen et al.• 1991).Reductive dechlorination. or direct removal of Cl atoms from the nng of aromatic compounds at a first stepis a significant process. because the dechlori~ated products are usually less toxic and are more readilydegraded either anaerobically or aerobically (Mikesell and Boyd. 1986).

335

336 H. TSUNOet al.

The expanded-bed anaerobic reactor with granular activated carbon (GAC) medium has been developed totreat wastewaters which contain high concentrations of inhibitory and/or refractory organic compounds aswell as biodegradable organic compounds (Wang et al., 1986; Gardner et al., 1988; Suidan et al., 1988;Tsuno et al., 1993). The process is characterized by a combination of physical and biological removalmechanisms ; adsorption onto GAC and biological degradation by microorganisms grown on GAC. Theadsorptive function of GAC contributes towards the reduction of the aqueous phase concentration of theinhibitory organic compound to below threshold level under which the inhibitory effect on the biologicaldegradation decreases. GAC has also been shown to be a preferred medium for the expanded-bed reactorbecause of its rough and creviced surface which helps the attachment and growth of microorganisms (Fox,1989).

In this study, the expanded-bed GAC anaerobic reactor was applied to treatment of wastewater whichcontains high concentration of PCP as well as acetate. Possibility of biodegradation of PCP is discussedfrom methane production rate, material balance, detection of intermediates of PCP biodegradation inextracted solution from GAC medium and effluent, and batch experiments on degradation of PCP and 2-CPwith microorganisms collected from the reactor.

MATERIALS ANDMETHODS

Schematic diagram of experimental apparatus used in this study is shown in Fig. I. The reactor is made of athick Plexiglass tube which has a diameter of 10 em and a volume of 10 L. GAC of 0.9 to 1.1 mm particlesize was put into the reactor at 1500 g. The liquid in the reactor was circulated from the top part to thebottom of the reactor to expand the GAC medium at 25% expansion. Synthetic wastewater composed ofPCP and acetate as well as nutrients and minerals, as shown in Table I, was fed into the circulation line at agiven flow rate. Water temperature in the reactor was controlled at 30 by the jacket installed around thereactor. At the start of the experiment, 100 mL of sludge obtained from an anaerobic digestion tank in amunicipal sewage treatment plant in Kyoto City was inoculated into the reactor.

10cmH

Organic NutrientsSubstrates

RecirculationPump

Figure I. Schematic diagramof experimental apparatus.

Anaerobicdegradation of pentachlorophenol 337

Table 1. Composition of synthetic wastewater

OrganicsC6CIsONa (PCP-Nal

CH3COONa

Nutrients

K2HP04

KH2P04

MgCI2 - 6H20

CaCI2 - 2H20

Vitamins and Minerals

Tap Water

100 mg

500 mg

0.348 9

0.227 9

0.41 9

0.25 9

trace

L

Operational conditions are summarized in Table 2. Hydraulic retention time (HRT) was set at 5 days duringthe experiment. Concentrations of PCP-Na and sodium acetate were set at 100 and 500 mgIL, respectively,at the start-up duration, and their concentrations were increased to 4 times of the initial concentrations onday 83 of the experiment. With this change. COD loading was increased from 0.64 to 2.3 g-COD/(kg­GACeday).

During the experiment. water samples were taken at inlet and outlet of the reactor once a week. and analyzedfor PCP-Na, COD and dissolved organic carbon (DOC). pH and ORP of the liquor in the reactor were alsochecked. Gas production rate was measured every day by a gas meter, and methane production rate wasCalculated from the data with the composition of the gas produced. PCP was determined by ultravioletabsorption method (Crosby. 1981) after filtration of sample. The other items were determined by the methoddeScribed in the STANDARD METHOD.

Before the experiment of continuous treatment. the adsorptive characteristic of PCP-Na on GAC was~hecked. A given weight GAC (l-5g) was added to I L of each solution containing 101,000 mg PCP-NaILIn a I L beaker. These beakers were stirred by magnetic stirrer with 100 rpm at 30·C. PCP-Na in eachsolution was traced over 100 hours. peP-Na concentration was determined by ultraviolet absorption method(Crosby. 1981) after filtration of sample with 0.5 I.lJIl pore size filter. Every adsorption experiment had aControl in which no GAC was added to the solution.

Table 2. Operational conditions for anaerobic GAC reactor

Run No. I ITTlme,days 0--------- 83---------375

Flow Rale.L/day 2 2

HRT,days 5 5

(2.2) (2.2)

400

2000

100

500

0.10 0.34

(0.23) (0.79)

g/(kgGAC-day) 064 2.3

Inlluent.mg/L

PCP-Na

CH,COONa

CODer Loading

kg/(m'- day)

E.xtraction of chlorophenols from the GAC medium was carried out with Soxhlet extractor usingdlchIoromethane as solvent. GAC of 5 g (wet weight) obtained from sampling port of the reactor on 353 dayOfthe experiment was taken into Soxhlet extractor and 100 ml of dichloromethane was added. Extraction\V.as continued for 3 hours at 50'C and cycle rate of 20 timeslh. Then dichloromethane phase was collected.liqUid-liquid extraction method was used for extraction of chlorophenols from the effluent of the reactor.l'he effluent sampled on 353 day of the experiment was filtrated using membrane filter (pore size of 0.45

338 H. TSUNOet al.

um). The filtrateof 500 mLwas takeninto a separatory funnelof I L volume, and 50 ml of dichloromethaneand a few drops of IN HCl were added. The separatory funnel was shaken for IS minutes at reciprocatingrate of 250 cycles/min. Then dichloromethane phase was collected. This procedure was repeatedtwo timesand dichloromethane phases were put together. Both extracts were analyzed by GC/MS after dehydrationwithsodiumsulfate. Analytical conditions of GC/MSmeasurement are shownin Table 3.

Table 3. Analytical conditionsof GC/MS measurement

Column : 2,100 mm X 2.6 1.0. mm Glass SpiralStationary Phase, OV-17 (2%)Support, Chromosorb W 80-100 mesh

Acid washedSlIanlzedTemperature; Range 50-250 "C, rate 10"C/mln

Hold for 20 min at 250 "CInjection 250 "CDetector 250 "C

Carrier gas : He, 40 mLlmlnMass Spectrum : EI, 70eV

Scan rate, 1 tlme/3 secScan range, 40-500 rnIe

Batch experiments on anaerobic degradation of PCP and 2-CP were carried out using microorganismscollected from the GAC medium in the reactor on 372 day of the experiment. 50 g (wet weight) of GACtaken from the reactor was stirred with 100 mL nutrient solutionjust like influent in a flask by magneticstirrer with 100rpm under anaerobic conditionfor I hour. And after IS minutesof stationary sedimentation.supernatant was collected anaerobically. This supernatant was used as a microorganisms mixed liquor.Solutioncontaining 10 mgIL of PCP-Na (or 30 mgIL of ~-CP) and 50 mgIL of sodium acetate as well asnutrients just like the influentcomposition was prepared. The solution of 90 ml was taken into a few vialsof100mLvolume. 10ml of the collectedmicroorganisms mixed liquorwas added into each vial with flushingof nitrogengas and the vial was immediately sealed with siliconestopperand aluminum cap. Control vialswere prepared by adding 10 mL of distilled water instead of microorganisms mixed liquor. All thisprocedure was performed anaerobically and aseptically. All vials was incubated on orbital shaker at 30·Cand 120rpm. All mixed liquor of each vial was taken out periodically and centrifuged at 10,000rpm for 10minutes to monitor the change of PCP (or 2-CP) concentration in liquid phase with incubation time. PCPwas measured by the ultraviolet absorption method and 2-CP was measured by GC/MS analysisafter liquid­liquid extraction using dichloromethane as solvent. Analytical conditions of GC/MS measurement are thesame as shownin Table 3.

RESULTS ANDDISCUSSIONS

Adsorptiye characteristics ofpcp-Na on GAC

The difference between PCP-Naconcentration in control and that in each experimentof the adsorptive testof PCP-Na on GAC corresponds to concentration of PCP-Na adsorbed to GAC. Isotherm curve ofadsorption is shown in Fig. 2. PCP-Na is shown to be a strongly adsorptive compounds. and the isothermcurvecan be describedby a Freundlich type equation;

q = 131 XC 0.28 (I)

where.q is mass of PCP-Naadsorbedof unit mass of GAC (mg/g-GAC), and C is concentration of PCP-Nain liquidphase (mgIL).

Anaerobic degradationof pentachforopheno! 339

q .131XC 028

104-................"f.-..................,.,.,j--.-........;.,...;j

1 10 100 1000PCP-Na in liquid Phase, C. mg/L

CT

ai~ 1001~·:"-_""""'1----"""--""';"c,o4(

"£to

'tE50.

figure 2. Relationship between PCP·Nain liquidphaseand GACphase.

C2ntim.lOUS treatment with anaerobic expanded-bed reactol:

Treatment performance during the experiment is shown in Fig. 3 and Fig. 4 by changes in concentrations ofPCP-Na and COD in influent and ef~uent. PCP-Na ~oncentration ~n effluent was kept under 0.50 mgILduring the experiment. The concentrauon showed no Increase even In start-up duration and even when the~nfluent concentration of PCP-Na was increased from loo ~o 400 mgIL. Total COD (T-COD)concentrationIn effluent increased to about300 mgILon day 30 of operatlon,.and thendecreased to around50 rngIL whileT-CODconcentration in influent was about500 mgIL. Immediately after the shock load, in which T-CODconcentration in influent was increased to around 1600 rngIL. T-COD concentration in effluent slightlyincreased for severaldays. However, afterthat it decreased and maintained around 100mgIL. Soluble CODconcentration in effluentwas lowerby 30 to 60 mgIL than the T-CODconcentration.

400

0J .1. n .1

f""

n. 9.1 JI',' ~Ll'" I'-"'

'-'

J2 Influent IEffluent

,.",

ruo

Do 100 ZOO 300

Time. days

Figure3. Otange in pentaChlorophenol conce:ntration in anaerobic GACreactor.

100

60

::::! 400ellE";300z

1

~ 2000.

500

Change in methane production rart during the experiment. is shown in Fig. S•.This index shows a proof ofanaerobic biodegradation, so theoretical methane pr~uc~on rates corresponding to each acetate load andPep load are also drawn by a solid line and a dotted hne 10 the ~gure. Methaneproduction began on about30 day of the experiment and increased to the rate correspondl~g to the acetate load. After increase ofOrganic load. methaneproduction rate responded successtil!ly and I?creased to the rate corresponding to thellCetate load. After day 150of the experiment, the .methane production rate exceeded ~e rate correspondingto the acetateload and reached the rate corresponding to the acetate plus pcp load.This may be the proofof

biologiCal degradation of PCPto methane.

340 H.TSUNO etal.

100 200 ~O 400Time, days

Figure4. ChangeinCODerconcentration in anaerobic GACreactor.

Material balance about PCP-Na during the experiment is shown in Figure 6. This figure was drawn under theassumption that acetate was completely transformed to methane and CO2, Area B shows the mass of PCP­Na degraded biologically and transformed to methane and CO2, About 60% ofPCP-Na loaded to the reactoris shown to be biologically degraded during 375 days operation. The remainder (Area A) means the mass ofPCP-Na which was adsorbed and retained to the GAC or transformed to intermediate organic products. Themass immobilized to microorganisms may be negligible. If all the difference of PCP-Na between input andoutput of the reactor during experiment, which amounts to, 250 g. were supposed to be adsorbed to GAC,the concentration of PCP-Na in liquid phase calculated by equation (1) is estimated to be 2.4 mg/L.However, PCP-Na concentration in effluent on 375 day of experiment was determined to be 0.4 mg/L. Thisdemonstrates that a part of PCP-Na that entered the reactor was degraded biologically.

400300

I4.----..,..---...,.---"""T'"-----.

i:::;ai3C;ex:

~ 2+----t-----+---f-----1

~~ 1-t---F3i1il1l1'!!_.~~i;;..jIII:SGl

~O

o 200Time, days

Figure,. Changein methane production rate in anaerobic GACreactor.

Chromatograms obtained by GClMS analysis of extracts from the GAC medium and effluent are shown inFig. 7 and Fig. 8. Tetrachlorophenol (TeCP), trichlorophenol (TCP), dichlorophenol (DCP) andmonochlorophenol (CP) as well as PCP were clearly detected in extract of the GAC medium in the reactor.Traces of PCP, TeCP ,TCP, DCP and CP were detected in extract of effluent of the reactor. These resultsindicate that reductive dechlorination of PCP occurred on the GAC in expanded-bed and dechlorinatedphenols were adsorbed to the GAC. Traces of dechlorinated phenols flowed out in effluent. Lower amountof DCP and CP compared with the other chlorophenols in extract of the GAC medium indicates that thesechlorophenols may be degraded more easily by microorganisms grown on the GAC medium under anaerobicconditions.

Anaerobic degradation of pentachlorophenol341

400

Influent

Gasi"Effluent

Effluent

O~~""''''''''-'''''''"'r'"'__~~...,...~o

300

01250

to2200

60.. 150CII>

.~ 100"SEa 50

100 200 300

Time,days

Figure6. Changein cumulative PeP-Na in anae~obic CAC reactor, A: Retained or trasfonned to inte1llledial

products. B: Bllldegraded 10CH. andCo,. C

128(# 20.0)162(#20.0)196(#10)232(#t.01266(#1.0)

PCP

CP DCP

"'--'--

F' ; I . I, i I

5 10 15 20 25lIME, MIN

Figure7.ChromalOgrams of exlTaCt of OAe medium in the reactorby GC/MS analysis.

rcpTeCP

128(#1.0)162(#1.0)196(#1.0)232(#1 .01266(#10)

rcp PCPCP TeCP

i~pc, , I i f

5 10 15 2Q 25TIME. MIN

Figure8. Chromalognuns of eltlTaCl ofeffluent from thereactorbyGClMs analysis.

Table 4. Estimation of biomass from DNA measurement of the GAC medium

121 322

DNA,rng'!/gGACj 0.10 0.17

Blomass,­rngVSS' /(gGACI 5 9

... ; It Waf .uumed that DNA content of

bacteria Wal 2 " In weight.

To estimate biological mass in the reactor. exrraction of DNA fro~ the GAC medium in the reactor and

DNA measurement was conducted on day 121 and 322 of operation by method of Kaneko (1974). The

342 H.TSUNOet al.

results are shown in Table 4. During the experiment. DNA content was 0.10-0.17 mg!g-GAC. which isestimated as S to 9 mgVSS/g-GAC. This is corresponded to 1700-3100 mgVSSIL-expanded-bed volume.

Batch experiments on de~dation ofPCP-Na and 2-CP

The change in PCP-Na concentration with time in the batch experiments with microorganisms collectedfrom the GAC medium in the reactor are shown in Fig. 9 and Fig. 10. The experiments were performed withand without addition of acetate. For the case without acetate. PCP-Na concentration did not decrease untilday 10 of the experiment and then gradually dropped from 14 to 12 mgIL. On the other hand. for the case ofthe addition of acetate of initially SO mg/L, PCP-Na concentration dropped immediately from 9.S to 45mgIL on day 1 of experiment and then decreased gradually to 3.8 mgIL on day 6. PCP-Na concentration incontrols in both cases did not change during experiments. These results indicate that PCP-Na was removedby the microorganisms collected from the GAC medium in the reactor. and that the removal can beaccelerated by the presence of acetate. Change of 2-CP concentration with time in the batch experiment isshown in Fig. 11. 2-CP concentration decreased linearly from initial 30 to 10 mgIL for 10 days of theexperiment. It's removal rate was 12.5 mg!(gVSSoday). 2-CP concentration in control did not change. It isshown that 2-CP can be degraded more readily by microorganisms collected from the GAC medium in thereactor.

1 _<,

!"ow..

.---1P Control ~

• Biomass.330 mgVSS/L

16

14

12...J

en10Erti 8zd. 6oCl. ..

2

oo 5 10 15 20 25 30

Time,daysFigure 9. Degradation of PCP·Naby microorganisms collected fromGACin the reactorin culturewithout

CH)COONa.

65

.....

1\\\\~

~ o Control ~• Blomass,620 mgVSS/L

1098

...J 7....~ 6rti 5zd. ..~ 3

21

oo 23 ..

Time, days

Figure 10. Degradation ofPCP·Na by microorganisms collected fromGACin the reactorin culturewithCH3COONa (initial concentration of SO mgIL).

Anaerobic degradation of pentachlorophenol 343

<,~

........I'-....

.......<,

~?Controlh• Biomass.160mgYSS/L

35

30~

0>25E

g2018"15o:t: 10oN

5

oo 2 4 6 8

Time, days10

Figure I I. Degradation of 2-chlorophenol by microorganisms collectedfromGACin the reactor in culturewithoutCH)COONa.

SUMMARY AND CONCLUSIONS

I~ this study. theexpanded-bed GACanaerobic reactor was applied to treatment of waste watercontainin~gb concentration of pcp-J:la as ~~ll as ace~ate. Treatment performance and possibility of PC~bIodegradation under anaerobic condition were discussed based on the experimental data. Main resultsobtained wereas follows;

1) T-COD concentration in effluent was kept less than 100my!-- except for.~e start-up duration and PCP­Np

a concentration ,was kept less than 0.50 mgIL ~nflnder theceoxopenmental conditions (HRT of 5 days, influentCP-Naconcentration of 100and400mgIL. and1 uent of 480 and 1600 mgIL).

2) Reactor couldrespond successfully to 4 timesshock-load without serious increase of T-COD in effluent.

3) Any increase at PCP-Na concentration in effluent could not be observed even in start-up duratio devenunderthe shockloadby increase of influent PCP-~a concentration from 100to 400 mgIL. Thisw~~econtribution by theadsorptive capacity of theGACmedium.4) Biological degradation of PCP-Na to methane was proved by methane production rate and materialbalance. About60%ofPCP-Naloaded into thereactor wastransformed to methane andCO2 for 375 days.

5) GCiMS analysis of extracts of the GAC medium and effluent indicated that reductive dechlorination ofPcp OCCUlTed in the reactor. Dechlorinated phenols hadbeenadsorbed on theGACmedium in thereactor.

6) Microorganisms collected from the reactor degraded !'C:-Na and 2-CPanaerobically. Acetate playedanimportant role in anaerobic degradation of pcp-Na; thatIS. It accelerated the degradation.

REFERENCES

AkiraKudo(1989). Decomposition of penllChlorophenol b~ ~acrobic digesti?n. War. ~cI, Tlch. •21(12), 1685·1688.APHA, AWWA, WPCF:STANDARD ME1lIOD, 16thEdition (1985), AmencanPublicHealthAssociation. Washington, DC.Crosby 0 0 (1981) E ' talchemistry ofpentaeh/orophenol. Purl Appl. Chtm.53, 1051.

, " . nVIl'Onmen f bi I ' 11 ' hib'Fox. P. (1989). An innovative reactordesign for the treaunent0 10 oglca y In Itory wastewater. Doctoral Thesis, University

of Illinois. 8 R I f GAC . . d ' .Gardner D A. S .dan, M T and Kobayashi. H. A. (/9 8). 0 e 0 acuvlty an particle SIZC during the fluidized-bed, • ' . W • f' fi .~ur waterstripperbottoms. WPCF. 60, 505·513,anaerobiC treaunent 0 re nery.., L. 1 (1984) Pc hI .Outhrie M A v:_~.. E ] W k."'h. It P, and Grady C. P. • t . ntec orophcnol blodearadatiOIl -Il an bi

• • •• n.J,IMo'lt . . , U __ aero Ie.WartrRn. 18,451-461. d' . ti Ii Doct al The ' K .

Kaneko, M. (/974). Biological activity of activated sludgean IlJ es ma on. or SIS, yolO University.

344 H. TSUNO et al.

Larsen. S.• Hendriksen. H. V. and Ahring. B. K. (1991). Potential for thermophilic (50'C) anaerobic dechlorination ofpentachlorophenol in different ecosystems. Appl. Environ. Microbiol.•57. 2085-2090.

Mikesell. M. D. and Boyd, S. A. (1986). Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobicmicroorganisms. Appl. Environ. Microbiol,52,861-865.

Suidan, M. T.. Najim, J. N., Pfeffer, 1. T. and Wang, Y. (1988). Anaerobic biodegradation of phenol: inhibition kinetics andsystem stability. Environ. Div. ASCE. 114, 1359-1376.

Tsuno, H.• Kawamura, M., Somiya, I. and Rou, Z. (1993). Anaerobic treatment of phenol by fluidized - bed GAC reactor. Proc.Environmental Engineering Research, 30, 27-38.

Wang, Y. T., Suidan, M. T. and Rittman, B. E. (1986). Anaerobic treatment of phenol by an expanded-bed reactor. WPCF.58,227-233.