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Aerobic fluidized-bed treatment of polychlorinated phenolic wood preservative constituents
IT'at. Res. Vol. 26. No. 6. pp. 765-770. 1992 Ptanted in Great Britain
0043-1354,92 $5.00 +0.00 Pergamon Press Ltd
AEROBIC FLUIDIZED-BED TREATMENT OF POLYCHLORINATED PHENOLIC WOOD PRESERVATIVE CONSTITUENTS JAAKKOA. PUHAKKA* and KIMMOJ.~RVINEN Institute of Water and Environmental Engineering. Tampcre University of Technology, P.O. Box 527, SF-33101 Tampere, Finland (First receh'ed June 1991; accepted in rerised form November 1991)
Abstract--Degradation of polyehlorinated phenols was studied in continuous-flow fluidized-bed reactors using pure oxygen for aeration and celite carrier for cell immobilization. High dilution rates and chlorophenols as the only source of carbon and energy were used for maintenance of the mixed biofilm cultures. The chlorophenol degradation performance was monitored as release of inorganic chloride and removal of total organic carbon and by direct gas chromatographic analyses. Continuous polychlorophenol biodegradation activity was maintained in a fluidized-bed reactor for 315 days. Chloride release and chlorophenol removal efliciencies of over 99% were achieved at substrate loading rates of up to 430 g 2,4,6-trichlorophenol/m~;d and 400 g 2,3.4.6-tetrachlorophenol/m~/d at 3-5 h hydraulic retention times. respectively. Immobilized mixed cultures biodegraded 2.3,4.6-tetrachlorophenol with partial efficiency at feed concentrations as high as 157 mg/I as indicated by inorganic chloride release. Inorganic chloride release in experiments with pr~tctical grade 2.3.4,6-tetrachlorophenol exceeded that which is possible by dechlorination of tetrachlorophcnol alone suggesting dcchlorination of unidentified chlorinated impurities of the preparation as well. Fluidizcd-bed treatment of simulated wocM preservative contaminated groundwater at a chlorophcnol loading rate of 217 g/m~/d and 5 h hydraulic retention time resulted in the fi~llowing rcmov:d cfficiencics:99.5% for 2.4,6-trichlorophcnol, 99.6% for 2.3,4.6-tctrachlorophenol and 92.5'/0 for pcnlachlorophenol. Key word~'--biodegradation, groundwater
chlorophenols,
dechlorination,
IN'I'ROI)UC'I'ION
Chlorophenols have been widely used against fungal timber and lumber deterioration. Pentachlorophenol has been the most abundantly produced and used fungicide for protection of wood. Estimated annual worldwide PCP production has gradually decreased from 150,000 tons to 50,000 tons during 1978 and 1981 (Nilsson et al., 1978; Crosby, 1981). in Finland, the most commonly used wood preservative, Ky-5, a technical sodium chlorophenolate preparation contained 12g of chlorophenol/I consisting of 78-83*/0 2,3,4,6-tctrachlorophenol, 7-15°/. 2,4,6trichlorophenol and 5-10% pentachlorophenol (Valo et al., 1984). This preparation was used over 40 years with total use amounting to over 30,000 tons (Kitunen, 1990). Due to environmental toxicity and persistence, the use of polychlorophenols for wood protection has been banned in many countries including, for example, Sweden. Finland and Japan.
"Present address: Department of Civil Engineering. University of Washington, Seattle. WA 98195. U.S.A, Abbreviations used: TCP. trichlorophenol; TcCP. tetrachlorophenol; PCP, pentachlorophenol. ICI, inorganic chloride; HRT. hydraulic retention time; TOC, total organic carbon.
fluidizcd-bed, immobilized bacteria,
Environmental effects of chlorophenols were not of concern by the time of widc scale use. Therefore, contamination of soil has taken place in the vicinity of wood preservation sites. Chlorophenols and especially those preparations used :is sodium salts further migrate into the soil eventually contaminating groundwater (Valo et al., 1984; Goerlitz et al., 1985). Biodegradation and biotransformation of chlorophenols has been extensively studied and the degradation pathways delineated (for review see e.g. Tiedje et al., 1987; Hiiggblom, 1990). Chlorophenol removal has been reported in conventional wastewater treatments such as activated sludge (e.g. Puhakka et al., 1991a). Use of immobilized-cell systems often results in long cell retention times together with high tolerance of toxic compounds (Klein and Kluge, 1981; Kobayashi and Rittmann, 1982; Westmeier and Rehm, 1985; Sofer et al., 1990). Both pure and mixed culture approaches have been used in studies on aerobic immobilized-cell treatment of ehlorophenolic water contaminants (Etzel and Kirsch, 1974; O'Reilly and Crawford, 1989; Valo et aL. 1989; Shieh et aL, 1990). The present study was undertaken to determine the ability of high-rate oxic fluidized-bed mixed culture
765
766
JAAKKO A. Pt,'t~KKA and K.IMMOJ.~I~VINEN Table I. Compoution of the
OXYGEN (3rag oo/I)
basal salts medium
BAR= 10cm I
EFFLUENT
Compound
mg/1
KzHPO, KHzPO, (NH,): SO, NaHCO3 FeSO4"THzO
380 200 10 25 5 20 5 2.5 1.0
MgSO4.7H:O
CaCO~ ZnSO4.7H:O Yeast extract
attachment. The biomass accumulation, scanning electron microscopic characterization as well as mono- and dichlorophenol degradation performance has been reported elsewhere (Shieh et al., 1990; Puhakka et al.. 1991b).
RECYCLE 420 mllmir
Experimental
Reactor microcarrier expansion was maintained at 50% by liquid recirculation to obtain complete mixing (Shieh and Keenan. 1986). The upflow velocity was provided with liquid recycle ratios from 300 to 420 depending on the HRT maintained. The empty bed HRT or chlorophenol concentration was used as the experimental variable in polychlorophenol degradation tests. The reactor biomass immobilized in carriers was not removed during experimentation. Thus. the results were obtained under continuous biomass accumulation, i.e. pseudo-steady-state conditions. Volumetric loading rates were based on fluidized-bed volumes of the reactors. Experiments on 2,4.6-TCP and 2.3,4,6TeCP were conducted in reactor I and on simulated groundwater in reactor 2.
FLUIDIZEDBED VOL.L~O ral
GLASS BEADS FEED 1-1.4mllmln
Fig. I. Scheme of fluidized-bed biorcactor.
Chlorophenol solutions
bioreactors to biodcgradc polychlorophcnolic w o o d preservative constituents. MATERIALS AND METllODS bTuidi2ed-bcd reactors
Continuous-flow reactor experiments were conducted using two pure oxygen aerated glass fluidized-bed reactors with internal recycle and spherical silica-based microcarriers (Manville Celite R-633) for cell immobilization as shown in Fig. I. Dissolved oxygen concentration was at least 3 mg/I. The detailed description of fluidized-bed reactor design is given by Shich et al. (1990). The reactors were operated at ambient temperature ranging from 24 to 29'~C. Growth on the walls of the reactors and in the tube connections was removed. Biomas$
Unacclimated activated sludge from a pilot-seale reactor treating simulated municipal sewage was used as the original seed material. Chlorophenol degrading biomass was enriched and maintained using mono- and dichlorophenols as the sole carbon and energy source for I year. HRTs of 5 h or less were applied during enrichment and experimentation to sch.'ct for microorganisms with improved surface
Reactor feed solutions consisted of a basal medium (Table I) and the defined chlorophenol(s) in tap water, which had a background TOC of 4-5 mg/I. Stock solutions of 2,4,6-TCP, 2.3,4.-6-TeCP, PCP and chlorophenol mixtures were prepared as their sodium salts. The simulated groundwater had the following composition: 15.1 + 0.3 mg 2,4,6-TCPil, 26.3 + 1.9 mg 2,3,4,6-TeCP/I and 3.8 + 0.6 mg PCP/I. Practical grade 2,4,6-TCP was produced by Fluka Chemic AG, Buchs, Switzerland and the preparation of 2,3,4,6-TeCP by American Tokyo Kasei Inc., Tokyo, Japan. According to our analysis the TeCP preparation contained 11% of PCP. Chemical determinations
Inorganic chloride (ICI) was determined with a chlorideion selective electrode (W. lngold AG, Urdorf, Switzerland, Type 15 213 3000) and an Ingold Ag/AgCI reference electrode (Type 373-90-WTE-ICE-S7) using an Orion pH/mV meter (Orion Research Inc., Kusnecht, Switzerland, Model SA 720) at 20 + I°C. Total organic carbon (TOC) was measured from acidified samples using a Unicarbo universal carbon analyzer (Elektrodynamo, Helsinki, Finland). Dissolved oxygen concentration in the reactor was monitored using a WTW oxygen electrode (Wissenschafi. Tech.
Table 2. Chloride release and TOC removal efliciencies from 2,4.6-trichlorophcnol at dilTcrent loading rates and hydraulic retention times (HRT) in fluidized-bcd reactor ICI release
Expt No. I 2 3
TCP-loading rate (rag/I/d) 260 325 430
inorganic chloride; N dcterrained.
ICI =
-
HRT (h) 5
(rag/I) 31.0
(SD) 0.8
TOC removal
(rag/I) 18.9
(SD) 1.5
N 10 4 28.8 1,9 18.9 0.9 10 3 32,8 0.5 ND 10 number of samples during pseudo-steady*stateexperiments; SD = standard deviation; ND = not
767
Fluidized-bed treatment of chlorophenols Werkst/tten, Weilheim, Germany, Model EO 96) and an oxygen meter (WTW, Model OXI 96). The chlorophenolic compounds from 0.5 ml samples were acetylated with 0.5 ml acetic anhydride using 25 ml of 0.1 M K~CO3 as buffer. The acetylated derivatives were extracted with 2.5 ml n-hexane and a 2.00 #1 extract was used for injection. 2,3,6-TCP was used as the internal standard. GC analyses were carried out on a Perkin-Elmer Sigma 200 gas chromatograph (Perkin-Elmer Instrument Division, Norwalk, Conn., U.S.A.) equipped with a eNi electron capture detector and the DP-5 column (30 m x 0.25 nun ID x 0.25 #m) supplied by J&V Scientific (Folsom, Calif., U.S.A.), with a nitrogen flow-rate of I.Sml/min. The
20
I 25
130m
TOC,,
Chloride 80 6O E 40 20 0
i
|
i
i
a
i
a
TOC
3O I
zo E 10
i
2 , 3 , 4 , 6 -TeCP 90 "-
60
E
30
.
.
.
.
.
.
.
v
v
-
-
_ _ -
I
PCP
~ E
8
4
~ |
0
30
i
60
a
90
i
120
Time (doys } Fig. 2. Fluidized.bed bioreactor performance at increasing 2,3,4,6-t©trachlorophenol loading rates. 171, Feed; O, effluent. WR
~/6--E
temperature program was: oven temperature at 100°C for I rain, then increased at YC/min to 250°C. The injection temperature was 2.50~C. RESULTS First, the degradation performance of 2,4,6-TCP was studied in fluidized-bed reactor using a 20 mg TOC/I feed concentration. The effect of TCP loading rate and HRT on chloride release and TOC removal is shown in Table 2. ICI releases indicated 100% dechlorination of 2,4,6-TCP in all three pseudosteady-state experiments The feed pH was 7.3-7.5 decreasing during treatment to 6.9-7.0. TOC removals were 82-83% in the first and second experiment, respectively. The observed ACI -/ATOC ratios (mg chloride/rag TOC) of 1.5 and 1,6 agree well with the theoretical value of 1.5. GC analyses of 6 effluent samples (3 from Expt 2 and 3 from Expt 3) gave 2,4,6-TCP concentrations of 0.1 mg/I or less. These results demonstrate that 2,4,6-TCP was readily degradable by the immobilized aerobic microbes and that a 40% increase in dilution rate did not affect the reactor performance. After the 2,4,6-TCP experimentation, the reactor feed was switched to 2,3,4,6-TeCP solution containing l 1% PCP as an impurity. The TeCP feed concentration was set at 20 mg TOC/I and fed at 5 h HRT for 3 weeks until effluent ICI concentration was stabilized at over 40 mg/I. The process performance was monitored for 2 weeks at this chlorophenol feed which was then gradually increased to 30 mg TOC/I while the HRT remained at 5 h. These feed patterns corresponded with loading rates between 230 and 400 g chlorophenol/m~/d, The results of overall reactor performance from 130 days of operation are presented in Fig. 2. TeCP and PCP mineralization reaction stoichiometries yield 2 and 2.5 mg ICI, respectively, per milligram of TOC removed. According to analyzed feed chlorophenol concentrations during the 130 day period the mean ICI release would have been 45.9mg/I while 56.6mg ICI/I was observed. This indicates that other than chlorophenol bound chlorine was released as well from the 2,3,4,6-TeCP solution produced from the practical grade preparation. Observed mean TOC removal of 21.7 mg/I was in conformity with the mean 99.3% 2,3,4,6-TeCP and 74.5% PCP removals which in terms of TOC indicate 22.9 mg TOC/I removal. Most of the effluent TOC accounted for is by the tap water background TOC (4-5 mg/I). The results also show variation at the substrate loading rate of 400 g chlorophenol/m3/d and the highest chlorophenol feed concentration indicating reactor operation near the maximum substrate utilization capacity or near the inhibitory feed concentration. The chlorophenol loading rate was further increased to 810 g/mJ/d and process performance was monitored by ICI release for 8 weeks. ICI formation results at different chlorophenoi loading rates are
768
JAAKKOA. PUHAKKAand KIMMOJ,~RV1NEN 120 100
o°
t.°
I"
4O
0
0
,6o
66o
s6o
tooo
Loading rate (g/m 3. (3")
Fig. 3. The effect of chlorophenol loading rate on chloride release. ©, Calculated maximum |CI releasesfrom 2,3,4,6TeCP plus PCP constituents of the CP-preparation: A,
nolle wood preservative constituents in oxic fluidizedbed reactors. They are consistent with extensive data on the biodegradability of these compounds (e.g. H~iggblom, 1990). Various techniques have been tested for aerobic treatment of polyehlorophenol contaminated water. Etzei and Kirsch (1974) reported 99% PCP removal from wood treatment wastewater containing 2060 mg PCP/I by a mixed aerobic suspension culture at HRTs of 6-12 h. Valo et al. (1989) immobilized chlorophenol-mineralizing rhodococci on a biofilter cartier for treatment of technical chlorophenol (a
observed ICI releases.
Chloride 40
summarized in Fig. 3. The straight line shows calculated maximum ICI releases from 2,3,4,6-TeCP and PCP constituents of the technical grade chlorophenol preparation at different loading rates. ICI release exceeded that of the complete TeCP plus PCP deehlorination at the 230-400 g/m~/d loading rates indicating dechlodnation of unidentifed impurities of the chlorophenol preparation as well. IC! release remained stable (70.9 _+8.0 mg/l; number of analyses 9) during the last 2 weeks of operation at 810 g/m'/d representing 67% chlorophenol dcchlorination. Stable operation indicates that the substrate convcrsion efficiency was limited rather by the maximum substrate utiliz~Ltion rate than the toxicity of substrate at the high loading rate. Finally, we studied a chlorophenol mixture which simulated groundwater contaminated with chlorophenolic wood preservative. This experiment was carried out in reactor 2 which had received monoand diehlorophenol feeds only. The reactor was fed a mixture of 2,4,6-TCP, 2,3,4,6-TeCP and PCP at chlorophenol loading rate of 217 g/m~/d and HRT of 5 h for 5 weeks. Figure 4 presents the results of the following pseudo-steady-state degradation of simulated groundwater at 5 h HRT. The mean chloride release was 31.4mg/I. The respective mean chlorophenol removals were as follows: 99.5% for 2,4,6TCP, 99.6 for 2,3,4,6-TeCP and 92.5% for PCP accounting for 26.8mg/I release of chlorophenol bound chlorine. This again indicates 15% additional ICI formation from unidentified simulated groundwater constituents. As a result of dechlorination, pH dropped from 7.1 to 6.7. The mean TOC removal of 14. I mg/I agrees well with the measured chlorophenol removals. The mean effluent TOC of 4.9 mg/I corresponds with the background TOC from the tap water used for feed dilution. The results show efficient and simultaneous removal of wood preservative chlorophenol constituents. Release of ICI and evolution of H * as seen by pH decrease indicate biodegradation. DISCUSSION The results of this investigation showed continuous high-rate dechlorination/degradation of ehlorophe-
30
10
DC~DD s
IDCCCD i
!
i
i
=
TOG 2O u
"" 10 5
o 16
~ . ~ G - - - - - O 0 0 O0 i
I
I
I
i
I
2,4,6-TCP
~ 12 v
8 4 ~.J
"~.Jx-J~../N.,J
= 2O
10 0
=
3
o
O ~" 2
"-' 4
~ O¢:L~:~ 6
Time
8
lO
12
(days)
Fig. 4. Simulated groundwater treatment performance in fluidized-bed biorcactor under pseudo-steady-state conditions, rl, Feed: O, emuent.
769
Fluidized-bed treatmcnt of chlorophcnols mixture of 2,4,6-TCP, 2,3,4,6-TeCP and PCP) at concentrations of 3.4--130 mg/l. The batch operation resulted in the elimination of chlorophenois from the influent and 40% release of chlorophenol bound chlorine as ICI. In another study, PCP (55 rag/I) degradation by polyurethane-immobilized Flavobacterium was studied in the presence of 0.5 g glutamate/l (O'Reilly and Crawford, 1989). At the residence time of 8.3 h, more than 90% of PCP was degraded over a period of 25 days after which the degradation efficiency started to decline. Anaerobic treatment of chlorophenols often results in partially or fully dechlorinated products requiring complementary aerobic treatment for complete mineralization. Krumme and Boyd (1988) used anaerobic upflow bioreactors to treat three monochlorophenols and 3,4,5-TCP and reached substrate conversion efficiency of > 9 0 % at substrate loading rates of up to 20 mg/I/d (corresponding HRT 2--4 days). Woods et al. (1989) studied chlorophenoi degradation in an upflow anaerobic sludge blanket reactor in the presence of high concentrations of readily biodegradable organic compounds. Among other chlorophenols, 2,4,6-TCP, 2,3,4,5-TeCP and PCP at concentrations of 0.I-I.0 mg/I were converted to lesser chlorinated compounds at the HRT of 13 h. Our results on aerobic fluidized-bed treatment of polychlorophcnols compare favorably with the above summarized results on previous aerobic and anaerobic treatments. First, no supplemental carbon or energy source was required for continuous operation and maintenance of the mixed chlorophenol degrading culture so that the possibility of o-methylation (Suzuki, 1983, Hiiggblom et al., 1989) would be expected to be minimal. Methylation increases the lipophilicity of chlorophenols and thus their tendency to bioaccumulation (Lechet al., 1978). In addition, the methylated compounds may be more toxic than their precursors (Neilson et al., 1984). In reactor I, chlorophenol degradation was first maintained with mono- and dichlorophenols for over I year (Shieh et aL, 1990) and then continued for 315 days with polychlorophenols as presented in this study. This shows that microbial cells entrapped within the pores and on the surface of the carrier were protected from shear forces and carrier collisions and remained active in a variety of operational conditions. Furthermore, the celite carrier had a high persistence to the mechanical friction caused by the 50% upflow velocity. Second, no inhibition due to high feed chlorophenol concentrations was observed. This is due to the high recycle ratio applied which in addition to providing completely mixed conditions effectively diluted the feed. Third, polychlorophenol loading rates were higher and HRTs were lower at removal efficiencies of >99% for 2,4,6-TCP and 2,3,4,6-TeCP than reported earlier. Polychlorophenol degradation performances were similar to those observed with mono-
and dichlorophenols in our earlier studies (Shieh et al., 1990; Puhakka et al., 1991b).
In aerobic waste treatment, often parallel biochemical mechanisms are in operation. Hence, the oxic mixed culture process is considered more stable against toxicity and varying operational conditions than pure culture processes. Scanning and transmission electron microscopy of reactor carrier material after an additional year of operation with polychlorophenol contaminated groundwater showed a diverse microbial community and an immobilized biomass concentration of 6-7g/1 (Puhakka et al., in preparation). Synergistic action of microorganisms may add to the advantages of mixed culture approaches. This was apparent in 2,4-DCP degradation by a two-component consortium of bacteria isolated from reactor 1 during dichlorophenol studies (Puhakka et al., 1991 b). CONCLUSION
This study shows that the use of oxic fluidized-bed reactors supplied with porous carrier for adsorptive and entraptive cell immobilization is an effective method for continuous, high-rate dechlorination and removal of polychlorophenols from water. Immobilized mixed cultures can be maintained with chlorophenols as the only source of carbon and energy. Over 99% biodegradation efficiencies are achievable at a substrate loading rate of 400 g 2,4,6TCP or 2,3,4,6-TeCP/m~/d and 3-5 h HRTs. Simultaneous treatment of a mixture of 2,4,6-TCP, 2,3,4,6-TeCP and PCP is also achievable. Thus, the oxic fluidized-bed process is an attractive treatment alternative for groundwater contaminated with wood preservation chemicals typically consisting of polychlorophenol congeners. Acknowledgements--This work was financially supported
by the Maj and Tor Nessling Foundation, Finland (K.J.) and the Academy of Finland (J.A.P.). We thank Professor Wen K. Shieh (University of Pennsylvania. U.S.A.) for initiation of the fluidized-bed treatment studies at the Tampere University of Technology and fruitful discussions during this study and Mr Juhani Tarhanen and Mr Jouni Rokkanen (University of Kuopio, Finland) for GC analyses. REFERENCES
Crosby D. G. (1981) Environmental chemistry of pentachlorophenol. Pure AppL Chem. 53, I051-I080. Etzel J. E. and Kirsch E. I. (19"74) Biological treatment of contrived and industrial wastewater containing pentachlorophenol. Dev. ind~t. Microbiol. 16, 28"7-295. Goerlitz D. F., Troutman D. E., Godsy E. M. and Franks B. J. (1985) Migration of wood-preserving chemicals in contaminated ground water in a sand aquifer at Pensacola, Florida. Enrir. Sci. Technol. 19, 955-961. H~iggblom M. (1990) Mechanisms of bacterial degradation and transformation of chlorinated monoaromatic compounds. J. Basic MicrobioL 30, 115-141.
770
J~KKO A. PU~KKA and Kl~,a~o J~vL,,~m
H:~ggblom M., Janke D., Middeidorp P. J. and SalkinojaSalonen M. (1989) O-methylation of chlorinated phenolic compounds in the genus Rhodococcus. Archs Microbiol. 152, 6-9. Kitunen V. H. 0990) The use and formation of CPs, PCPPs and PCDDs, PCDFs in mechanical and chemical wood processing industries. Ph.D. thesis, Department of General Microbiology, University of Helsinki. Heisinki. Finland. Klein J. and Kluge M. (1981) Immobilization of microbial cells in polyurethane matrices. Biotechnol. Left. 3, 65-70. Kobayashi H. and Rittmann B. (1982) Microbial removal of hazardous organic compounds. Era'Jr. Sci. Technol. 16, 170A-183A. Krumme M. L. and Boyd S. A. (1988) Reductive dechlorination of chlorinated phenols in anaerobic upflow bioreactors. War. Res. 22, 171-177. Lech J. J., Glickman A. H. and Statham C. N. (1978) Studies on the uptake, disposition and metabolism of pentachlorophenol and pentachloroanisole in rainbow trout. In Em,ironmental Science Research. 12. Pentachlorophenol: Chemistry, Pharmacology and Era,ironmental ToxiciO" (Edited by Rao K. R.), pp. 107-113. Plenum Press, New York. Neilson A. H.. Allard A.-S., Reiland S., Remberger M., Tarnholm A., Viktor T. and Lendner L. (1984) Tri- and tetrachloroveratrole, metabolites produced by bacterial o-methylation of tri- and tetrachloroguaiacol: an assessment of their bioconcentration potential and their effects on fish reproduction. Can. J. Fish. Aquat. Sci. 4I, 1502-1512. Nilsson C.-A., Norrstrom A., Andersson K. and Rappe C. (1978) Impurities in commercial products related to pentachlorophenol. In Ent,ironmental Science Research 12. Pentachlorophenol: Chemi.vtr~', Pharmacology and Em,ironmental Toxicity (Edited by Rao K. R.), pp. 313-324. Plenum Press, New York. O'Reilly K. T. and Crawford R. L. 0989) Degradation of pcntachlorophcnol by polyurethane-immobilized Flavobacterium cells. Appl. era'Jr. Microbiol. 55, 2113-2118.
Puhakka J. A., Rintala J., Wartiovaara J. and Heinonen P. (Eds) (1991a) Forest lnduJtry Wastewaters. Wat. Sci. Technoi. 24. Puhakka J. A., Melin E., Jirvinen K., Tuhkanen T. and Shieh W. K. (1991b) Oxic fluidized-bed treatment of dichlorophenols. Wat. Sci. l'echnol. 24, [71-177. Shieh W. K. and Keenan J. D. (1986) Fluidized-bed biofilm reactor for waste water treatment. In Advances in Biochemical Engineering/Biotechnology (Edited by Fiechter A.), pp. 131-169. Springer, Berlin. Shieh W. K., Puhakka J. A., Melin E. and Tuhkanen T. 0990) Immobilized-ceU degradation of chlorophenols. J. em'ir. Engng, Am. Soc. civ. Engrs 116, 683-697. Sorer S. S., Lewandowski G. A., Lodaya M. P., Lakhwala F. S., Yang K. C. and Singh M. (1990) Biodegradation of 2-chlorophenol using immobilized activated sludge. Res. J. War. Pollut. Control Fed. 62, 73-80. Suzuki T. (1983) Methylation and hydroxylation of pentachlorophenol by Mycobacterium sp. isolated from soil. J. Pestic. Sci. 8o 419-428. Tiedje J. M., Boyd S. A. and Fathepure B. Z. 0987) Anaerobic degradation of chlorinated aromatic hydrocarbons. Dev. indust. MicrobioL 27, 1[7-127. Valo R. J., H~iggblom M. M. and Salkinoja-Salonen M. S. (1989) Bioremediation of chlorophenol containing simulated ground water by immobilized bacteria. Wat. Res. 23, 253-258. Valo R. J.. Kitunen V., Salkinoja-Salonen M. and R~iis~nen S. (1984) Chlorinated phenols as contaminants of soil and water in the vicinity of two Finnish sawmills. Chemosphere 13, 835-844. Wcstmeier F. and Rehm H. J. (1987) Biodegradation of 4-chlorophenol in municipal wastewater by adsorptive immobilized Alcaligenes sp. A7-2. Appl. Microbiol. Biotechnol. 26, 78-83. Woods S. L., Ferguson J. F. and Benjamin M. M. 0989) Characterization of chlorophenol and chloromethoxybenzene biodegradation during anaerobic treatment. Envir. Sci. Technol. 23, 62-68.
0043-1354,92 $5.00 +0.00 Pergamon Press Ltd
AEROBIC FLUIDIZED-BED TREATMENT OF POLYCHLORINATED PHENOLIC WOOD PRESERVATIVE CONSTITUENTS JAAKKOA. PUHAKKA* and KIMMOJ.~RVINEN Institute of Water and Environmental Engineering. Tampcre University of Technology, P.O. Box 527, SF-33101 Tampere, Finland (First receh'ed June 1991; accepted in rerised form November 1991)
Abstract--Degradation of polyehlorinated phenols was studied in continuous-flow fluidized-bed reactors using pure oxygen for aeration and celite carrier for cell immobilization. High dilution rates and chlorophenols as the only source of carbon and energy were used for maintenance of the mixed biofilm cultures. The chlorophenol degradation performance was monitored as release of inorganic chloride and removal of total organic carbon and by direct gas chromatographic analyses. Continuous polychlorophenol biodegradation activity was maintained in a fluidized-bed reactor for 315 days. Chloride release and chlorophenol removal efliciencies of over 99% were achieved at substrate loading rates of up to 430 g 2,4,6-trichlorophenol/m~;d and 400 g 2,3.4.6-tetrachlorophenol/m~/d at 3-5 h hydraulic retention times. respectively. Immobilized mixed cultures biodegraded 2.3,4.6-tetrachlorophenol with partial efficiency at feed concentrations as high as 157 mg/I as indicated by inorganic chloride release. Inorganic chloride release in experiments with pr~tctical grade 2.3.4,6-tetrachlorophenol exceeded that which is possible by dechlorination of tetrachlorophcnol alone suggesting dcchlorination of unidentified chlorinated impurities of the preparation as well. Fluidizcd-bed treatment of simulated wocM preservative contaminated groundwater at a chlorophcnol loading rate of 217 g/m~/d and 5 h hydraulic retention time resulted in the fi~llowing rcmov:d cfficiencics:99.5% for 2.4,6-trichlorophcnol, 99.6% for 2.3,4.6-tctrachlorophenol and 92.5'/0 for pcnlachlorophenol. Key word~'--biodegradation, groundwater
chlorophenols,
dechlorination,
IN'I'ROI)UC'I'ION
Chlorophenols have been widely used against fungal timber and lumber deterioration. Pentachlorophenol has been the most abundantly produced and used fungicide for protection of wood. Estimated annual worldwide PCP production has gradually decreased from 150,000 tons to 50,000 tons during 1978 and 1981 (Nilsson et al., 1978; Crosby, 1981). in Finland, the most commonly used wood preservative, Ky-5, a technical sodium chlorophenolate preparation contained 12g of chlorophenol/I consisting of 78-83*/0 2,3,4,6-tctrachlorophenol, 7-15°/. 2,4,6trichlorophenol and 5-10% pentachlorophenol (Valo et al., 1984). This preparation was used over 40 years with total use amounting to over 30,000 tons (Kitunen, 1990). Due to environmental toxicity and persistence, the use of polychlorophenols for wood protection has been banned in many countries including, for example, Sweden. Finland and Japan.
"Present address: Department of Civil Engineering. University of Washington, Seattle. WA 98195. U.S.A, Abbreviations used: TCP. trichlorophenol; TcCP. tetrachlorophenol; PCP, pentachlorophenol. ICI, inorganic chloride; HRT. hydraulic retention time; TOC, total organic carbon.
fluidizcd-bed, immobilized bacteria,
Environmental effects of chlorophenols were not of concern by the time of widc scale use. Therefore, contamination of soil has taken place in the vicinity of wood preservation sites. Chlorophenols and especially those preparations used :is sodium salts further migrate into the soil eventually contaminating groundwater (Valo et al., 1984; Goerlitz et al., 1985). Biodegradation and biotransformation of chlorophenols has been extensively studied and the degradation pathways delineated (for review see e.g. Tiedje et al., 1987; Hiiggblom, 1990). Chlorophenol removal has been reported in conventional wastewater treatments such as activated sludge (e.g. Puhakka et al., 1991a). Use of immobilized-cell systems often results in long cell retention times together with high tolerance of toxic compounds (Klein and Kluge, 1981; Kobayashi and Rittmann, 1982; Westmeier and Rehm, 1985; Sofer et al., 1990). Both pure and mixed culture approaches have been used in studies on aerobic immobilized-cell treatment of ehlorophenolic water contaminants (Etzel and Kirsch, 1974; O'Reilly and Crawford, 1989; Valo et aL. 1989; Shieh et aL, 1990). The present study was undertaken to determine the ability of high-rate oxic fluidized-bed mixed culture
765
766
JAAKKO A. Pt,'t~KKA and K.IMMOJ.~I~VINEN Table I. Compoution of the
OXYGEN (3rag oo/I)
basal salts medium
BAR= 10cm I
EFFLUENT
Compound
mg/1
KzHPO, KHzPO, (NH,): SO, NaHCO3 FeSO4"THzO
380 200 10 25 5 20 5 2.5 1.0
MgSO4.7H:O
CaCO~ ZnSO4.7H:O Yeast extract
attachment. The biomass accumulation, scanning electron microscopic characterization as well as mono- and dichlorophenol degradation performance has been reported elsewhere (Shieh et al., 1990; Puhakka et al.. 1991b).
RECYCLE 420 mllmir
Experimental
Reactor microcarrier expansion was maintained at 50% by liquid recirculation to obtain complete mixing (Shieh and Keenan. 1986). The upflow velocity was provided with liquid recycle ratios from 300 to 420 depending on the HRT maintained. The empty bed HRT or chlorophenol concentration was used as the experimental variable in polychlorophenol degradation tests. The reactor biomass immobilized in carriers was not removed during experimentation. Thus. the results were obtained under continuous biomass accumulation, i.e. pseudo-steady-state conditions. Volumetric loading rates were based on fluidized-bed volumes of the reactors. Experiments on 2,4.6-TCP and 2.3,4,6TeCP were conducted in reactor I and on simulated groundwater in reactor 2.
FLUIDIZEDBED VOL.L~O ral
GLASS BEADS FEED 1-1.4mllmln
Fig. I. Scheme of fluidized-bed biorcactor.
Chlorophenol solutions
bioreactors to biodcgradc polychlorophcnolic w o o d preservative constituents. MATERIALS AND METllODS bTuidi2ed-bcd reactors
Continuous-flow reactor experiments were conducted using two pure oxygen aerated glass fluidized-bed reactors with internal recycle and spherical silica-based microcarriers (Manville Celite R-633) for cell immobilization as shown in Fig. I. Dissolved oxygen concentration was at least 3 mg/I. The detailed description of fluidized-bed reactor design is given by Shich et al. (1990). The reactors were operated at ambient temperature ranging from 24 to 29'~C. Growth on the walls of the reactors and in the tube connections was removed. Biomas$
Unacclimated activated sludge from a pilot-seale reactor treating simulated municipal sewage was used as the original seed material. Chlorophenol degrading biomass was enriched and maintained using mono- and dichlorophenols as the sole carbon and energy source for I year. HRTs of 5 h or less were applied during enrichment and experimentation to sch.'ct for microorganisms with improved surface
Reactor feed solutions consisted of a basal medium (Table I) and the defined chlorophenol(s) in tap water, which had a background TOC of 4-5 mg/I. Stock solutions of 2,4,6-TCP, 2.3,4.-6-TeCP, PCP and chlorophenol mixtures were prepared as their sodium salts. The simulated groundwater had the following composition: 15.1 + 0.3 mg 2,4,6-TCPil, 26.3 + 1.9 mg 2,3,4,6-TeCP/I and 3.8 + 0.6 mg PCP/I. Practical grade 2,4,6-TCP was produced by Fluka Chemic AG, Buchs, Switzerland and the preparation of 2,3,4,6-TeCP by American Tokyo Kasei Inc., Tokyo, Japan. According to our analysis the TeCP preparation contained 11% of PCP. Chemical determinations
Inorganic chloride (ICI) was determined with a chlorideion selective electrode (W. lngold AG, Urdorf, Switzerland, Type 15 213 3000) and an Ingold Ag/AgCI reference electrode (Type 373-90-WTE-ICE-S7) using an Orion pH/mV meter (Orion Research Inc., Kusnecht, Switzerland, Model SA 720) at 20 + I°C. Total organic carbon (TOC) was measured from acidified samples using a Unicarbo universal carbon analyzer (Elektrodynamo, Helsinki, Finland). Dissolved oxygen concentration in the reactor was monitored using a WTW oxygen electrode (Wissenschafi. Tech.
Table 2. Chloride release and TOC removal efliciencies from 2,4.6-trichlorophcnol at dilTcrent loading rates and hydraulic retention times (HRT) in fluidized-bcd reactor ICI release
Expt No. I 2 3
TCP-loading rate (rag/I/d) 260 325 430
inorganic chloride; N dcterrained.
ICI =
-
HRT (h) 5
(rag/I) 31.0
(SD) 0.8
TOC removal
(rag/I) 18.9
(SD) 1.5
N 10 4 28.8 1,9 18.9 0.9 10 3 32,8 0.5 ND 10 number of samples during pseudo-steady*stateexperiments; SD = standard deviation; ND = not
767
Fluidized-bed treatment of chlorophenols Werkst/tten, Weilheim, Germany, Model EO 96) and an oxygen meter (WTW, Model OXI 96). The chlorophenolic compounds from 0.5 ml samples were acetylated with 0.5 ml acetic anhydride using 25 ml of 0.1 M K~CO3 as buffer. The acetylated derivatives were extracted with 2.5 ml n-hexane and a 2.00 #1 extract was used for injection. 2,3,6-TCP was used as the internal standard. GC analyses were carried out on a Perkin-Elmer Sigma 200 gas chromatograph (Perkin-Elmer Instrument Division, Norwalk, Conn., U.S.A.) equipped with a eNi electron capture detector and the DP-5 column (30 m x 0.25 nun ID x 0.25 #m) supplied by J&V Scientific (Folsom, Calif., U.S.A.), with a nitrogen flow-rate of I.Sml/min. The
20
I 25
130m
TOC,,
Chloride 80 6O E 40 20 0
i
|
i
i
a
i
a
TOC
3O I
zo E 10
i
2 , 3 , 4 , 6 -TeCP 90 "-
60
E
30
.
.
.
.
.
.
.
v
v
-
-
_ _ -
I
PCP
~ E
8
4
~ |
0
30
i
60
a
90
i
120
Time (doys } Fig. 2. Fluidized.bed bioreactor performance at increasing 2,3,4,6-t©trachlorophenol loading rates. 171, Feed; O, effluent. WR
~/6--E
temperature program was: oven temperature at 100°C for I rain, then increased at YC/min to 250°C. The injection temperature was 2.50~C. RESULTS First, the degradation performance of 2,4,6-TCP was studied in fluidized-bed reactor using a 20 mg TOC/I feed concentration. The effect of TCP loading rate and HRT on chloride release and TOC removal is shown in Table 2. ICI releases indicated 100% dechlorination of 2,4,6-TCP in all three pseudosteady-state experiments The feed pH was 7.3-7.5 decreasing during treatment to 6.9-7.0. TOC removals were 82-83% in the first and second experiment, respectively. The observed ACI -/ATOC ratios (mg chloride/rag TOC) of 1.5 and 1,6 agree well with the theoretical value of 1.5. GC analyses of 6 effluent samples (3 from Expt 2 and 3 from Expt 3) gave 2,4,6-TCP concentrations of 0.1 mg/I or less. These results demonstrate that 2,4,6-TCP was readily degradable by the immobilized aerobic microbes and that a 40% increase in dilution rate did not affect the reactor performance. After the 2,4,6-TCP experimentation, the reactor feed was switched to 2,3,4,6-TeCP solution containing l 1% PCP as an impurity. The TeCP feed concentration was set at 20 mg TOC/I and fed at 5 h HRT for 3 weeks until effluent ICI concentration was stabilized at over 40 mg/I. The process performance was monitored for 2 weeks at this chlorophenol feed which was then gradually increased to 30 mg TOC/I while the HRT remained at 5 h. These feed patterns corresponded with loading rates between 230 and 400 g chlorophenol/m~/d, The results of overall reactor performance from 130 days of operation are presented in Fig. 2. TeCP and PCP mineralization reaction stoichiometries yield 2 and 2.5 mg ICI, respectively, per milligram of TOC removed. According to analyzed feed chlorophenol concentrations during the 130 day period the mean ICI release would have been 45.9mg/I while 56.6mg ICI/I was observed. This indicates that other than chlorophenol bound chlorine was released as well from the 2,3,4,6-TeCP solution produced from the practical grade preparation. Observed mean TOC removal of 21.7 mg/I was in conformity with the mean 99.3% 2,3,4,6-TeCP and 74.5% PCP removals which in terms of TOC indicate 22.9 mg TOC/I removal. Most of the effluent TOC accounted for is by the tap water background TOC (4-5 mg/I). The results also show variation at the substrate loading rate of 400 g chlorophenol/m3/d and the highest chlorophenol feed concentration indicating reactor operation near the maximum substrate utilization capacity or near the inhibitory feed concentration. The chlorophenol loading rate was further increased to 810 g/mJ/d and process performance was monitored by ICI release for 8 weeks. ICI formation results at different chlorophenoi loading rates are
768
JAAKKOA. PUHAKKAand KIMMOJ,~RV1NEN 120 100
o°
t.°
I"
4O
0
0
,6o
66o
s6o
tooo
Loading rate (g/m 3. (3")
Fig. 3. The effect of chlorophenol loading rate on chloride release. ©, Calculated maximum |CI releasesfrom 2,3,4,6TeCP plus PCP constituents of the CP-preparation: A,
nolle wood preservative constituents in oxic fluidizedbed reactors. They are consistent with extensive data on the biodegradability of these compounds (e.g. H~iggblom, 1990). Various techniques have been tested for aerobic treatment of polyehlorophenol contaminated water. Etzei and Kirsch (1974) reported 99% PCP removal from wood treatment wastewater containing 2060 mg PCP/I by a mixed aerobic suspension culture at HRTs of 6-12 h. Valo et al. (1989) immobilized chlorophenol-mineralizing rhodococci on a biofilter cartier for treatment of technical chlorophenol (a
observed ICI releases.
Chloride 40
summarized in Fig. 3. The straight line shows calculated maximum ICI releases from 2,3,4,6-TeCP and PCP constituents of the technical grade chlorophenol preparation at different loading rates. ICI release exceeded that of the complete TeCP plus PCP deehlorination at the 230-400 g/m~/d loading rates indicating dechlodnation of unidentifed impurities of the chlorophenol preparation as well. IC! release remained stable (70.9 _+8.0 mg/l; number of analyses 9) during the last 2 weeks of operation at 810 g/m'/d representing 67% chlorophenol dcchlorination. Stable operation indicates that the substrate convcrsion efficiency was limited rather by the maximum substrate utiliz~Ltion rate than the toxicity of substrate at the high loading rate. Finally, we studied a chlorophenol mixture which simulated groundwater contaminated with chlorophenolic wood preservative. This experiment was carried out in reactor 2 which had received monoand diehlorophenol feeds only. The reactor was fed a mixture of 2,4,6-TCP, 2,3,4,6-TeCP and PCP at chlorophenol loading rate of 217 g/m~/d and HRT of 5 h for 5 weeks. Figure 4 presents the results of the following pseudo-steady-state degradation of simulated groundwater at 5 h HRT. The mean chloride release was 31.4mg/I. The respective mean chlorophenol removals were as follows: 99.5% for 2,4,6TCP, 99.6 for 2,3,4,6-TeCP and 92.5% for PCP accounting for 26.8mg/I release of chlorophenol bound chlorine. This again indicates 15% additional ICI formation from unidentified simulated groundwater constituents. As a result of dechlorination, pH dropped from 7.1 to 6.7. The mean TOC removal of 14. I mg/I agrees well with the measured chlorophenol removals. The mean effluent TOC of 4.9 mg/I corresponds with the background TOC from the tap water used for feed dilution. The results show efficient and simultaneous removal of wood preservative chlorophenol constituents. Release of ICI and evolution of H * as seen by pH decrease indicate biodegradation. DISCUSSION The results of this investigation showed continuous high-rate dechlorination/degradation of ehlorophe-
30
10
DC~DD s
IDCCCD i
!
i
i
=
TOG 2O u
"" 10 5
o 16
~ . ~ G - - - - - O 0 0 O0 i
I
I
I
i
I
2,4,6-TCP
~ 12 v
8 4 ~.J
"~.Jx-J~../N.,J
= 2O
10 0
=
3
o
O ~" 2
"-' 4
~ O¢:L~:~ 6
Time
8
lO
12
(days)
Fig. 4. Simulated groundwater treatment performance in fluidized-bed biorcactor under pseudo-steady-state conditions, rl, Feed: O, emuent.
769
Fluidized-bed treatmcnt of chlorophcnols mixture of 2,4,6-TCP, 2,3,4,6-TeCP and PCP) at concentrations of 3.4--130 mg/l. The batch operation resulted in the elimination of chlorophenois from the influent and 40% release of chlorophenol bound chlorine as ICI. In another study, PCP (55 rag/I) degradation by polyurethane-immobilized Flavobacterium was studied in the presence of 0.5 g glutamate/l (O'Reilly and Crawford, 1989). At the residence time of 8.3 h, more than 90% of PCP was degraded over a period of 25 days after which the degradation efficiency started to decline. Anaerobic treatment of chlorophenols often results in partially or fully dechlorinated products requiring complementary aerobic treatment for complete mineralization. Krumme and Boyd (1988) used anaerobic upflow bioreactors to treat three monochlorophenols and 3,4,5-TCP and reached substrate conversion efficiency of > 9 0 % at substrate loading rates of up to 20 mg/I/d (corresponding HRT 2--4 days). Woods et al. (1989) studied chlorophenoi degradation in an upflow anaerobic sludge blanket reactor in the presence of high concentrations of readily biodegradable organic compounds. Among other chlorophenols, 2,4,6-TCP, 2,3,4,5-TeCP and PCP at concentrations of 0.I-I.0 mg/I were converted to lesser chlorinated compounds at the HRT of 13 h. Our results on aerobic fluidized-bed treatment of polychlorophcnols compare favorably with the above summarized results on previous aerobic and anaerobic treatments. First, no supplemental carbon or energy source was required for continuous operation and maintenance of the mixed chlorophenol degrading culture so that the possibility of o-methylation (Suzuki, 1983, Hiiggblom et al., 1989) would be expected to be minimal. Methylation increases the lipophilicity of chlorophenols and thus their tendency to bioaccumulation (Lechet al., 1978). In addition, the methylated compounds may be more toxic than their precursors (Neilson et al., 1984). In reactor I, chlorophenol degradation was first maintained with mono- and dichlorophenols for over I year (Shieh et aL, 1990) and then continued for 315 days with polychlorophenols as presented in this study. This shows that microbial cells entrapped within the pores and on the surface of the carrier were protected from shear forces and carrier collisions and remained active in a variety of operational conditions. Furthermore, the celite carrier had a high persistence to the mechanical friction caused by the 50% upflow velocity. Second, no inhibition due to high feed chlorophenol concentrations was observed. This is due to the high recycle ratio applied which in addition to providing completely mixed conditions effectively diluted the feed. Third, polychlorophenol loading rates were higher and HRTs were lower at removal efficiencies of >99% for 2,4,6-TCP and 2,3,4,6-TeCP than reported earlier. Polychlorophenol degradation performances were similar to those observed with mono-
and dichlorophenols in our earlier studies (Shieh et al., 1990; Puhakka et al., 1991b).
In aerobic waste treatment, often parallel biochemical mechanisms are in operation. Hence, the oxic mixed culture process is considered more stable against toxicity and varying operational conditions than pure culture processes. Scanning and transmission electron microscopy of reactor carrier material after an additional year of operation with polychlorophenol contaminated groundwater showed a diverse microbial community and an immobilized biomass concentration of 6-7g/1 (Puhakka et al., in preparation). Synergistic action of microorganisms may add to the advantages of mixed culture approaches. This was apparent in 2,4-DCP degradation by a two-component consortium of bacteria isolated from reactor 1 during dichlorophenol studies (Puhakka et al., 1991 b). CONCLUSION
This study shows that the use of oxic fluidized-bed reactors supplied with porous carrier for adsorptive and entraptive cell immobilization is an effective method for continuous, high-rate dechlorination and removal of polychlorophenols from water. Immobilized mixed cultures can be maintained with chlorophenols as the only source of carbon and energy. Over 99% biodegradation efficiencies are achievable at a substrate loading rate of 400 g 2,4,6TCP or 2,3,4,6-TeCP/m~/d and 3-5 h HRTs. Simultaneous treatment of a mixture of 2,4,6-TCP, 2,3,4,6-TeCP and PCP is also achievable. Thus, the oxic fluidized-bed process is an attractive treatment alternative for groundwater contaminated with wood preservation chemicals typically consisting of polychlorophenol congeners. Acknowledgements--This work was financially supported
by the Maj and Tor Nessling Foundation, Finland (K.J.) and the Academy of Finland (J.A.P.). We thank Professor Wen K. Shieh (University of Pennsylvania. U.S.A.) for initiation of the fluidized-bed treatment studies at the Tampere University of Technology and fruitful discussions during this study and Mr Juhani Tarhanen and Mr Jouni Rokkanen (University of Kuopio, Finland) for GC analyses. REFERENCES
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770
J~KKO A. PU~KKA and Kl~,a~o J~vL,,~m
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