2,4-Dichlorophenoxyacetic Acid

Transcription

1 APPLID AND NVIRONMNTAL MICROBIOLOGY, May 1986, p Vol. 51, No /86/595-6$2./ Copyright C) 1986, Amerian Soiety for Mirobiology ffets of Dissolved Oxygen Conentration on Biodegradation of 2,4-Dihlorophenoxyaeti Aid T. A. SHALRt AND G. M. KLCKA* nvironmental Chemistry Researh Laboratory, Dow Chemial U.S.A., Midland, Mihigan Reeived 1 November 1985/Aepted 2 February 1986 Bath experiments were onduted to examine the effets of dissolved oxygen onentration on the degradation of 2,4-dihlorophenoxyaeti aid (2,4-D) by an enrihment ulture of 2,4-D-utilizing bateria. A modified Monod equation was found to desribe the relationship between the speifi growth rate and the onentrations of both the organi substrate and dissolved oxygen. Values for the maximum speifi growth rate, yield, and Monod oeffiient for growth on 2,4-D were.9 h-1,.14 g/g, and.6 mg/liter, respetively. The half-saturation onstant for dissolved oxygen was estimated to be 1.2 mg/liter. These results suggest that dissolved oxygen onentrations below 1 mg/liter may be rate limiting for the biodegradation of hlorinated aromati ompounds suh as 2,4-D, whih have a requirement for moleular oxygen as a osubstrate for metabolism. Aerobi miroorganisms utilize oxygen primarily as the terminal eletron aeptor for aerobi respiration. In addition, moleular oxygen is required as a osubstrate for the mirobial degradation of wide variety of organi hemials, inluding hydroarbons and aromati ring ompounds (6-8). Although aerobi miroorganisms appear to have a high affinity for dissolved oxygen, the effets of oxygen limitations on mirobial ativities have been noted. Muh of the work on the effets of dissolved oxygen onentration have been onerned with its effets on mirobial growth (4, 5, 13, 14, 27) and respiration rate (2). In general, baterial respiration does not appear to be affeted above a ritial dissolved oxygen onentration. The ritial dissolved oxygen onentration has been defined as the onentration at whih the respiration rate of the ells is one-half of the maximum rate observed at saturating levels; it is generally lower for dispersed ultures than for floulant ultures (11). Longmuir (2) reported ritial dissolved oxygen onentrations in the range of.1 to.38 mg/liter for a variety of yeast and baterial ultures. For floulant mirobial ultures, the ritial dissolved oxygen onentration is generally higher and is typially in the range of.5 mg/liter (11). Above these ritial oxygen levels, inreases in oxygen onentration have little effet on mirobial respiration rates. Mirobial growth rates have also been shown to be influened by the availability of dissolved oxygen (4, 5, 13, 14, 27). The effets of oxygen onentration on the growth rate have been examined by using ontinuous-ulture tehniques for a number of miroorganisms growing on substrates suh as aetate (14), glyerol (4, 27), and gluose (5, 13). The relationship between the oxygen onentration and mirobial growth rate has been shown to be onsistent with Monod kinetis. Hao et al. (13) reported half-saturation onstants for oxygen (Kdo) in the range of.14 to.73 mg/liter for several Sphaerotilus speies growing on gluose as the sole arbon soure. Sinlair and Ryder (27) examined the growth of Candida utilis on glyerol in ontinuous * Corresponding author. t Present address: Department of Chemistry, University of California Riverside, Riverside, CA ulture under dissolved-oxygen- and arbon-limiting onditions and reported a Kd value of.84 mg/liter. Additional studies with C. utilis by Johnson (14) and Button and Garver (4) indiated Kdo values of.42 and.45 mg/liter for growth on aetate and glyerol, respetively. Reently, Chen et al. (5) examined the kineti effets of oxygen on the aerobi growth of Azotobater vinelandii and sherihia oli on gluose and reported Kd values of.17 and.13 mg/liter, respetively. Relatively little is known about the influene of dissolved oxygen on the mirobial degradation of organi hemials. Larson et al. (19) reported that the half-life for the biodegradation of nitrilotriaeti aid in natural water samples inreased from 1.3 to 5.8 days as the dissolved oxygen onentration dereased from saturating levels to approximately.3 mg/liter. From these results, a half-saturation onstant for dissolved oxygen of approximately 1.4 mg/liter an be alulated. Tiedje and Mason (29) also noted that the degradation of nitrilotriaeti aid was limited under miroaerophili onditions in soils. Rates of the reation were omparable in soil perfusion olumns inubated in the presene of air or nitrogen mixtures ontaining 1.% oxygen but were substantially redued when the oxygen onentration in the gas phase was redued to.1%. The availability of oxygen has also been shown to influene the rates of hydroarbon degradation (12, 3), whih is onsistent with the role of moleular oxygen as a osubstrate for mirobial hydroarbon degradation. Hambrik et al. (12) noted that the degradation of otadeane and naphthalene in estuarine sediment slurries was highly dependent on the availability of dissolved oxygen. Sediment suspensions inubated at -22 mv mineralized approximately.6% of the applied naphthalene over a 35-day period. However, when the redox potential of the sample was inreased to + 13 mv, an additional 22.6% of the ompound was mineralized after a 35-day inubation period. Ward and Brok (3) similarly reported that hexadeane was rapidly mineralized in freshwater lake sediments under aerobi onditions, whereas negligible degradation ourred under anaerobi onditions. Although the availability of oxygen has been shown to limit the mirobial degradation of a number of organi hemials, relatively few studies have attempted to desribe 95 Downloaded from on July 7, 218 by guest

2 VOL. 51, 1986 these effets quantitatively. Consequently, the present study was onduted to examine the effets of dissolved oxygen onentration on the biodegradation of 2,4-dihlorophenoxyaeti aid (2,4-D). 2,4-D was hosen as a model beause it is representative of a variety of hlorinated aromati ompounds. In addition, moleular oxygen is known to be required as a reatant for the mirobial metabolism of 2,4-D for the initial leavage of the ether side hain and both the hydroxylation and subsequent fission of the aromati nuleus (22). MATRIALS AND MTHODS Isolation and maintenae of organisms. A mixed baterial ulture apable of utilizing 2,4-D as the sole arbon soure was isolated from a sample of industrial sewage by using a ontinuous-ulture enrihment tehnique as previously desribed (16). The ulture vessel was onstruted from a 2-liter Pyrex resin kettle with an overflow tube plaed at the 1-liter level. Culture medium was supplied to the reator from a 4-liter reservoir via glass and silione rubber tubing. The flow rate of the medium was ontrolled by a peristalti pump (model 72-5-; Manostat Corp., New York, N.Y.). The ulture was aerated with filtered and humidified air at a rate of 25 ml/min and agitated with a star-head magneti stirrer. The reator was maintained in a onstant temperature inubator at 25 C. The ontinuous ulture was maintained on a mineral salts medium ontaining 2 mg/liter of 2,4-D as the sole arbon soure. The medium ontained (per liter): Na2HPO4, 7.6 g; KH2PO4, 3.6 g; NH4Cl,.5 g; MgSO4-7H2,.1 g; CaC12. 2H2,.2 g; FeSO4. 7H2,.1 g; ZnSO4- H2,.5 g; and Na2MoO4 2H2,.2 g. 2,4-D was added to the medium from a stok solution (2 g/liter) prepared in.2 N NaOH. The medium was adjusted to ph 7.5 with H3PO4. The reator was initially operated on a disontinuous basis until onsistent substrate removal was noted. At that point, the medium was ontinuously supplied to the reator at a flow rate of approximately 4 ml/h to maintain a onstant hydrauli residene time of 1 days. Samples were removed daily from the reator for 2,4-D analysis and periodially for ell mass determinations. One stable operation of the reator was noted, the ontinuous ulture provided the soure of inoulum for subsequent kineti experiments. Kineti experiments. Bath ulture experiments were onduted in water-jaketed aeration ylinders (Fig. 1). The units were onstruted from glass ylinders (46 by 6 m) and had a working volume of 1 liter. A side arm allowed for reirulation of the ontents of the ylinder and failitated the diret measurement of dissolved oxygen onentrations or removal of samples for 2,4-D analysis. Kineti experiments were onduted in 1.5-liter aeration ylinders ontaining 1 liter of the mineral salts medium desribed above. The ylinders were supplemented with various onentrations of 2,4-D and inoulated with 1 ml of the enrihment ulture. Cultures were inubated at various dissolved oxygen onentrations by supplying air or a mixture of oxygen in nitrogen gas at a flow rate of 5 ml/min. Inubation temperatures were maintained at a onstant 25 C by irulating water from a ontrolled bath through the water jakets on the aeration ylinders. The ultures were analyzed periodially for dissolved oxygen and 2,4-D onentrations, as desribed below. Analytial methods. 2,4-D was analyzed by high-pressure liquid hromatography. Culture samples were initially filtered through.45-,um Arodis disposible filter units FFCTS OF OXYGN ON 2,4-D DGRADATION 951 (Gelman Sienes, In., Ann Arbor, Mih.) and aidified to ph 2 with onentrated HCI. Chromatography was performed with a system omposed of an automati sample proessor (model 71B; Waters Assoiates, In., Milford, Mass.), a solvent delivery system (model 59, Waters), and an LDC Spetromonitor III variable-wavelength detetor adjusted to 214 nm. Separations were ahieved on a Partisil 5-ODS-3 RAC II reverse-phase analytial olumn (1 by 4.6 mm; Whatman In., Clifton, N.J.) with a solvent system omposed of aetonitrile-water (35:65) ontaining.57 M NaCIO4 and.5 M KH2PO4 and aidified to ph 3.3 with H3PO4. The flow rate was.8 ml/min at approximately 1, lb/in2. The output of the detetor was onneted to a reording integrator (model 73; Waters). 2,4-D onentrations were alulated on the basis of peak area measurements by omparison with an external standard prepared in mineral salts buffer (ph 2). The detetor response was linear over the onentration range of interest (5 to 25,,g/liter), with a detetion limit of approximately 5,ug/liter. Dissolved oxygen onentrations were measured diretly in the aeration ylinders with an oxygen eletrode (model 97-8; Orion Researh, In., Cambridge, Mass.) and digital ionanalyzer (model 51, Orion). The eletrode was alibrated with water-saturated air before eah set of measurements. Gas mixtures (2-N2) were analyzed with a gas partitioner hromatograph (model 12; Fisher Sientifi Co., Pittsburgh, Pa.). The instrument was used in the two-olumn mode; olumn 1 (2. m by 3.2 mm) was paked with Columpak PQ (8 to 1 mesh, Fisher), and olumn 2 (3.4 m by 4.8 mm) was paked with Moleular Sieve 13X (6 to 8 mesh, Fisher). Samples were introdued into the olumns through a 2.5-ml sample loop. Components of the mixture were eluted isothermally at 5 C with a helium arrier gas at a flow rate of 15 ml/min. The omponents were deteted with a thermal ondutivity detetor at a bridge urrent of Water - Jaket FIG. 1. Modified aeration ylinder. Sample Port Downloaded from on July 7, 218 by guest

3 952 SHALR AND KLiKA 225 ma. The output of the detetor was onneted to a reording integrator (model 3392A; Hewlett-Pakard Co., Palo Alto, Calif.). All values were alulated from peak area measurements by omparison with an external standard of air-zero gas (Sott Speialty Gases, Troy, Mih.). Cell mass onentrations were determined by gravimetri analysis as previously desribed (2). Data analysis. The results of the kineti experiments were analyzed with an IBM 37 omputer by using the Dow advaned omputer simulation language (DACSL [1]). The kinetis of 2,4-D degradation were analyzed by using Monod equations (21) for growth and substrate utilization: dx (S dt KmaxK + 5 d ds (Lmax *X' ( S A dt Y JKK5+S) where Umax is the maximum speifi growth rate oeffiient, X is the ell mass onentration, Y is the yield oeffiient, S is the 2,4-D onentration, KS is the Monod onstant for 2,4-D, and kd is the endogenous deay oeffiient. To simplify the model alulations, the kd value was assumed to be.2 h-1, whih is in the range of values frequently observed for heterogenous mirobial populations (11). The effets of dissolved oxygen onentration on 2,4-D degradation were analyzed by using modified Monod equations (4, 13, 14, 27): dx /S\ DO\ y- = Lmax'XIK +-~+O -kd -X dt = &X*XT + S tkdo + DO) -ds (PLmax* X\(S \/ DO dt ky JIKs + SAKdo + DOJ where DO is the dissolved oxygen onentration and Kdo is the half-saturation onstant for dissolved oxygen. The kineti parameters (Pmax, Y, K5, and Kdo) were estimated by omputerized numerial integrations by fitting the equations for growth and substrate utilization to the shape of the substrate onentration versus time urves. The Adams-Moulton algorithm was used for the integrations. Note that the differential forms of the equations were used during the omputer analysis. Chemials. Analytial grade 2,4-D (99.95%) was obtained from the Dow Chemial Company, Midland, Mih. Gas mixtures ontaining.5, 1.3, 2.6, and 5.% oxygen in nitrogen were purhased from Sott Speialty Gases. The aetonitrile (distilled-in-glass grade) used for liquid hromatography was obtained from Burdik and Jakson Laboratories, Muskegon, Mih. All other hemials were reagent grade. RSULTS Kinetis of 2,4-D biodegradation. Initial studies were performed to evaluate the kinetis of 2,4-D degradation by the laboratory enrihment ulture. Biodegradation rates were examined in 1-liter bath ultures inoulated with approximately.8 mg of ells and supplemented with initial 2,4-D onentrations ranging from.2 to 2. mg/liter. Dissolved oxygen onentrations in the ultures were maintained at saturating levels (8. mg/liter). The results of the study are shown in Fig. 2. XrX-. ~~~~~~~~~~Y =.149g/g.. APPL. NVIRON. MICROBIOL FIG. 2. Kinetis of 2,4-D degradation by the enrihment ulture. Bath tests were onduted in 1.5-liter aeration ylinders ontaining 1 liter of mineral salts medium,.8 mg of ells, and various onentrations of 2,4-D. Cultures were inubated at 25 C and aerated with air at a flow rate of 5 ml/min. Kineti parameters were estimated by fitting the equations for growth and substrate utilization to the experimental data, as desribed in the text. The data were analyzed with a omputer simulation model to obtain values for the kineti parameters (tlmax, Y, and Ks). During omputer analysis, values for the kineti parameters and the initial ell mass onentration (X) were varied individually to obtain the best fit of the experimental data. Final values for eah of the parameters were obtained by averaging the results of omputer simulations of the individual bath experiments. Average values for the parameters were as follows: Lmax =.9 h'-; Xo =.2 mg/liter; Y =.14 g/g; and K, =.6 mg/liter. The validity of the average values was tested by simultaneously applying the oeffiients to desribe the kinetis of 2,4-D biodegradation over the entire range of initial substrate onentrations. There was exellent agreement between the measured and predited 2,4-D onentrations (Fig. 2). Note that the X value estimated with the omputer simulation model was somewhat lower than the measured suspended solids onentration of the inoulum. This was presumably beause of a differene in the onentrations of ative and total miroorganisms in the mixed ulture. However, the exellent agreement observed over the entire range of initial substrate onentrations suggests that the average values adequately desribed the kinetis of 2,4-D biodegradation by the enrihment ulture. ffet of dissolved oxygen onentration on 2,4-D biodegradation. The effet of dissolved oxygen onentration on the rate of 2,4-D degradation by the laboratory enrihment ulture was examined in 1-liter bath ultures ontaining.2 mg/liter of 2,4-D and.6 mg/liter of ells. The ultures were maintained at onstant dissolved oxygen onentrations ranging from.25 to 8.2 mg/liter by aeration with air or a mixture of oxygen (.5 to 5.%) in nitrogen gas. The results of the study are shown in Fig. 3. The rates of 2,4-D degradation were essentially idential in ultures inubated with dissolved oxygen onentrations above 2 mg/liter. In ontrast, biodegradation rates were redued when the dissolved oxygen onentration was less than 1 mg/liter. To determine the half-saturation onstant for dissolved oxygen (Kdo), the experimental data were analyzed with the omputer simulation model by using the oeffiients for 2,4-D degradation obtained from the results Downloaded from on July 7, 218 by guest

4 VOL. 51, 1986 A -J o ) U s FFCTS OF OXYGN ON 2,4-D DGRADATION 953 B 21 DO = 2. mg/l \.16 Kd= 1.2 mg/l -16 \ mx=.9 h-i ) \ X =.16 mg/l Y=.14g/g C 12 K,.6 mg/l ie\ C 8\ N~ 4 C: C a C T a) a 2 DO = 1. mg/l Kdo= 1.2 mg/l 16 -\, m,. =.9 h-1 XO =.16 mg/l Y =.14 g/g K, =.6 mg/l O of previous kineti experiments. The be 1.2 mg/liter. I D DO =.54 mg/l.kd= 1.2 mg/l X 16 \max =.9 h m *M XO=.16 mg/l Y =.14 g/g K, =.6 mg/l 12 o a)\ o FIG. 3. ffets of dissolved oxygen onentration on biodegradation of 2,4-D by the enrihment ulture. Bath tests were onduted in 1.5-liter aeration ylinders ontaining 1 liter of mineral salts medium,.6 mg of ells, and.2 mg of 2,4-D. Cultures were inubated at 25 C and aerated with air or a mixture of oxygen in nitrogen gas to maintain onstant dissolved oxygen onentrations (milligrams per liter) as follows: 8.2 (A), 2. (B), 1. (C),.54 (D), and.25 (). Kineti parameters were estimated by fitting the equations for growth and substrate utilization to the experimental data, as desribed in the text. of 2,4-D degradation over a wide range of initial substrate onentrations. The rate of 2,4-D biodegradation was shown to be influened by the availability of dissolved oxygen, whih is onsistent with the role of moleular oxygen as a osubstrate for the initial leavage of the ether side hain and both the hydroxylation and subsequent fission of the aromati nuleus (22). Biodegradation rates were substantially redued Kd was estimated to when the dissolved oxygen onentration was less than 1 mg/liter. In ontrast, there was little inrease in the removal rate at dissolved oxygen onentrations above 2 mg/liter. Analysis of the experimental data indiated that the rate of DISCUSSION Bath experiments to examine the kinetis of 2,4-D utili- 2,4-D degradation was a hyperboli funtion of the dissolved zation by the mixed enrihment ultiure were initially on- oxygen onentration (Fig. 4). The half-saturation onstant duted to provide a framework for Ifurther analysis of the for oxygen (Kd) was estimated to be 1.2 mg/liter. effets of dissolved oxygen onenttration on degradation The Kdo value for 2,4-D biodegradation was onsiderably rate. The biodegradation of 2,4-D ait saturating dissolved higher than the range of values reported -for mirobial oxygen onentrations was shown to be onsistent with respiration (2) or growth on substrates suh as gluose, Monod kinetis. Values for the kineti oeffiients were ULmax glyerol, and aetate (4, 5, 13, 14, 27), in whih oxygen is =.9 h-1, Y =.14 g/g, and Ks =.6 mg/liter and were required prinipally as the terminal eletron aeptor for the similar to those previously reported by Shamat and Maier ytohrome oxidase. For example, respiration rates for a (26). The validity of the kineti parameters was onfirmed by variety of yeast and baterial ultures do not appear to be simultaneously applying the oeffiieints to predit the rates affeted until the dissolved oxygen onentrations approah Downloaded from on July 7, 218 by guest

5 954 SHALR AND KLtKA APPL. NVIRON. MICROBIOL. TABL 1. Half-saturation onstants (Kdo) for various oxygenases Oxygenase Soure Kd,t Referene (mg/liter) Catehol-1,2-dioxygenase Pseudomonas arivilla C Catehol- 2,3-dioxygenase Pseudomonas sp. OC ,4-Dihydroxy-phenylaetate-2,3-dioxygenase "Pseudomonas ovyalis" (Pseiudomonas putida).8 15 Melilotate-hydroxylase Pseudomonas sp Orinol-hydroxylase Pseudomonas putida Phenol-hydroxylase Trihosporon utaneuim Protoatehuate-3,4-dioxygenase Pseudomonas aeruiginosa Protoatehuate-4,5-dioxygenase Pseudomonas sp Tryptophan-2,3-dioxygenase Pseudomonas aidovorans =.5 (D/ 11 = 11max ~Kdl DO Dissolved Oxygen Conentration (mg/l) FIG. 4. Relationship between dissolved oxygen onentration and mirobial growth rate alulated by using the Monod equation. The values of the parameters used for alulation were P-max =.9 h-' and Kdo = 1.2 mg/liter. a range between.1 and.38 mg/liter (2). In ontrast, the Kdo value for 2,4-D biodegradation was similar to the Kdo value of 1.4 mg/liter alulated from the results of Larson et al. (19) for the degradation of nitrilotriaeti aid in natural water samples. Beause the mirobial metabolism of nitrilotriaeti aid under aerobi onditions requires the ativity of an oxygenase (9), these observations suggest there may be fundamental differenes in the affinities of oxygen-utilizing enzymes for moleular oxygen. Kdo values have been measured for a number of mirobial oxygenases (Table 1). Note that with the exeption of the atehol dioxygenases, relatively high Kdo values have been reported for a number of mirobial oxygenases responsible for either the initial hydroxylation of the aromati ring or fission of the aromati nuleus. Therefore, dissolved oxygen onentrations in the range of 1 mg/liter.may be rate limiting for the mirobial degradation of many aromati ompounds whih have a requirement for moleular oxygen as a osubstrate. ACKNOWLDGMNTS We aknowledge the work of Stanley Dagley, who has ontinued to emphasize the importane of moleular oxygen in the mirobial metabolism of organi ompounds in the biosphere. We thank Walter Maier for many helpful disussions during the development of this projet, Lori Likly for assistane with the omputer models, Celia Mendoza for analytial troubleshooting, and Linda Roy for assistane in preparing the manusript. LITRATUR CITD 1. Agin, G. L., and G.. Blau Appliation of DACSL (Dow advaned ontinuous simulation language) to the design and analysis of hemial reator systems, p In R. S. H. Mah and G. V. Reklaitis (ed.), Seleted topis on omputeraided proess design and analysis. AICh Symposium Series 214, vol. 78. Amerian Institute for Chemial ngineers, New York. 2. Amerian Publi Health Assoiation Standard methods for the examination of water and wastewater, 15th ed. Amerian Publi Health Assoiation, Washington, D.C. 3. Brady, F. O., M.. Monao, H. J. Forman, G. Shutz, and P. 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