Environ. Scl. Technol, 1987, 21, 280-288
Kim, B. R., Ph.D. Dissertation, University of Illinois, Urbana, IL, 1977. Crittenden, J. C.; Wong, B. W. C.; Thacker, W. E.; Snoeyink, V. L.; Hinrichs,R. L. J.-Water Pollut. Control Fed. 1980, 52, 2780.
Crank, J. Q. J. Mech. Appl. Math. 1957,10, 220-231. Finlayson, B. A. The Method of Weighted Residuals and Variational Principles; Academic: New York, 1972; Chapter 5. Stroud, A. H.; Secrest, D. Gaussian QuadratureFormulas; Prentice-Hall: Englewood Cliffs, NJ, 1966; Chapter 3. Hindmarsh, A. C. ACM-SIGNUMNewsletter, 1980, 15, 10-11.
DiGiano, F. A.; Dovantzis, K.; Speitel, G. E., Jr. Environmental Engineering, Proceedings of the 1984 Specialty Conference of ASCE, Los Angeles, CA; Pirbazari, M.; Devinny, J. S., Eds.; American Society of Civil Engineers: New York, 1984.
(35) Speitel, G. E., Jr.; Dovantzis, K.; DiGiano, F. A. J. Environ. Eng. Diu. (Am. SOC. Civ. Eng.), in press. (36) Andrews, G. F.; Tien, C AZChE J . 1981,27, 396-403. (37) Ying, W.; Weber, W. J., Jr. Proceedings of the 33th Purdue
Industrial Waste Conference; Ann Arbor Science: Ann Arbor, MI, 1978; pp 128-141.
Received for review February 10, 1986. Accepted October 27, 1986. Although the informationdescribed in this paper has been funded wholly by the U.S. Environmental Protection Agency under Assistance Agreement EPA Cooperative Agreement CR810462 to the Advanced Environmental Control Technology Research Center, it has not been subjected to the Agency's required peer and administrative review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
Verification of the Model of Biofilm on Activated Carbon H. Ted Chang" and Bruce E. Rittmann
Department of Civil Engineering, University of Illinois, Urbana-Champaign, Illinois 6 1801
rn Laboratory-scale column reactors were used to verify the model of biofilm on activated carbon (BFAC model). The effluent from the reactor was recycled so that the reactor approached a completely mixed regime. Twelve Coefficientswere measured independently from the column studies, while three parameters had to be assumed so that the model simulated the substrate concentration results very well. The BFAC model was verified with biofilms grown on spherical activated carbon (BACM) packed in the column reactor. The detailed sequence of the bioregeneration, modeled by the BFAC model, showed that the substrate flux in the activated carbon sequentially was positive during adsorption, was negative during bioregeneration, and approached zero toward steady state. Although the BFAC model accurately described the substrate concentration and the sequence of bioregeneration, it predicted lower effluent suspended biomass than the experimental results. Further research is needed to understand the mechanisms of the shearing loss of biofilms. Introduction
Chang and Rittmann (I)presented a model for the kinetics of biofilm on activated carbon (the BFAC model). The model incorporated the mechanisms of film transfer, biodegradation, and adsorption of a substrate, as well as biofilm growth. Model formulation and solution were tailored to the unique features of the BFAC system: namely, diffusion across a moving boundary, diffusion with nonlinear reaction, and diffusion in layered media. Solution was by the global orthogonal collocation method (GOCM) (I), which provides time-dependent values of substrate concentration, biofilm thickness, and substrate fluxes. This paper presents experimental results for the verification of the BFAC model. The verification includes experimental evidence for and mechanistic explanations of an important characteristic of a BFAC system, bioregeneration. Bioregeneration, defined here as the removal of previously adsorbed substrate from the adsorbent surface through biological means, has been demonstrated in previous studies (2-6). Because bioregeneration involves 280
Environ. Sci. Technol., Vol. 21, No. 3, 1987
Table I. Composition of Mineral Medium salt concn, mg/L KH2P04 8.5 KzHPO4 21.75 Na2HP04 17.7 MgS04 11.0
salt
concn, mg/L
FeC1, NaHCO, KNO, CaClz
0.15 1.0 3.215 27.5
Table 11. Composition of Growth Medium salt KHzPO4 FeC1, NaHC03
concn, mg/L
salt
concn, mg/L
155.0 12.0 100.0
KN03 CaC1,
1172.0 200.0
the sequential adsorption of a solute, growth of a biofilm, and desorption of the solute, it gives the BFAC model a rigorous test. An experiment with biofilm grown on nonadsorbing media, glass beads, was used as a control. Readers interest in detailed procedures and results for the determination of coefficients is directed to Chang (7). Materials and Methods
Supporting Media. For the purpose of verifying the mathematical model, bead-shaped activated carbon (BACM, Kureha Chemical Industry Co., Ltd., Tokyo, Japan) was chosen as one supporting medium for the biofilm attachment. The carbon is sturdy, and its bead-shaped configuration matches the assumption of diffusion to and in a spherical particle (I). The activated carbon was sieved with U.S. standard sieves, and particles that passed through No. 30 (0.59 mm) and were retained on No. 40 (0.42 mm) sieves were used. The geometric mean diameter, 0.50 mm, was used in the mathematical modeling. The carbon was washed several times with distilled/deionized water (DIDW) to remove carbon fines and stored in an oven at 110 "C before use. Glass beads with diameters ranging from 0.9 to 1.23 mm (geometric mean 1.05 mm) also were chosen as a supporting medium for biofilm growth. The nonadsorbing characteristics of glass beads provide a control for biofilm activity alone. The glass beads were not sieved but were
0013-936X/87/0921-0280$01.50/0
0 1987 American Chemical Soclety
Table 111. Composition of PM Buffer"
salt KHZPO4 KPHPOd
concn, mg/L 8.5 21.75
salt NazHP04 MgS04
concn, mg/L 17.7 11.0
"ate: PMT buffer, which was used as dilution water for the samples from column reactor, contained a surfactant at 0.1 mL/L concentration.
washed with chromic acid, rinsed clean with DIDW, and stored in an oven at 110 "C prior to use. Nutrient Salts, Nutrients required for biological growth were supplied in two nutrient salt compositions: mineral medium and growth medium (8). The compositions of the mineral medium and growth medium are shown in Table I and 11, respectively. Nitrate is used as nitrogen source, instead of ammonia, to avoid nitrifier growth in the reactor. Mineral medium was used in the feed solution for column studies. However, when high concentrations of substrate were used to grow a high density of suspended biomass, growth medium was used to supply the salts and buffer the solution at pH 7.1. Two other mineral solutions were used in this research. Phosphate and magnesium buffer (PM buffer) was used to wash cell suspension and to dilute the samples in the suspended-growth experiment for cell enumeration. The composition of P M buffer is shown in Table 111. In the column studies, clumps of bacteria were dispersed with an anticlumping agent (Tween 80, ICN Nutritional Biochemicals, Irvine, CA) to allow accurate enumeration. The nonionic surfactant, poly(oxyethy1ene)sorbitan monooleate, was added to PM buffer at a concentration of 0.1 mL/L to disperse the clumps (9, 10). The solution is designated as PMT buffer and was used as the dilution water for the samples taken from the effluent of the column reactor. Sterile Substrate Solutions. A rigorous sterilization procedure was developed to prepare microorganism-free substrate solutions. Autoclave (hot) and filter (cold) sterilizations were the two basic techniques employed to prepare large quantities of sterile solution. For smaller amounts of sterile solution (ca. 1L), the solution with the desired concentration of the substrate was pumped through a 0.2-pm membrane filter (GA-8 type, Gelman Sciences Inc., Ann Arbor, MI) into a container that has been autoclaved. The filter membrane was held in a holder (Swinnex, Millipore Corp., Bedford, MA), which could be autoclaved together with the container. To prepare large amounts of substrate solution (ca. 25 L) relatively rapidly for a column study, a combination of autoclave and filter sterilization was employed. The feed tank was thoroughly cleaned, assembled, and autoclaved while it was empty. Then, most of the DIDW was autoclaved in 8-L glass aspirator bottles and aseptically transferred into the feed tank. Desired volumes of stock substrate, radiolabeled substrate, and nutrient solutions, to make up to the desire final concentration of feed, were then filtered through a 0.2-pm filter membrane and into the sterilized feed reservoir. Pressurized air, which provided mixing and aeration in the sterile container, also was filtered through an Aqua-Pure filter cartridge (AP10, AMF Inc. Meriden, CT) and a 0.2-pm membrane. Autoclaved DIDW then was added to make up the desired volume. All the waters used in this study were deionized, distilled, and passed through a Milli-Q water purification system (Millipore Corp., Bedford, MA), Bacterial Culture. Initially, a column reactor exactly the same as that described in a later section was set up and
packed with glass beads. An inoculum was obtained from the Saline Ditch, 15 m downstream of the UrbanaChampaign Sanitary District's outfall in Urbana, IL. The sample was diluted 1:lOOOwith PM buffer, seeded into the column, and left overnight to allow bacteria to attach to the glass beads. The column was then fed with 3 mg/L phenol in mineral medium until the bacteria population reached a steady state. The effluent yielded at least three distinct colonies on standard plate count agar (11). A rod-shaped bacterium, which had the highest density on the agar, was picked for this research. The bacterium was streaked and picked several times on new plates and inoculated again in a column reactor for growth on 3 mg/L phenol. The glass beads then were transferred into a test tube and vortexed to shear off the biomass (8). The sheared bacteria were suspended in PM buffer, diluted to the desired density, and stored in the refrigerator (4"C) before being used as the seed in subsequent studies. The cell density in the seed was about 1X lo6 viable cells/mL. Analytical Techniques. Phenol was chosen as the model substrate because it is adsorbable, easily biodegradable at low concentration, and a relevant priority pollutant. Regular (nonradiolabeled) phenol was obtained from Mallinckrodt Inc. (Paris, KY) in analytical-reagent grade. Radiolabeled phenol was procured from Amersham Corp., Arlington Heights, IL (C6*H50H,CFA.125) Concentrations of phenol and phenol-derived products were measured with scintillation counting. Measurement of all forms of 14Cwas performed according to the following steps. One milliliter of aqueous sample was transferred with a gas-tight syringe (Hamilton Co., Reno, NV) to glass scintillation vial (Research Products International Corp., Mt. Prospect, IL). Ten milliliters of scintillation cocktail (ACS cocktail, Amersham Corp., Arlington Heights, IL) was then added, and the vial was capped and shaken vigorously. After 30 min to allow conditions in the vial to reach equilibrium, each vial was counted for 10 min with a Beckman L-100 liquid scintillation counter. A dilution-water blank was treated in the same manner and used for background correction. The concentration was computed with
- cpmb cpmO - cpmb
s, = socpme
(1)
where S, and Soare the concentration in the sample and initial substrate (M,/L3),respectively. cpm,, cpmo, and cpmb are counts per minute for sample, initial solutions, and blank, respectively. The I4C radioactivity after biological transformation can be categorized into original substrate, soluble products coming directly from metabolism of original substrate, soluble products coming from respiration of biological mass, carbon dioxide, and biomass (12). The second and third components are the soluble metabolic products (SMP). The first three components were measurable together as the soluble organic matter. Soluble organic matter, COz,and biomass were differentiated by a threestep approach. First, the radioactivity of the soluble organic matter was measured by the following procedure. Four milliliters of effluent sample was taken with a 5-mL gas-tight syringe and filtered through 0.2-pm filter membrane to remove suspended biomass. One milliliter of filtrate was transferred into a scintillation vial, and 1drop of concentrated hydrochloric acid was added to acidify the sample pH value to lower than 2.0. The vial was then swirled 200 times to drive off carbon dioxide (7). Ten milliliters of scintillation cocktail was added to the vial, and the raEnviron. Sci. Technol., Vol. 21, No. 3, 1987
281
dioactivity was counted, as described above. Second, the sum of the radioactivities of soluble organic matter and carbon dioxide was counted by the following procedure. One milliliter of filtrate was added to a scintillation vial. Instead of acidifying the sample, 1drop of Carbo-Sorb I1 (6003073, United Technologies, Packard Instrument Co., Downers, Grove, IL) was added to the vial to absorb carbon dioxide in the sample. The capacity of Carbo-Sorb I1 is such that one drop can absorb 5.47 mg of carbon dioxide, which is much higher than was present in the effluent sample. Carbo-Sorb I1 itself does not exhibit radiation or chemiluminescence (7). The vial then was counted for 10 min in the normal manner. The third quantity measured was total radioactivity, which included all five components. One milliliter of effluent sample was transferred to a scintillation vial without filtration. Carbo-Sorb I1 and cocktail were added before counting. The concentration of soluble material was computed directly with eq 1. The concentration of carbon dioxide was computed by subtracting the soluble organic matter concentration from the sum of the soluble organic matter and carbon dioxide concentrations (second measurement). Biomass concentration was computed by subtracting the second measurement from the total concentration (third measurement). All concentrations are recorded as normalized radioactivities against influent substrate radioactivity. C02, suspended biomass, and total radioactivities are converted to milligrams of C per liter by a factor of 2.30 mg of C per normalized radioactivity. Standard plate counts (SPC) were used to enumerate the number of viable cells in column tests and suspended growth experiments. The agar used was standard plate count agar (BBL Microbiology Systems, Cockeysville, MD). Dilution waters were PMT and PM buffers (Table 111). The dilution procedure was performed according to standard methods (11). The correct dilution factor was determined by estimating the cell numbers with a hemacytometer ( A 0 Scientific Instruments, Buffalo, NY) and light microscope. One hundred microliters of the sample was spread on one plate with a glass spreader that was flame sterilized between each spreading. Cell numbers were counted as the colony number on the plate after incubation for 12-24 h at 30 "C. Total suspended solids were used to measure cell dry weight in suspended growth experiments. One 0.45-pm membrane filter (WCN type, Whatman, Ltd.) was placed in an aluminum pan and dried overnight in an oven at 110 "C. The weight of the dried filter membrane and pan was the tare weight. One hundred to 1000 mL of sample, depending on the biomass concentration, was vacuum filtered through the membrane filter. The membrane and the pan were then weighed after being dried for 2 h at 110 "C. The increase in weight was the dry weight of the biomass. Adsorption Kinetic Coefficients. Two batch tests were required to evaluate three coefficients in the adsorption kinetics-K,, n, and D,. Isotherm tests were conducted with 10 screw-capped serum bottles. Granular activated carbon was pulverized into powdered activated carbon with a Tekmar mill (Tekmar Co., Cincinnati, OH). Powdered activated carbons were weighed directly in oven-dried serum bottles, capped, and sterilized. Lowconcentration solutions of radiolabeled phenol in mineral medium were filter sterilized before being added into the bottles. Each bottle contained 100 mL of phenol solution and varying amounts of activated carbon, so that a reasonable spread of the equilibrium concentrations was ob282
Environ. Sci. Technol., Vol. 21, No. 3, 1987
tained. Two bottles without activated carbon were treated as blanks. All bottles were shaken in a rotary shaker for at least 7 days before the equilibrium concentrations were measured in the filtrate passing through a 0.45-pm membrane filter. The pH for the solutions was 7.1 f 0.2. Bacterial contamination was checked with plate counting, and no contamination was found. The logarithmic form of the Freundlich isotherm was employed to compute the coefficients: 1 In q = In K , - In S n K , was computed from the y interception, and n was computed from the slope of the line obtained by linear regression (23). To determine the surface diffusivity (D,) of substrate in activated carbon, finite-batch kinetic tests were conducted (14). Radiolabeled phenol in mineral medium was prepared with care. No sterilization was necessary because of the short period of the experiments. The size of the granular activated carbon was the same as those used in column studies. Mixing was achieved with an overhead propeller stirrer to avoid breaking up the carbon particles. The test was initiated when activated carbon was added into the solution. Samples were taken at 5-40-min intervals and measured for substrate concentration without filtration. An entire run lasted about 4 h. A finite-batch adsorber model is required to describe the changes of the substrate concentration (14). The model was solved with a global orthogonal collocation method (GOCM) (7), using Legendre polynomials in spherical coordinates to approximate the concentration profile in the carbon. The detailed solution procedure is not presented here, but it follows the method of Thacker (14). Best fit D, and kf values were obtained with the generalized reduced gradient (GRG) method (15). The objective function was to minimize the sum of squared residual (SSR) between predicted and experimental results:
+
where Sexpt and Spred are substrate concentrations of experiment and model prediction, respectively. N is the number of data points. Different starting points were used to initiate the search procedure to avoid having the subroutine trapped at a local minimum. Biofilm Kinetic Coefficients. Three batch suspended-growth tests were conducted to determine the four biokinetic coefficients-k, K,, Y, and b. Biofilm density Xfwas estimated from simultaneously measuring biofilm thickness and biofilm mass on glass beads taken from one of the reactors (8). To determine the decay coefficient, b, radiolabeled bacteria were collected from the effluent of the column reactor. They were harvested by filtering the effluent through a 0.2-pm membrane filter and washed several times with PM buffer. The radiolabeled cells were washed into a flask and made up with buffer to have an initial radioactivity of suspended biomass around 500 cpm. The solution was mixed and aerated with a magnetic stirrer. Samples were taken every 1-2 days to measure the suspended biomass radioactivity. The following equation was used to compute the decay coefficient (8): In (XdX,) b= (4) t where X o and X,are biomass radioactivities at time zero and t , respectively. The shear loss coefficient (b,) was not
determined experimentally but was used as a fitting parameter for the BFAC model. The values used will be found in later sections. A batch test with high substrate concentration was conducted to determine the maximum specific substrate utilization rate (k) and yield coefficient (Y).The initial phenol concentration was 200 mg/L, a concentration high enough so that the effect of decay was negligible. Under these circumstances, the models for the substrate utilization and growth of bacteria in batch reactor can be solved to give (8)
Y = -AX/ AS
(5)
where AX is the isnueaseof the dry weight of the cell and AS is the change in the substrate concentration. The specific growth rate (pm)was determined from the slope of the growth curve at the log growth phase in the yield experiments. The following equation was employed to compute pm (16):
- In (X,/XO)
-
(6) t The maximum specific utilization rate coefficient was computed with the following equation (17): Pm
k = pm/Y
(7)
To determine K,, the change of the real substrate concentration in the reactor had to be measured so that interferences from other soluble organics were avoided. Very high densities of nonradiolabeled bacteria were used to exert immediate utilization. To produce a high density of cells, a seed suspension was added to a sterile solution containing 200 mg/L phenol (nonlabeled) in growth medium. With a magnetic stirrer providing mixing and aeration, cell growth reached a peak after 1-2 days, at which time the cells were harvested by centrifugation, decantation, and filtering onto a 0.45-pm membrane filter. The cells were washed several times with P M buffer and then washed off into a Wheaton square bottle (Wheaton Scientific, Millvile, NJ), in which the desired cell density was obtained by dilution with P M buffer. The cell suspension was extremely turbid, and the density could reach as high as loll viable cells/mL, as enumerated by plate counting. After a radiolabeled solution was added to the suspension of unlabeled cells, samples were taken at 0.5-20-min intervals. The utilization of the substrate in the sample was stopped instantaneously with a drop of concentrated hydrochloric acid. Substrate concentration was measured with liquid scintillation counting, and biomass concentration was measured at the start of the experiment as the dry weight of the suspended solid retained on a 0.45-pm filter membrane. The slope of the substrate utilization curve at time zero was taken to compute the K , value:
K , = -so[1 + kXo(
dS
-l
At t = 0, no labeled product materials were present, which meant that scintillation counting measured only the values of s. Biofilm grown on glass bead (BFCM1 test) was used to determine biofilm density. A t the end of the experiment with the glass bead reactor, 100 glass beads were transferred, one by one with tweezer to preclude inclusion of interstitial water, to a tared aluminum pan. The beads containing biofilm were weighed before and after drying in the 110 OC oven. Since water comprises about 99% of the total biofilm mass (8), the weight loss upon drying is
assumed to be the summation of biofilm and adsorbed water. Clean glass beads were immersed in clean water and picked out to measure the amount of adsorbed water by the same methods. The biofilm volume was obtained by the difference of the two measurements. Biofilm mass was obtained by first measuring the nonpurgable radioactivity on 100 glass beads and then converting radioactivity to cell mass. The radioactivity per viable cell was assayed by measuring suspended radioactivity and plate counts in the effluent of the column reactor. Radioactivity of 100 clean glass beads was measured and treated as the correction. The correction was close to the background radioactivity. The viable cell number was converted to active cell weight by assuming the cell dry weight equal to 2 X mg/cell (18). The biofilm density was computed by dividing the biofilm cell weight by the biofilm volume. The biofilm density thus obtained was 3.22 mg of active cells/cm3. This value is a t the lower range found by Rittmann and McCarty (a), but higher than that found by Namkung et al. (19). Since total biofilm was comprised of viable cells, dead cells, and extracellular materials, it was assumed that only half of the biofilm mass was active biomass (8). Because biokinetic coefficients were obtained on the basis of the total biomass, the total biofilm density, 6.44 mg of cells/cm3, was used in the model simulation. Mass Transfer Coefficients. The molecular diffusion coefficients in water ( D ) and in biofilm (Of) and the film transfer coefficients (kf) were determined from the literature. The approximate relationship of Wilke and Change (20) was used to estimate D of phenol in dilute solution:
where #b = association parameter = 2.6 for water, Mb = molecular weight of solvent = 18 g for water, T = absolute temperature in K,fib = absolute viscosity of the soluiton in cP, and V, = molar volume of the solute as liquid at its normal boiling point. Molar volumes of solutes can be estimated from the atomic volume of their atoms (21). For phenol, v b = 118.4 cm3/mol. Therefore, D = 8.44 X lo4 cm2/s = 0.730 cm2/day. Diffusion coefficients in the biofilm (Of)are smaller than D because the bacteria and their extracellular materials pose extra diffusional resistances. The ratio D f / D = 0.8, found by Williamson and McCarty (22) using artificially formed biofilm, was used in this research. The ratio falls at the upper range of 0.5-0.8 for naturally grown heterotrophic biofilms (23). The formula of Williamson et al. (24), which was used by Thacker (14) in the simulation of packed activated carbon adsorber, was used to compute the film transfer coefficient (itf):
(3-
k f = 2 . 4 0 ~~
(SC)-O.~'
(10)
where us = superficial flow velocity through column (I,/!!'), Re is Reynolds number = pdpu,/p, Sc is Schmidt number = p / ( p D ) , p = mass density of the flowing fluid, d, = diameter of the packed material, and p = absolute viscosity of water [M/(L!!')].The applicable ranges for the formula are 0.08 I R e / € I125 and Sc = 1000. The ranges of the R e / € and the Sc encountered in this research were 11.08-23.4 and 926-1188, respectively, and were in the applicable ranges of the above equation. Design of Experimental System. Because of the complexity of the model, the experimental system was Environ. Scl. Technol., Voi. 21, No. 3, 1987
283
Table IV. Kinetic Parameters Used in the Model Simulation for 3 mg/L Phenol parameter substrate medium
k K8
Y b b," Xf &On
Lmn Df ke D8
xw
KP n PP
units mg/L
BFACl
3 phenol BACM mg/[(mg of cell)-day] 13.7 mg/cm3 0.00024 mg of cell/mg 0.34 day-' 0.068 day-' 0.10 mg of cell/cmg 6.44 mg of cell/cm* 1 x 10-6 0.25 m cm2/day 0.584 cm/day 623.7 cm2/day 4 x 10"' g 0.4988 38.05 2.504 g/cm3 0.686
BFCMl 3 phenol glass beads 13.7 0.00024 0.34 0.068 1.05 6.44 1 x 10-6 0.015 0.584 380.4
All parameters except these were determined according to the methods given under Materials and Methods. Estimates of b,, XaO, and Lm were obtained from the column results, as described under Results and Discussion.
-----
ShlcoNE W I N G TERON W I N G
Figure 1. Expertmentat system for model verification.
designed to reduce the number of variables and to conform to the assumptions of the model as close as possible. The experimental system is shown in Figure 1. The inlet tubing to the reactor consisted of silicon and Teflon tubing. A break tube was placed before the feed pump to prevent back-contamination of the microorganisms from the column to the feed reservior. The feed pump was a peristaltic pump (Minipuls, Gilson Medical Electronics, Middleton, CA), which accurately delivered flow rates of 6 L/day. The recycle line consisted of glass connectors and silicon and Teflon tubing. The recycle pump also was a peristaltic pump (Masterflex, Cole Parmer Instrument Co., Chicago, IL) but had higher capacity than the feed pump. The recycle ratio was set a t 21.7 to reach a nearly completely mixed condition in the column reactor. Hence, the complex hydrodynamics of a fixed-bed once-through reactor were avoided. The column was a chromatographic preparative glass column (Beckman Instrument, Inc., Irvine, CA) having 1.5-cm inside diameter. The height of the packing could be adjusted with a plunger assembly (Beckamn Instrument Inc.) so that any amount of packing material could be held tightly. Feed flow upward was spread uniformly across the cross-section of the column by two diffusion discs placed on each at the influent and effluent ends. Effluent passed to a 500-mL aspirator bottle, which provided another break in the effluent stream to avoid back-contamination from air-borne microrganisms. Column Studies. To start a column study, the column assembly was autoclaved while disassembled. All the glassware, filter membranes, filter holders, feed containers, and DIDW were autoclaved at 125 OC and under 20 psi steam pressure for 20 min. Two to five milliliters of bacterial suspension was seeded into the column after it was assembled. The column was allowed to sit overnight to allow the bacteria to sorb to the supporting media and the activated carbons saturated with liquid. 284
Environ. Sci. Technol., Vol. 21, No. 3, 1987
The column was then flushed with PM buffer to remove suspended and loosely bound bacteria. The feed was then initiated, and the time was designated zero. The feeding system (feed tank and inlet tubing) and sterile feed solution were prepared 1day before the initiation of the experiment. Effluent samples were collected in a disposable test tube at 2-5-h intervals. The samples were analyzed for soluble organic matter, carbon dioxide, and biomass concentrations with liquid scintillation counting. Standard plate counting was performed to check the contamination-free condition of the feed system and to enumerate the suspended cell number in the effluent. All the analytical techniques followed the procedures described in previous sections. The amount of activated carbon or glass beads and the feed flow rate for the column studies were predetermined with the aid of the mathematical model (I). Several trial experiments were then conducted to reach the carbon weight of around 0.5 g and feed flow rate of 6 L/day. An isotherm test was conducted with powdered BACM as the adsorbent and SMP from degradation of acetate as the adsorbate (7). The result showed that approximately 99% of the SMP was adsorbed by BACM for a dosage from 0.20 to 10.54 mg of SMP as C/g of BACM.
Results and Discussion Verification of the BFAC Model. Two column tests using BACM and glass beads as supporting media were designated BFACl and BFCM1, respectively. The kinetic coefficients required as the input to the model were determined as described above and are listed in Table IV. The characteristics of the column reactors are listed in Table V. The apparent dried particle density for the activated carbon was assumed to be 0.686 g/mL (14). Three parameters in Table IV could not be measured independently. They were initial suspended biomass concentration (Xso),initial biofilm thickness (Lm),and the shear loss coefficient (bs). Xsoand Lm were assumed to be 1X mg of cell/cm3 and 0.25 pm, respectively, for the BFACl test. Changing Xsohad only negligible effect on the performance of the model, because sensitivity analysis (7) showed that biofilm was the dominant form of biological growth in the reactor and X,was very low in the reactor. The initial biofilm thickness was chosen to be less than a monolayer coverage of bacteria, since the inocula-
Table V. Characteristics of Column Reactor and Supporting Media for Model Simulation of 3 mg/L Phenol" parameter
units
medium
R
cm cm3 cm min
V
H
e t
Rr
cm/min cm-I
u0
a
BFACl
BFCMl
BACM 0.0249 12.38 0.5 2.97 0.4 21.7 53.57 7.096
glass beads 0.0526 21.64 5.0 5.19 0.4 21.7 53.57 13.98
'1
MODEL
lENT EXPERIMENT
c .g0.8 L
0
c
L
1
1
J
0.2
a
c
a 0.0 01
20
40
60
I 80
100
I1
BFAC SFAC ---BFCM a BFCM rn
MODEL EXPERIMENT MODEL EXPERIMENT
I
0.5
0.6
-v
,
I
F 3 . 0
tention time based on empty bed; t = bed porosity in the packed bed; R, = recycle ratio; u, = superficial flow velocity; a = specific surface area based on total reactor volume. MODEL ENT EXPERIMENT
' J3'5
" V = empty reactor volume; H = bed height; 0 = hydraulic de-
BFAC BFAC BFCM BFCM
4.0
110
Time, hours Flgure 2. Non-steady-state substrate concentrations for experiments and model simulations for BACM (BFACI test) and glass beads (BFCMI test).
tion did not provide sufficient bacteria to give full coverage. These two initial conditions were kept as constant as possible, and the model was run repeatedly with different values of the shear loss coefficient, b,. The b, values that gave the best simulation of all the substrate concentration data are reported in Table IV. The test using glass beads as supporting media for biofilm growth, BFCM1, was treated as control when phenol was the sole carbon source in the feed. The coefficients used in the model simulation were listed in Tables IV and V. Since glass beads did not adsorb phenol, the biofilm along model [BFCM model (7)] was used to simulate the process. The behavior of the BFCM model was equivalent to the BFAC model with no adsorption. However, the BFCM model was simpler and cheaper to run when no adsorption occurred. The model-generated and experimental substrate concentrations, for BFAC and BFCM tests, are shown in Figure 2. Both models simulated the experimental results very well throughout the entire courses of the tests. The behavior of the substrate curve can be understood better by considering also the concentration of the COz in the effluent. The production of the C02 represents the complete mineralization of phenol. The concentrations of C02 for the BFAC and BFCM tests are shown in Figure 3. BFAC Results. The performance of the BFAC system can be described by four parts. First, the effluent concentration jumped up to about 0.3 mg/L a t time zero, a characteristic of completely mixed flow reactor. Then the substrate concentration increased steadily to about 1.2 mg/L at 32 h. During this period, biological growth inside the reactor and biodegradation of the phenol were not detected (Le., no C02production in Figure 3). The reactor was behaving similarly to an activated carbon adsorber,
0.0
0
eo
40
60
80
100
I
110
Tlme, hours Flgure 3. Non-steady-state COPconcentrations for experiments and model simulations for BACM (BFAC1 test) and glass beads (BFCM1 test).
and the substrate concentration curve was the same as a typical breakthrough curve of an activated carbon adsorber. The model predicted the effluent substrate concentration extremely well, and there was no possible interference from SMP in the measurement of the phenol by the liquid scintillation counting, because no SMP was being produced. The second part ran from 32 to 41 h, a period during which the substrate curve just started to deviate from the breakthrough curve of an adsorber. The effluent substrate concentration leveled off and then began to decrease, while carbon dioxide concentration (Figure 3) increased rapidly: both phenomena confirm the active utilization of the phenol and that biofilm was growing significantly during the second period. The model gave slightly higher substrate concentrations than experimental results (Figure 2), but the differences were in the range of the experimental error. C02 concentrations were modeled accurately through the second stage. Although the biofilm could release some SMP at this stage, the SMP load was estimated at 0.46 mg of SMP as C/g of BACM on the basis of a net SMP production of 0.09 mg of SMP as C/mg of phenol degraded (25). Hence, the SMP was probably completely adsorbed by the BACM (7). The third part of the substrate curve ran from 41 to 80 h. The substrate concentration decreased steadily from 41 to 50 h and then leveled off. However, the production of the COP from phenol mineralization was at its peak (Figure 3) when the substrate concentration leveled off. By the end of the third part, the production of the COO declined and reached a nearly constant level. Although the production of SMP probably was significant after 50 h, the SMP was almost completely adsorbed (load = 4.67 mg of SMP as C/g of BACM) and did not appear in the effluent. The fourth part of the substrate curve ran from 80 h until the termination of the test. Substrate concentration reached a steady-state level, which was equivalent to about 90% removal. Even though the phenol concentration reached a steady-state level between 60 and 70 h, the COz concentration did not level off until around 80 h. During this stage, the model consistently predicted slightly lower substrate concentrations than the experimental results. Because the production of the SMP was more significant when biofilm was well developed (load = 7.4 mg of SMP as C/g of BACM at 110 h) than at the beginning of the test and because the BACM had adsorbed the SMP for more than 30 h, the SMP may have contributed to the effluent soluble carbon. However, the amount was small, and hence, the soluble carbon was only slightly higher than the phenol concentrations predicted by the BFAC model. Environ. Sci. Technol., Vol. 21, No. 3, 1987
285
5 4.0
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1
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c
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40
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Time, hours Flgure 4. Non-steady-state soiubie puls COP carbon concentrations for experiments and model simulations for BACM (BFAC1 test) and glass beads (BFCMI test).
BFCM Results. The substrate curve of the BFCM system differs from the BFAC curve in that the jump at time zero is much higher (Figure 2). This is due to the fact that the glass beads adsorbed little, if any, phenol. Most phenol passed through the completely mixed flow reactor immediately. After 30 h, substrate concentration decreased rapidly due to the active utilization by the growing biofilm and approached a steady state after 60 h. The BFCM model predicted no phenol removal from 0 to 25 h, when the biofilm growth was not significant. However, the measurements showed about 5% removal. One possible cause was adsorption of the phenol by the tubing and glass beads. The model predicted quite well the experimental results from 30 to 50 h. The amount of the SMP produced by the degradation of the phenol was probably not high enough to interfere with the measurement of the phenol when growth was being initiated. However, the model consistently predicted phenol concentrations lower than measured after 55 h. The difference can be attributed to SMP, which was measured along with the phenol with scintillation counting. Comparison of BFAC to BFCM. The most prominent contrast between the BFAC and BFCM results was the peak of the CO, curve for the BFACl system. The peak of the BFAC curve at 50 h indicates that more phenol was available to the biofilm than was available at steady state or to the biofilm of BFCM1. In fact, the amount of the C02 at the peak was approximately double the C02 production at steady state or from feed phenol only. This extra C02 production can be explained by the desorption of the previously adsorbed (from 0 to 32 h) phenol from the activated carbon. The desorbed phenol was subsequently mineralized by the biofilm. This phenomenon, bioregeneration, reached a peak value at 50 h and then started to decline due to the decrease in the previously adsorbed phenol. The model-generated C02 curve agreed well with the C02 concentration in the BFCMl test. However, the model-predictedC 0 2concentration was consistently higher than the experimental result after 50 h. C02 data for BFACl and BFCMl converged to the same level at steady state, where 50% of the influent phenol C was converted to COz C. The result suggested that biofilm utilization dominated phenol removal in both reactors during this period and activated carbon adsorption was not important at steady state. Figure 3 showed that in the BFACl reactor the C02 concentration was higher during bioregeneration than at steady state. The overall picture of bioregeneration can be shown more clearly by the effluent soluble plus C02 carbon concentration (Figure 4). The figure shows that 286
0.25
I
3 m g / i P H E N O L ' A N D BACM
BFAC EXPERIMENT BFCM MODEL BFCM EXPERIMENT
Environ. Sci. Technoi., Voi. 21, No. 3, 1987
5
0.15
E
O.1° 0.05
t
\
4
-.25 20
40
60
80
100
110
Time, hours Flgure 5. Model-predicted flux of substrate into BACM (BFACI test). Positive flux meant substrate diffused into carbon, and negative flux meant substrate diffused out of carbon.
during 44 to 56 h, the effluent soluble plus C02 carbon concentration exceed the influent level (2.295 mg of C/L) for the activated carbon support. The only possible source of this carbon was from the desorption of the previously adsorbed phenol. The BFAC model calculated the soluble plus C02carbon very well until 50 h, when bioregeneration was near its peak. However, the model predicted higher soluble plus C02 carbon after 50 h. The difference was in the C02 carbon, which was overpredicted by the BFAC model. C02 could be lost during sample handling and scintillation counting (26). For comparison, the effluent soluble plus CO, carbon for the BFCMl test also is plotted in Figure 4. When no adsorption (and, hence, no bioregeneration) could occur, the effluent carbon concentration never exceed the influent level, and no peak was observed for the BFCMl test. The BFCM model calculates the experimental soluble plus C02 carbon concentration extremely well. The steady-state total carbon concentrations for both systems were about 87 % of the influent level. The reasons for less than 100% recovery are the loss of C02 during sample handling and scintillation counting (26). While Figures 3 and 4 demonstrate bioregeneration, the flux of the phenol across the biofilm/activated carbon interface (JJ can best illustrate the phenomenon. The advantages of the GOCM made the computations for the concentration profiles and the fluxes of the substrate possible (1). A positive flux means phenol was diffusing from the biofilm into the activated carbon, while a negative flux means phenol was diffusing from the activated carbon into the biofilm. The latter case represented the desorption of the phenol, i.e., bioregeneration. The model-predicted fluxes, Jq [mg/(cm2.day)], at different times are plotted in Figure 5. The curve shows a positive flux of the phenol into the activated carbon from 0 to 40 h. The flux was a maximum in the beginning of the test, because biofilm growth was negligible, the diffusion resistance was minimal, and the activated carbon had no adsorbate. The flux decreased as phenol started to accumulate inside the activated carbon, thus reducing the gradient of adsorbate density at the exterior surface of the activated carbon. The most important point in Figure 5 is that the flux changed from a positive value to a negative value at 40 h. This required a reversal of the adsorbate density gradient inside the activated carbon. The resulting negative adsorbate density graident caused the phenol to diffuse out the activated carbon. The cause for the reversal of the adsorbate density gradient was growth of the biofilm. As the biofilm grew thicker, the biofilm consumed phenol; thus, the substrate concentration at the biofilmjactivated
'I
i-.-
be formulated before the basic mechanisms and their interactions are understood. Research into the mechanisms controlling biofilm losses is of key importance if a completely mechanistic model is to be verified.
-BFAC MODEL ' BFAC EXPERIMENT --- BFCM MODEL
0, E 1.5
0
BFCM EXPERIMENT
m
m
Conclusions m
m I
I
Time, hours Flgure 6. Non-steady-state suspended carbon concentratlons for experiments and model simulations for BACM (BFACI test) and glass beads (BFCM1 test).
carbon interface became lower. With biological activity increasing, phenol concentrations in the bulk liquid also continued to decrease. As bulk and biofilm concentration declined, they became lower than the concentration at equilibrium with the previously adsorbed phenol. At this point, phenol simply diffused out of the activated carbon, and activated carbon was bioregenerated by the biofilm. Although the BFAC model simulated the substrate concentration very well (Figure 2); it predicted high C02 concentrations after 50 h (Figure 3). Figure 6 Qhowsthat the BFAC model, using b, = 0.1 day-l, also consistently predicted low biomass concentrations after about 45 h and failed to simulate the peak of the suspended biomass during the bioregeneration. The relatively poor fib for C02 and biomass are closely related for the BFAC. A higher value of the shear loss coefficient after about 45 h would have increased the predicted suspended biomass in the effluent, making the experimental and modeling results more coincident for effluent biomass (Figure 4). Such an increased biofilm loss would have decreased the accumulated biofilm mass in the reactor and decreased the C02 production from respiration; thus, the COz results from the model and experiments (Figure 3) also would be more coincident. The good agreement of C02 and biomass for the glass beads study indicates that the model correctly simulates the phenomena if a reasonable shear loss term is available. The large peak in effluent-suspended biomass during bioregeneration cannot be explained by a simple increase in b,. A similar peak in suspended biomass also was observed during bioregeneration in another study (27), in which the experimental condition was different from this study in that activated carbon was saturated with radiolabeled substrate at the beginning of the experiment. Modeling (6)related to this second study was improved significantly for the entire bioregeneration phase when the biofilm shear loss rate was treated as a variable proportional to the specific growth rate (30),as well as to the shear stress. B i o f i i loss due to the fluid shear seems to be a complex function of a variety of hydrodynamic and biological factors, such as surface texture of the supporting medium, degree of surface coverage, shear stress of the flow (28), biofilm thickness (B), and biofilm specific growth rate (30). One simple first-order loss coefficient, b,, was not able to take into account the complex changes on GAC medium, although it seemed to be adequate for the glass beads. Unfortunately, a scenario for increasing b, for the BFAC run cannot be prescribed satisfactorily, because mechanisms affecting the shear loss of a biofilm have not yet been well-defined. An appropriate functional form for b, cannot
(1) With assumed values of Lfoand Xso and a best fit value of b,, the BFAC mathematical model simulated well the experimentalresults for the transient growth of biofilm on activated carbon and on glass beads and the bioregeneration of the activated carbon. (2) The transient responses of a BFAC system were composed of four parts: (1)the adsorption and breakthrough of substrate; (2)growth of biofilm and utilization of substrate; (3) bioregeneration of activated carbon; (4) steady state. (3) The peak of the C02 curve was the result of the bioregeneration of the previously adsorbed phenol. This peak should not be confused with the peak of the substrate concentration curve, and it occurred after the peak of the substrate curve. (4) Solution of the BFAC model by GOCM computed the substrate flux across the biofilm/activated carbon interface and quantitatively illustrated the mechanism of bioregeneration. Fluxes across the biofilm/carbon interface showed a complete reversal, with phenol diffusing out of the activated carbon during the period of the bioregeneration, when biofilm lowered the phenol concentration at the biofilm/activated carbon interfaces as it grew thicker. (5) The main biofilm loss mechanism was the shearing of the biofilm by the flowing liquid. However, proper modeling of the shear loss of the biofilm seemed to depend on several factors, such as biofilm thickness, surface characteristics of the attaching medium, biofilm growth rate, and hydrodynamics in column. One simple first-order biofilm loss coefficient was not adequate for modeling the BFAC process, although it worked well for biofilm on glass beads. Although the growth rate related approach of Speitel and DiGiano (30)is a reasonable first step toward describing biofilm losses during bioregeneration, the need for further research into biofilm loss mechanism must be emphasized. Glossary b specific decay coefficient (1/T ) specific shear-loss coefficient (1/T ) b, b' total biofilm loss coefficient (l/T), b' = b b, count per minute of liquid scintillation counting
+
Df
D, Jq
k kf Kq
K, L
Lfo
M
Mb MS Mq Mx n 4 Re sc
particle diameter ( L ) molecular diffusivity in bulk liquid (L2 T ) molecular diffusivity within biofilm (L / Z!)' surface diffusivity ( L 2 / T ) substrate flux from biofilm into activated carbon [M,/ G 2 T )1 maximum specific rate of substrate utilization [Ma/ (MXT)1 liquid film mass transfer coefficient (L/T ) Freundlich isotherm coefficient half-velocity concentration (M,/L3) length initial biofilm thickness ( L ) mass, in general molecular weight of solvent ( M ) mass of substrate (M,) mass of activated carbon (M,) mass of bacteria (M,) Freundlich isotherm coefficient surface concentration (Ms/Mq) Reynolds number = pdpus/p Schmidt number = p / ( p D )
d
Environ. Sci. Technol., Vol. 21, No. 3, 1987 287
substrate concentration in the effluent (Ms/L3) substrate concentration in the biofilm (M8/L3) substrate concentration at liquid/biofilm interface (Ms/L3) substrate concentration in the feed (Ms/L3) absolute temperature (K) time (T) initial time (T) superficial flow velocity through reactor (LIT) molar volume of solute A as liquid at its normal boiling point (L3/mol) biomass concentration at time zero biofilm density ( M x / L 3 ) true yield coefficient of biomass (MJM,) reactor porosity absolute viscosity of water [ M / ( L T ) ] absolute viscosity of solvent [M/(LT)I maximum specific growth rate density of water ( M / L 3 ) association parameters
(1/n
Literature Cited Chang, H. T.; Rittmann, B. E. Environ. Sci. Technol.,
preceding paper in this issue. Schultz,J. R.; Keinath, T. J. J.-Water Pollut. Control Fed. 1984, 56, 143. Chudyk, W. A.; Snoeyink, V. L. Environ. Sci. Technol.1984, 18, 1.
Suidan, M. T.; Cross, W. H.; Fong, M.; Calvert, W. J. Environ. Eng. Diu. (Am.SOC.Civ. Eng.) 1981,107,563-579. Khan, K. A.; Suidan, M. T.; Cross, W. H. J.-Water Pollut. Control Fed. 1981,53, 1519-1532. Speitel, G. E., Jr.; Dovantzis,K.; DiGiano,F. A. J. Environ. Eng. Diu. (Am. SOC.Civ. Eng.), in press. Chang, H. T. Ph.D. Dissertation, University of Illinois,
Urbana, IL, 1985. Rittmann, B. E.; McCarty, P. L. Biotechnol. Bioeng. 1980, 22, 2359-2373. Severin, B. F.; Suidan, M. T.; Engelbrecht,R. S. J.-Water Pollut. Control Fed. 1984, 56, 881. Severin,B. F.; Suidan, M. T.; Rittmann, B. E.: Engelbrecht, R. S. J.-Water Pollut. Control Fed. 1984,56, 164-169. Standard Methods for the Examination of Water and Wastewater,15th ed.; American Public Health Association:
Washington, DC, 1981. Namkung, E.; Rittmann, B. E. Water Res. 1986, 20, 795-806. Weber, W. J., Jr. Physicochemical Process for Water Quality Control; Wiley: New York, 1972; Chapter 5.
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(14) Thacker, W. E. Ph.D. Dissertation, University of Illinois, Urbana, IL, 1980. (15) Lasdon, L. S.; Waren, A. D.; Ratner, M. GRG User’s Guide; Feb 1982. (16) Stratton, R. G. M.S. Dissertation, University of Illinois, Urbana, IL, 1981. (17) Metcalf and Eddy, Inc. WastewaterEngineering: Treatment, Disposal, and Reuse, 2nd ed.; Revised by G. Tchobanoglous; McGraw-Hill: New York, 1979; Chapter 9. (18) Gaudy, A. F.; Gaudy, E. T. Microbiology for Environmental Scientists and Engineers;McGraw-Hill: New York, 1980; p 226. (19) Namkung, E.; Stratton, R. G.; Rittmann, B. E. J.-Water Pollut. Control Fed. 1983, 55, 1366-1372. (20) Wilke, C. E.; Chang, P. AIChE J. 1955, 1, 264-270. (21) Perry, R. H.; Chilton, C. H. Chemical Engineers Handbook, 5th ed.; McGraw-Hill: New York, 1973; pp 3-229. (22) Williamson,K. J.; McCarty, P. L. J.-Water Pollut. Control Fed 1976,48, 281-296. (23) Siegrist, H.; Gujer, W. Water Res. 1985, 19, 1369-1378. (24) Williamson,J. E.; Bazaire, K. E.; Geankoplis, C. J. Ind. Eng. Chem. Fundam. 1963,2, 126. (25) Lu, C. J. M.S. Dissertation, University of Illinois, Urbana, IL,1985. (26) Chang, H.T.; Rittmann, B. E., submitted for publication in J.-Water Pollut. Control Fed. (27) Speitel, G. E., Jr.; DiGiano, F. A. J.-Am. Water Works in press. ASSOC., (28) Rittmann, B. E. Biotechnol. Bioeng. 1982, 24, 501-506. (29) DiGiano, F. A.; Dovantzis, K.; Speitel, G. E., J. Environmental Engineering, Proceedings of the 1984 Specialty Conference of ASCE, Los Angeles, CA; Pirbazari, M.; Eds.; American Society of Civil Engineers: Devinny, J. S.,
New York, 1984. (30) Speitel, G. E., Jr.; DiGiano, F. A., submitted for publication in J. Environ. Eng. Diu. (Am. Soc. Civ. Eng.). Received for review February 10, 1986. Accepted October 27, 1986. Although the information described in this paper has been funded wholly by the US.Environmental Protection Agency under Assistance Agreement EPA Cooperative Agreement CR810462 to the Advanced Environmental Control Technology Research Center, it has not been subjected to the Agency’s required peer and administrative review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
