Biotechnol. Rag. 1993, 9, 46-53
Simultaneous Biodegradation of Toluene and p-Xylene in a Novel Bioreactor: Experimental Results and Mathematical Analysis Jang-Young Lee, Yong-Bok Choi, and Hak-Sung Kim* Department of Biotechnology, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Taejon, 305-701, Korea
Simultaneous biodegradation of toluene and p-xylene was performed in a novel bioreactor, and the performance was analyzed on the basis of both experimental and mathematical simulation results. The novel bioreactor employed a silicon tubing as a supplier of the aromatic solvents t o be treated, and the transfer rate of toluene and p-xylene into the culture broth was easily controlled. Batch, sequencing batch, and continuous mode of operation were carried out in the novel bioreactor to compare the performance of the biodegradation of solvent mixture. Sequencing batch operation worked well, but several operational difficulties were found. Continuous operation was shown to present higher biodegradation rates and operational stability. The continuous biodegradation process was mathematically simulated, and operational conditions were optimized on the basis of the simulation results.
Introduction Aromatic hydrocarbons are discharged from chemical and process industries as major components of mixedorganic wastes which contaminate the environment. Among these waters, benzene, toluene, and xylenes (collectively known as BTXs) are classified as major pollutants with high frequencies of occurrence on the EPA list of priority pollutants (7,8, 12, 12). Much effort has been directed toward the treatment of these solvents in the waste stream, and various methods have been reported (3,5,6,9). These treatment methods reported so far can be classified into three categories: physical, chemical, and biological methods. Among these methods, biological methods have advantages over other methods in that operational cost is relatively low and complete degradation of aromatic solvents is possible (3, 5, 6). Since BTXs are highly volatile and only slightly soluble in water, these solvents might be discharged from the waste steam in both the gaseous and liquid phases. For the biodegradation of gaseous BTXs, bioscrubber and trickling filter processes have been developed, and the main shortcoming of these processes is a low degradation capacity. In addition, an efficient control of operating conditions such as moisture content in the bed and transfer rate of solvents to be treated have been known to present difficulties (8, 9). Little attention has been paid to the biodegradation processes for these solvents in the liquid effluent stream. When liquid solvents are directly fed into the processes under aerobic conditions, both the degradation rate and removal efficiency can be expected to be low because a large portion of solvents fed into the process would be discharged in the exit gas without biodegradation due to air stripping and evaporation. As organic solvents are usually discharged in a mixed state, a single microorganism with catabolic activity on all of the components in the mixture or a microbial consortium is required for the complete degradation of the mixture of organic solvents. Biodegradationof BTXs in the liquid
* Author to whom all correspondence should be addressed. 8756-7938/93/3009-0046$04.00/0
effluent stream using an axenic culture of Pseudomonas putida has been investigated in our laboratory. This microorganism is known to degrade toluene, m-xylene, and p-xylene (15-17). In this study, a mixture of toluene and p-xylene as a model system was simultaneously degraded using an axenic culture of P. putida in the novel bioreactor developed in this work. Silicon tubing was immersed in a completely mixed and aerated bioreactor, and the liquid mixture of toluene and p-xylene was circulated within the tube from a solvent reservoir using a diaphragm pump. In the system, the solvent mixture diffused out of the tube wall and was transferred into the culture broth where degradation by microorganisms occurred. A schematic diagram of the bioreactor is shown in Figure 1. Biodegradation of the solvent mixture was carried out in batch, sequencing batch, and continuous operation in the novel bioreactor. Continuous biodegradation was mathematically simulated,and performance was analyzed on the basis of both experimental and simulation results. Details are reported here.
Theoretical Consideration Transfer Rate of Toluene and pXylene through Silicon Tubing. Volumetric transfer rates of toluene and p-xylene through silicon tubing in the bioreactor are experimentally determined on the basis of the following theoretical consideration. The material balance for toluene over the bioreactor during batch operation is
By assuminga steady state for toluene in the culture broth (input rate from the silicon tubing = output rate by air stripping) in the absence of microorganism, eq 1becomes
R, = FaCg V Thus, toluene transfer rate per culture volume is obtained by measuring a steady-state concentration of toluene in the exit gas. Similarly, the volumetric transfer rate of p-xylene can be determined as described above.
@ 1993 Amerlcan Chemlcal Society and American Institute of Chemical Englneers
Biotechnol. hog.., 1993, Vol. 9, No. 1 --).
P = Pm
K , + C, + Ci/KiKO+ C,
N*t llllll I ‘ 1 1 tt”il-4- I t
Since the specific growth rate is dependent on which substrate is limiting the growth, modeling can be performed under the different growth-limiting conditions as follows. 1. Only the Carbon Source (a Mixture of Toluene and pXylene) Limits Growth. In this case, dissolved oxygen is assumed to be present in excess (C, >> KO),so that eq 5 can be expressed as P = P,
K, + C, + Ci/Ki
Material balance for biomass at the steady state in continuous culture gives
Figure 1. Schematic diagram of bioreactor.
Degradation Rate of Toluene and p-Xylene. The toluene degradation rate per unit culture volume is determined from eq 1: Facgt dCt QtX = Rt - --(3) V dt In batch and sequencing batch operation, the toluene degradatiQn rate can be estimated by measuring both the toluene concentration in the exit gas and the change in dissolved toluene concentration with time. On the other hand, the toluene degradation rate in continuous operation is easily obtained by measuring the steady-state toluene concentration in the exit gas. The degradation rate of p-xylene is also determined using the same method as that for toluene. Removal Efficiency of Toluene and pXylene. The removal efficiency of toluene of p-xylene is calculated by dividing the total degradation rate by the total transfer rate for each solvent. Removal efficiency of toluene is given by (4)
Mathematical Model for Continuous Biodegradation. The aerobic degradation of toluene andp-xylene by P. putida used in this work has been known to proceed via an oxidative pathway, and the enzymes involved in this degradative pathway do not show substrate specificity for toluene and p-xylene (15-17). In our work, it was experimentally confirmed that toluene and p-xylene are taken up and degraded simultaneously at the same rate by this microorganism. On the basis of the above facts, modeling of continuous biodegradation was carried out by considering a mixture of toluene and p-xylene as a single carbon source. As molecular oxygen acts not only as a terminal electron acceptor in the aerobic energy yielding process but also as a cosubstrate for catabolism of toluene and p-xylene in the aerobic degradation pathway, it might be assumed that oxygen as well as the carbon source can be a growth-limiting substrate (15-1 7). Thus, direct coupling of the two uptake processes, carbon source and oxygen uptake, can be introduced into the unstructured growth model (2). The nitrogen source was observed to be sufficient so as not to limit growth under our experimental conditions. As the growth of the bacterium used in this work was found to be inhibited by toluene and p-xylene, growth kinetics for the bacterium was expressed using Haldane’s inhibition model (1, 13):
(7) Rearrangement of eq 7 gives the dissolved concentration of carbon source at the steady state as
- Pm)2 - ~ O ~ K J K , c, = (Pm - D ) + +(D2DIKi (8a) or
~ - ~ O ~ K J K ,(8b) c, = (pm - D ) - d m(I-Kpm)2 i Calculation of the biomass concentration using eq 8a results in a negative value under the operating conditions; thus, eq 8b should be used instead. The carbon source balance over the reactor can be established as
R,V = FaCg+ FC,
dCC + (QJ,) V + ZV
To correlate the toluene and p-xylene concentrations in the exit gas with those in the culture broth, Henry’s law was employed for each solvent: C, = htCt
As it was found that ht is almost equal to h, from the experimental results, eqs 10a and 10b can be combined as C, = hC, (11) Since observed growth yields on toluene and p-xylene were shown to be identical under the same cultivation conditions, the specific degradation rate of the solvent mixture, Q,, can be expressed in a more general form as P
Q, = y,
By introducing eq 11and 12 into eq 9, we obtain the steadystate biomass concentration under the carbon source limited condition as follows:
X , = -(R, - hDaC,- D ) D
2. Only Dissolved Oxygen Limits Growth. In this case, the carbon source is assumed to be present in excess
Biotechnol. frog., 1993, Vol. 9, No. 1
so that C,
>> K,, and eq 5 at the steady state becomes CL = D "=L C
Thus, the steady-state dissolved oxygen concentration can be expressed as
(15) CL,-D The dissolved oxygen balance can be established as DX, dC, k,a(C,* - C,) = DC, + (16) Yo +dt From eq 16,the steady-state biomass concentration under oxygen-limited conditions is
D By a similar procedure, the dissolved concentration of carbon source (a mixture of toluene and p-xylene) at the steady state is given by
R, - D X J Y, c, = hD, + D 3. Simultaneous Limitation by Carbon Sourceand Dissolved Oxygen. This situation takes place at the shifting point between carbon-limited and oxygen-limited conditions. Calculation of variables such as biomass and concentration of solvent mixture under this condition yields the same values regardless of the growth-limiting substrate. Thus, the operational conditions at which simultaneous limitation occurs can be determined by equating eq 13 and 17. Experimental Section Materials. Toluene and p-xylene were from Sigma Chemical Company (St. Louis, MO). Yeast extract was purchased from Difco. All other chemicals were of reagent grade. Silicon tubing (i.d. 0.157 cm, 0.d. 0.318 cm) was obtained from Dow Corning (Midland, MI). Microorganism and Cultivation. Pseudomonas putida ATCC 23973 was used. The medium composition for storage was 1g/L tryptone, 5 g/L yeast extract, and 10 g/L NaC1. When degradation experiments were conducted, the following medium was used: 5.8 g/L KzHPO4,4.5 g/L KHzP04, 2.0 g/L (NH412S04, 0.34 g/L MgCly6H20, 0.02 g/L CaC12, 0.002 g/L NazM00~2H20,0.002g/L FeS04. 7Hz0,0.0016 g/L MnCk4H20. Bioreactor. The bioreactor was a baffled, impelleragitated type (KLF 2000, Bioengineering, Switzerland). A predetermined length of silicon tubing was installed at the bottom of the bioreactor, and a liquid mixture of toluene and p-xylene was circulated at a fixed flow rate within the tube from a reservoir using a diaphragm pump (Figure 1). A dissolved oxygen probe (Ingold, Switzerland) was installed to monitor the dissolved oxygen concentration in the bioreactor. The culture volume was fixed at 1L and the temperature was controlled at 32 "C. Impeller speed and aeration rate were varied as required. Biodegradation of Toluene and p X y l e n e i n the Bioreactor. Toluene and p-xylene were simultaneously degraded in batch, sequencing batch reactor (SBR), and continuous stirred tank reactor (CSTR) operation. In batch operation, precultured microorganism was inoculated into the bioreactor containing culture medium and a liquid mixture of toluene and p-xylene was circulated
$ v L
Impeller speed (rpm)
Figure 2. Effect of impeller speed on the transfer rates of toluene (w) and p-xylene (0).
within the silicon tubing. Impeller speed and aeration rate were controlled at the predetermined values. In SBR operation, one-third of the culture broth was replaced with fresh medium when the concentration of ammonium sulfate as nitrogen source in the culture broth fell below 10 mg/L. In CSTR operation, fresh medium was fed at the predetermined flow rate into the bioreactor, and the culture broth was withdrawn to maintain a constant liquid level using a peristaltic pump. Culture broth and exit gas were periodicallyanalyzed to determine the concentrations of toluene, p-xylene, biomass, and ammonium sulfate. The transfer rates of toluene and p-xylene were varied by changing the tubing length in the bioreactor, and each solvent was transferred at the same rate by varying the composition of the solvent mixture. Analysis. The concentrations of toluene and p-xylene were determined using a gas chromatograph (HP5890 Model, Hewlett-Packard Co., Palo Alto, CA) equipped with a flame ionization detector. A stainless steel column (6 f t X in. i.d.1 packed with Chromosorb HWP (100120mesh) was used. The temperatures of both the injector and the detector were maintained at 250 "C, and the column temperature was initially maintained at 170 "C for 3.5 min, raised by 20 "Clmin to 215 "C, and fixed at 215 "C for 3 min. The flow rate of nitrogen as carrier gas was 15 mL/min. When determining the concentrations of toluene and p-xylene in the exit gas of the bioreactor, the exit gas was collected using a homemade gas collector equipped with a Teflon-coated rubber stopper, and 100 pL of collectedgas was injected into the gas chromatograph using a microsyringe. The concentrations of toluene and p-xylene in the culture broth were determined after filtering the culture broth through a polycarbonate filter (0.45 wm pore size, Millipore). Dry cell weight was determined by weighing the filtered bacteria after drying at 105 "C for 4 h. Ammonium sulfate was determined using the phenol-hypochlorite reaction method (14).
Results and Discussion Transfer Rates of Toluene and pXylene. Toluene and p-xylene are transferred in the liquid phase through the tubing into the culture broth via tube walls. Thus, transfer rates of toluene and p-xylene are thought to be affected by some operating parameters such as impeller speed, aeration rate, circulation rate of liquid solvent within the tubing, and composition of solvent mixture in the reservoir. As shown in Figure 2, transfer rates of toluene
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5 4 m v -
z2 6 I1
1 I Fraction of p-xylene in solvent mixture
Figure 3. Effectof compositionof solvent mixture on the transfer rate of toluene (B) and p-xylene ( 0 ) . and p-xylene varied most significantly with impeller speed, and toluene was transferred much faster than p-xylene under specified conditions. There was no effect of aeration rate and circulation of liquid solvent on the transfer rate (data not shown). As toluene and p-xylene are taken up and degraded a t the same rate by the microorganism used in this work, it is preferred that the transfer rate of each solvent through a silicon tube be maintained a t the same value in order to easily control the concentration of each solvent in the exit gas below the maximum allowable level. The effect of the molar ratio of toluene andp-xylene within silicon tubing on the transfer rate was investigated, and as is shown in Figure 3,the transfer rate of each solvent became identical a t the molar ratio of 1:1.8 (toluenep xylene). In this work, the molar ratio of toluene and p-xylene was fixed at 1:1.8,and impeller speed, aeration rate, and circulation rate of solvent mixture were maintained at 600 rpm, 1 L/min, and 30 mL/min, respectively. Since silicon tubing is known to be sufficiently hydrophobic not to resist to the diffusion of hydrophobic compounds and transfer rate is severely affected by agitation speed, the transfer of solvent mixture from the tube surface to the culture broth is thought to be limiting. Silicon tubing was observed to be swollen in the presence of liquid solvents, and the volume of the tube expanded about 1.5 times. The transfer rates of toluene and p-xylene were measured on the basis of the swollen conditions of the tube. Degradation of Toluene and p-Xylene in Batch Operation. The degradation of toluene and p-xylene mixtures by P. putida was carried out in batch operation. When the transfer rate of solvent mixture was fixed a t 0.2 g/L/h, toluene and p-xylene were simultaneously degraded at equal rates by the microorganism, and microbialgrowth continued up to 75 h of cultivation time as can be seen from Figure 4. The concentrations of toluene andp-xylene in the exit gas decreased gradually with microbial growth during the exponential growth phase and were maintained a t about 0.24 ppm until microbial growth ceased. Dissolved concentrations of toluene and p-xylene also showed trends similar to those in the exit gas and were not detected after the exponential growth phase of the microorganism. It is likely that toluene and p-xylene transferred to the culture broth are effectively utilized by the microorganism and the remainder is rapidly stripped from the culture broth with an air stream. Since the solvent mixture was continuously supplied to the culture broth, the microbial
Figure 4. Biodegradation of solvent mixture in batch operation: biomass concentration;0 , toluene concentrationin the exit gas; A,p-xylene concentrationin the exit gas. Transfer rate of solvent mixture was 0.2 g/L/h. growth rate was maintained a t a constant level even after the exponential growth phase, showing a high degradation rate. But a decrease in both the growth rate and the degradation rate was observed after 75 h of cultivation, and at this time, ammonium sulfate as a nitrogen source was found to be almost depleted. Degradation of Toluene and pXylene in Sequencing Batch Operation. In order to prolong the operation time, biodegradation of toluene and p-xylene was performed in sequencing batch operation. When the concentration of ammonium sulfate in the culture broth fell below 10 mg/L, one-third of the culture broth in the bioreactor was replaced with fresh medium. As shown in Figure 5a, when the transfer rate of solvent mixture was 0.2 g/L/h, microbial growth ceased at 37 h of cultivation after medium replacement, which is defined as cyclic time, resulting in the increase of the concentrations of toluene and p-xylene in the exit gas. In this case, the removal efficiency of solvent mixture was calculated to be about 92% ,and dissolved oxygen concentration was maintained a t 30% of air saturation. As the transfer rate of solvent mixture was increased to 0.39 g/L/h, the lag period increased to 10 h, whereas the removal efficiency was increased to 96% and the cyclic time was shortened to 17.5h, as can be seen in Figure 5b. The dissolved oxygen concentration approached zero under this condition. When the transfer rate of solvent mixture was further increased to 0.81 g/L/h, the lag period also increased to 20 h, which implies inhibition of microbial growth at elevated transfer rates of the solvent mixture, and the removal efficiency and cyclic time decreased to 83% and 8 h, respectively, as can be seen in Figure 5c. Dissolved oxygen concentration was also maintained near zero as observed at 0.39g/L/h. From the above results, it is likely that a decrease in removal efficiency with an increasing transfer rate of solvent mixture is a result of oxygen limitation, and this was mathematically analyzed in more detail in continuous operation. The reason for the decrease in cyclictime with increasing transfer rate of solvent mixture is not clear, but an increase in both the microbial growth rate and damage of the cell membrane with an increasing transfer rate of solvent might be a cause for the higher uptake rate of nitrogen source and the consequently decreased cyclic time. The observation that growth yield on the nitrogen source decreased
Bbtechnol. Prcg., 1993, Vol. 9, No. 1 6
E E g
Transfer rate of solvent mlxture (g/Uh)
Figure 6. Observed growth yield as a function of the transfer rate of solvent mixture: (a) growth yield on ammonium sulfate; (b) growth yield on solvent mixture.
Figure5. Biodegradationof solvent mixture in sequencing batch operation: W, biomass concentration; 0 , toluene concentration in the exit gas; A,p-xylene concentrationin the exit gas. Transfer rates of solvent mixture were (a)0.2, (b) 0.37, and (c) 0.81 g/L/h.
sharplywith an increasing transfer rate of solvent mixture as shown in Figure 6a also supports the above explanation. Sequencing batch operation has several advantages over batch operation, including the fact that a high degradation capacity can be maintained almost permanently if cyclic time is precisely predicted. However, it is somewhat difficult to calculate the exact replacement time of culture broth because knowledge regarding the microbial physiology on inhibitory substrate is still insufficient. Application of sequencing batch operation to the bioreactor developed in this work needs further investigation. Parameter Estimations. In order to simulate the continuous biodegradation of toluene and p-xylene in the bioreactor developed in this work, the parameters that appeared in the mathematical model were estimated. Kinetic constants regarding the aerobic growth of microorganism on an equimolar mixture of toluene and p-xylene were determined by cultivating the microorganism in a 300-mL side-armed flask capped with a Teflon-
coated rubber stopper. Different amounta of the equimolar mixture of toluene and p-xylene were directly added to the flask containing culture medium, and the specific growth rate was determined by measuring the optical density of the culture broth during the exponential growth phase. The specific growth rate obtained was plotted against initial concentration of solvent mixture (data not shown). The kinetic parameters regarding the growth rate on each solvent were almost the same, and the following parameters were estimated I . C ~= 0.437
h-' (SD = 0.008)
Ki= 1.98 g/L (SD = 0.098) K, = 0.006 g/L (SD = 0.004) where SD represents standard deviations for the parameters estimated. Another kinetic constant to be obtained is the Monod constant for dissolved oxygen, but it is very difficult to obtain an accurate value. Thus, we roughly estimated& to be about O.OOO1 g/L by plotting the specific growth rate as a function of dissolvedoxygen concentration in batch culture. In addition to the above kinetic parameters, growth yield factors must be estimated to simulate the growth of microorganismunder different growth-limitingsubstrates. From the experimental results of continuous cultivation, it was found that the observed growth yield on solvent mixture decreased exponentially with the increasing
Bbtechnol. Prog.., 1993, Vol. 9, No. 1
Table I. Estimated Parameters Used in Mathematical Simulation parameter
co* D. F. h k koa Ki KC
value 7 mg/L 60 h-l 60 L1h 0.14 0.266 61.2 h-'
1980 mg/L 6 mg/L
value 0.1 mg/L
n V Yo
-0.453 1L 0.37 mg of cells/
mg of oxygen see Figure 6b 0.437 h-l
RC = 0.72
Rc = 0.39
transfer rate of the solvent mixture (see Figure 6b), and growth yield coefficients with respect to each solvent were almost identical. Thus, the observed growth yield at different transfer rates of the solvent mixture was estimated by nonlinear regression as follows:
Dilution rate ( h-' )
(19) Empirical constants k and n were found to be 0.266 and -0.453, respectively. In order to calculate the biomass concentration under oxygen-limited conditions, the oxygen yield coefficient Yo for the microorganism is required. This coefficient was estimated by measuring the oxygen uptake rate and specific growth rate during the exponential growth phase in batch culture (data not shown) and was found to be about 0.37 g of cellslg of oxygen. The coefficient koa concerning oxygen transfer rate was calculated to be 61.2 h-l by measuring the oxygen transfer rate using a dissolved oxygen probe under the operating conditions. The concentrations of toluene and p-xylene in the exit gas were replaced with those in the culture broth using eq 11. Experimental data were linearly regressed, and the correlation constant h was estimated to be 0.14. Estimated parameters are summarized in Table I and were used for the simulation of continuous biodegradation. Degradation of Toluene and p X y l e n e in CSTR. Simultaneous biodegradation of toluene and p-xylene was carried out in continuous culture by feeding the essential nutrients such as a nitrogen source and trace elements into the bioreactor, and the performance of the bioreactor was investigated with respect to the transfer rate of solvent mixture and the dilution rate. The steady-state biomass concentration was investigated at different dilution rates and transfer rates of the solvent mixture. As can be seen from Figure 7a, the steady-state biomass concentration rapidly decreased with increasing dilution rate, which resulted from the fact that the transfer rate of solvent mixture through a silicon tube is independent of dilution rate. Predicted values were well coincident with the experimental results. When the transfer rate of solvent mixture was fixed at 0.17 g/L/h, the dissolved oxygen concentration was maintained at 40% air saturation, and the concentration of nitrogen source was sufficient so as not to limit the growth of microorganisms regardless of the dilution rate. Consequently, it was thought that the growth of microorganismswas limited by the carbon source under this condition. Removal efficiency of the solvent mixture was found to be about 93%. In order to increase the steady-state biomass concentration, the transfer rate of solvent mixture was further increased by changing the length of the silicon tubing. At a transfer rate of solvent mixture of 0.39 g/L/ h, the biomass concentration was increased at each dilution rate from 0.17 g/L/h, but when the transfer rate of solvent mixture was further enhanced to 0.72 g/L/h, no significant increase in the biomass concentration was observed compared with that obtained at 0.39 gILlh. The dissolved
D = 0.04
_"_ - DD- == 0.2 0.3
Transfer rate of solvent mixture (g/Uh)
Figure 7. Effect of operating parameters on biomass concentration: (a) dilution rate; (b) transfer rate of solvent mixture. Solid lines represent the predicted values and symbola represent experimental data. Dotted lines in b indicate the biomass concentrationthat could be attained, assumingno oxygen limitation.
oxygen concentration was maintained near zero, and the concentration of nitrogen source was enough to support the microbial growth when the transfer rate of solvent mixture was increased from 0.17 g/L/h. Thus, it is thought that the growth of microorganism was limited by oxygen at a transfer rate of solvent mixture higher than 0.39 g/L/h over the whole range of dilution rates. In order to analyze this observation in more detail, biomass concentration was plotted as a function of transfer rate of solvent mixture at a given dilution rate. The dotted lines shown in Figure 7b correspond to the biomass concentration which could be obtained assuming no oxygen limitation occurred, and this evidently supports the above fact. The point from which oxygen limitation occurs indicates the shifting region of growth-limiting substrate from carbon to oxygen. Figure 8 shows the specific degradation rate of solvent mixture at different dilution rates and transfer rates of solvent mixture. The specific degradation rate increased with increasing dilution rate, which can be attributed to the fact that the uptake rate of toluenelp-xylene as the sole source of carbon and energy by microorganism increases with increasing specificgrowth rate. The specific degradation rate considerably increased with an increasing transfer rate of solvent mixture at a fixed dilution rate. The specificdegradation rate is equivalent to the metabolic quotient expressed by pi Ye;thus, if the biomass yield coefficient Y,is constant, this must be constant at a fixed dilution rate provided that the steady state is maintained
Biotechnol. hog., 1993, Vol. 9, No. 1
D = 0.05
D = 0.2 E
D = 0.2
D = 0.1
D = 0.3
5 0.00.0 4
Transfer rate of solvent mixture (g/Uh)
Transfer rate of solvent mixture (g/L/h) Figure 8. Specific degradationrate as a functionof the transfer rate of solvent mixture at a given dilution rate in continuous operation. Solid lines representthe predicted values and symbols represent experimental data. ( p = D). Therefore, an increase in the specific degradation rate with increasing transfer rate of solvent mixture at a fixed dilution rate means that the biomass yield coefficient varied with the transfer rate of solvent mixture. Experimentally, the biomass yield coefficient was calculated at different transfer rates of solvent mixture, and as can be seen in Figure 6b, the observed biomass yield decreased exponentially from 0.6 to 0.3 g of biomasslg of solvent mixture as the transfer rate of solvent mixture was increased from 0.17 to 0.72 g/L/h. The reason for this phenomenon is not yet clear, but one possible explanation is that, when toluene and p-xylene are present at high concentrations, these penetrate into the cell membrane and act on the membrane protein of the microorganism, which causes them to malfunction in some way, and consequently the biomass yield on solvent mixture decreases. p-Xylene has been reported to penetrate into a cell membrane, change the shape of the cell, and cause the membrane content to shed into the culture broth in the case of P. putida (4). The effect of these aromatic solvents on microbial physiology is now under investigation in detail in our laboratory. The degradation rate obtained in this work was considerably higher than that in conventional biological methods, and this can be attributed to the fact that toluene and p-xylene were effectively transferred to the microorganism in the bioreactor system. For example, at a transfer rate of solvent mixture of 0.39 g/L/h, the volumetric degradation rate and removal efficiencywere about 0.354 g/L/h and 97%, respectively, at the dilution rate of 0.05 h-l. In conventional methods, the maximum degradation rate was reported to be about 0.04 g/L/h in the case of toluene (8, 9, 131,and this seems to be due to a low transfer rate of toluene from the gaseous to the aqueous phase because aromatic hydrocarbons such as BTX were usually fed into the process as a gas phase together with the air stream. The removal efficiency and the concentration of solvent mixture at different dilution rates and transfer rates of solvent mixture were shown in Figure 9a,b, respectively, and the experimental results agreed well with the calculated ones over the whole range of transfer rates. The removal efficiency increased as the transfer rate of solvent mixture increased up to the region where the growthlimiting substrate shifts from carbon to oxygen and then significantly decreased. This might be explained by the
Transfer rate of solvent mixture (g/Uh)
Figure 9. Performance of the bioreactor as function of the transfer rate of solvent mixture: (a) removal efficiency; (b) concentration of solvent mixture in the exit gas. Solid lines represent predicted values and symbols represent experimental data. fact that the biodegradation was restricted by oxygen a t a higher transfer rate of solvent mixture as the growth of microorganism was limited by oxygen. Accordingly, toluene and p-xylene were not further degraded without available oxygen, and the concentration of solvent mixture in the exit gas increased considerably. Under carbon source limited conditions, solvent concentration in the exit gas remained constant with an increasing transfer rate of solvent mixture because the removal efficiencyalso increased as the transfer rate of solvent mixture increased. The decrease in removal efficiency and increase in concentration of solvent mixture in the exit gas with increasing dilution rate at a fixed transfer rate seem to be due to a decrease in biomass concentration at higher dilution rates. In view of the above points, it follows that operational parameters of the biodegradation processes should be optimized by taking into consideration both the removal efficiency and the level of each solvent in the effluent stream. Thus, at a fixed transfer rate of solvent mixture, the remaining operational parameters such as dilution rate are determined in such a way that the removal efficiency is at a maximum and at the same time concentrations of toluene and p-xylene in the effluent stream are lower than the maximum allowable levels. a CC
Notation total surface area of air bubbles (cm2) dissolved concentration of solvent mixture (g/L)
Biotechnol. Prog., 1993,Vol. 9,No. 1
concentration of solvent mixture in the gas phase (gm toluene concentration in the gas phase (g/L) p-xylene concentration in the gas phase (g/L) dissolved oxygen concentration in media (g/L) saturated dissolved oxygen concentration (g/L) dissolved toluene concentration (g/L) dissolved p-xylene concentration (g/L) dilution rate defined by F/ V (h-l) dilution rate defined by Fa/V (h-l) feeding rate of media (L/h) air flow rate (L/h) empirical constant empirical constant empirical constant empirical constant oxygen transfer coefficient (cm/h) Haldane’s inhibition constant for solvent mixture (g/L) Monod constants for solvent mixture and oxygen, respectively (g/L) empirical constant specific degradation rate of solvent mixture (g/g/ h) specific oxygen uptake rate (g/g/h) specific toluene degradation rate (g/g/h) columetric toluene transfer rate (g/L/h) volumetric transfer rate of solvent mixture (g/L/ h) culture volume (L) biomass concentration (g/L) biomass concentration under the oxygen limited condition (g/L) biomass concentration under the carbon source limited condition (g/L) observed growth yield on oxygen observed growth yield on solvent mixture specific growth rate (h-l) maximum specific growth rate (h-l)
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