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Abstract--Various nutrient media and reactor configurations were explored in order to grow the white
rot fungus Phanerochaete chrysosporium, induce its active enzyme system, develop kinetic data for the
degradation of 2-chlorophenol and use chemical engineering analysis to design an effective reactor.
Preliminary experiments indicated that the biodegradation rate was improved by a factor of 40
when the fungus was immobilized. As a result, the project focused on a packed-bed reactor employing
a silica-based porous support for the fungus, and a well-mixed reactor employing alginate beads as the
immobilizing medium. Both were very effective in degrading 2-chlorophenol at inlet concentrations up to
520 ppm. Apparent Michaelis-Menten kinetic rate constants were developed for both reactors, which
to our knowledge are the first reactor design parameters to be published for this fungus for treating a
hazardous waste.
Key words--hazardous waste treatment, Phanerochaete chrysosporium, white rot fungus, 2-chlorophenol,
reactor design
75
76 GORDON A. LEWANDOWSKIet aL
Kirk, 1985; Leisola and Fiechter, 1985). However, the hol) were also used in some of the batch fermentor experi-
intensity o f agitation provided in those studies is ments. A stock solution of Tween 80 was obtained by
dissolving 10 g of Tween 80 (Sigma Chemical Co., St Louis,
significantly lower than that experienced by micro- Mo.) into 100 ml of distilled water. A veratryl alcohol stock
organisms suspended in fermentors stirred by turbine solution was prepared by adding 0.7 g of veratryl alcohol
impellers. This high shear condition is frequently (99% purity) (Aldrich Chemical Co. Inc., Milwaukee, Wis.)
encountered in industrial practice. to I00 ml of distilled water.
Although there is much information in the litera- Analytical methods
ture concerning its growth patterns and biochemistry, Specific electrodes were used to measure fixed nitrogen
there are almost no kinetic data capable o f allowing (as ammonia), dissolved oxygen and pH. The o-toluidine
an engineered reactor design [although a patent exists colorimetric method was used for glucose determination
(Zender, 1963; Mattenheimer. 1971).
for immobilizing the fungus on a rotating biological
Analysis of 2-chlorophenol (the model compound used in
contactor (Chang et al., 1985)]. The determination the present study) was accomplished using a Varian 3700
o f reaction rate constants for continuous flow reac- gas chromatograph with flame ionization detector. Nitrogen
tors was the purpose o f our research. The a p p r o a c h was the carrier gas. A 1/~1 aqueous sample was injected onto
involved a preliminary screening o f reactor designs a 6ft x 1/8" stainless steel column operating at 140°C
and containing 10% SP2100 on 100/120 Supelcoport (with
that were c o m p a r e d on the basis o f first-order rate an accuracy of about _ 1 ppm). The temperature of the
constants for biodegradation o f 2-chlorophenol. This injector was 210°C. The retention time for 2-chlorophenol
c o m p o u n d was chosen as a model toxicant in all o f in the column was 94 s. The amount of 2-chlorophenol
our experiments. Preliminary screening was followed in the sample was determined by integration of the area
under the chromatogram using an HP3390A integator
by a more detailed examination o f the two most
(Hewlett-Packard). The system was calibrated using stan-
promising reactor designs: a packed-bed reactor con- dards of known concentration.
taining a silica-based porous support for the fungus, The stripping rate of 2-chlorophenol was determined
and a well-mixed reactor in which the fungus was using dead fungus in a batch fermentor. This was accom-
immobilized in alginate beads. plished by growing the fungus in a 10-1. batch fermentor, as
described below, and then heat sterilizing the fermentor and
its contents in an autoclave at 121°C for 20min. After
PROCEDURES cooling, an aliquot of the standard 2-chlorophenol solution
Organism, media and materials was added to the fermentor so that the 2-chlorophenol
concentration was 500 ppm. The fermentor was sparged
Phanerochaete chrysosporium BKM F-1767 (ATCC
with filtered air at an aeration rate of 2 l/min. The off-gas
24725) was maintained on yeast-malt agar. In reactor
was passed through three flasks in series, each containing 21.
operation, the following media were used in the growth and
of water, in order to absorb the 2-chlorophenol. Every 12 h,
enzyme induction phases:
the water in the absorption flasks was changed, and samples
were analyzed from the fermentor and gas traps. A material
Growth medium Induction medium
balance was then performed on the system in order to
KH2PO 4 2.0 g 2.0 g calculate the first-order stripping rate constant (see Table I).
MgSO4 0.5 g 0.5 g This rate constant could also be predicted from thermo-
CaC12 0.1 g 0.1 g dynamic considerations by assuming 25% saturation of
NH4C1 0.12 g 0.012 g the air leaving the reactor. In the absence of aeration no
Glucose 10.0 g 2.0 g 2-chlorophenol degradation was observed, thus indicating
Thiamine 0.001 g 0.001 g that no significant adsorption of 2-chlorophenol on to the
Water 1.0 liter 1.0 liter fungus took place during our experiments.
induction (Jeffries et al., 1981; Fenn et al., 1981; Fenn ments had indicated that under the low pH conditions
and Kirk, 1981; Reid 1983a, b). After day 7, 2-chloro- employed in the reactor, there was no significant biological
phenol was introduced into the fermentor at a concen- contamination during the run length.
tration of 200 ppm, along with veratryl alcohol at a con- A 4" i.d. x 16" long glass cylinder was used to determine
centration of 0.04mM. In other experiments, 10ml if there was an unaccounted scale-up factor (such as
of the Tween 80 stock solution and 10 ml of the veratryl wall effect) on the performance of the packed-bed reactor.
alcohol stock solution were introduced in the 10-1. fermentor Reactor inoculation and operation were conducted as
together with 800 ppm 2-chlorophenol. In these experi- before. The total packed volume was 1800era 3, and the
ments, the resulting concentrations of Tween 80 and aeration rate was 2.31/min.
veratryl alcohol in the fermentor were 0.05 g/l and 0.04 mM, A third set of experiments was run utilizing balsa wood
respectively. chips cut to size (5 x 5 x 5 mm, with a void fraction of 0.48)
which could provide a carbon source (cellulose) to the
Packed-bed reactor fungus as well as support. The same 2" column used for the
A 2" i.d. x 10" long glass tube was used as the packed bed Manville beads was inoculated and operated as before.
reactor (see Fig. 1). Initially, 1/4" ceramic saddles were used However, the column was only packed to half of its height,
as the packing material. However, the fungus dumped for a total packed volume of 250 ml. The aeration rate
together unevenly, particularly at the top of the column. As was 3 l/rain. The induction solution had only 10% of the
a result, another set of experiments was conducted utilizing glucose growth requirement (1 g/l), and was introduced
silica-based porous beads (Manville Celite Catalyst Carrier, from the bottom at 0.4 ml/min. The inlet 2-chlorophenol
R-635) in place of the ceramic packing. The beads were concentration was 300 ppm.
cylindrical in shape, about 1/8" in dia and 1/4" long, with With both the packed-bed and alglnate-immobilized
a mean pore size of 20 microns and a void fraction of 0.38. reactors (described below), it was difficult to measure the
The total packed volume was 400 cm 3. The temperature of biomass concentration during the kinetic runs, and reliance
the system was maintained constant at 39°C. A fungal was placed instead on the inoculation procedure.
culture grown in a shaker flask for 7 days (90 mg of dry
biomass) was homogenized in a blender and used as inocu- Well-mixed reactor with immobilized cells
lum. The fungus was allowed to grow for 5 days on the A 2% sodium alglnate solution was blended with approx.
packing material by operating the reactor in a recirculation 90-100 mg of fungus (on a dry basis). The suspension was
mode. Electron scanning micrographs had shown that then extruded through a syringe pump (with an 18 gauge
the fungus grows into the porous interior of the catalyst needle) at the rate of 6 ml/min, into a 0.2 M calcium chloride
carrier (Fig. 2). After the five-day growth period, a feed solution at room temperature. The droplets formed into
stream containing 460 ppm of 2-ehlorophenol in induction 3.5-4 mm beads upon contact with the calcium chloride
media was introduced from the bottom at flow rates of solution. All 2000 beads (approximate count) produced in
0.2--0.9 ml/min. An overflow was used to maintain a con- this fashion were then placed in a 2" i.d. x 10" long upflow
stant liquid volume. The dissolved oxygen concentration reactor, with a working volume of 400 cm 3. This was the
was maintained at about 5 mg/l at the top of the column by same reactor used previously for the packed-bed experi-
utilizing up upflow aeration rate of 0.51/rain. With the ments. A feed stream containing 520 ppm of 2-chloropbenol
Manville support there appeared to be relatively uniform in induction media was introduced from the bottom at
growth and excellent 2-chlorophenol removal. It generally the rate of 0.1-1 ml/min (there was no separate growth
required about 5 days of reactor operation with induction phase). The aeration rate was approx. 0.31/min, which was
medium before steady-state was reached. During this sufficient to suspend the beads and achieve the condition of
period, the outlet concentration was monitored. When it a well-mixed reactor. The alglnate beads remained intact for
was clear that a constant outlet concentration had been about 3 weeks before the alginate structure began to deteri-
reached, the experiment was terminated. Preliminary experi- orate under the attack of the fungus.
Sampler
Temperature
DO
lcato Bath
Controller
arnpler
Air I I I I
Alr Filter Rotameter
ouo i ii I
lnductlon
Medium
Fig. 1. Schematic diagram of experimental set-up for packed-bed reactor.
78 (~ORDON A. LEWANDOWSKI~"! al.
Fig. 2. Photomicrographs of a spherical Manville celite catalyst carrier showing fungal hyphae that have
grown into the porous bead.
Reactor design using a white rot fungus 79
Table 2. Performance of the 400 cm 2 packed-bed reactor Table 3. Performance of the 1800cm 3 packed-bed
with Manville celite catalyst carrier reactor with Manville c.elite catalyst carrier
Flow rate 2-CP conc. in 2-CP conc. in Flow rate 2-Cp conc. in 2-CP conc. in
(ml/min) inflow (ppm) outflow (ppm) (ml/min) inflow (ppm) outflow (ppm)
02 460 0. I 0.5 500 0.1
03 460 23 1.0 500 104
0.4 460 37 2.0 500 160
05 460 39 2.5 500 182
0.7 460 114 3.0 500 204
0.8 460 160 40 500 233
0.9 460 166 50 500 292
6.O 500 323
Vm = 45 ppm/h.
10.0 500 375
K ~ - 132 ppm.
12.0 500 397
14.0 500 410
Table 3 shows the steady-state effluent concen- ,v'm= 26 ppm/h.
trations of 2-chlorophenol from the 4" dia packed- K m = 133 ppm.
bed reactor at different flow rates. These data were
regressed as before using the Michaelis-Menten wood as a packing material) needs to be explored
model, and the best-fit values of Vm and Km are listed further.
in the table. Figure 5 shows the fit. Once again, S m
Well-mixed reactor with immobilized cells
was allowed to float, and its regressed value was
438 ppm (instead of 500 ppm). Table 4 shows the steady-state effluent concen-
When the regressed contants are compared with trations of 2-chlorophenol from the well-mixed
those for the 2" dia reactor, virtually the same value reactor at different flow rates. Once again, the degra-
of Km was obtained (as would be expected for dation rate was described by the Michaelis-Menten
the same enzyme system). The different values of equation, and a standard mass balance written for the
Vm could be attributed to different biomass concen- reactor (as before, the stripping rate was negligible
trations in the two reactors. relative to the rate of biodegradation, and operating
As before, the inhibition model was also tested. at steady-state eliminated any significant adsorption
The corresponding values for kL, k~ and K l were effects):
5 ppm/h, 14 ppm, and - 4 0 8 ppm, respectively, with
QS~n - QS,,u~ + rc v = O.
a negative inhibition constant as before.
Only one flow rate was used with the balsa The data were regressed, resulting in the best-fit
wood reactor (the steady-state effluent concen- values of Vm and K m reported in Table 4. Figure 6
tration was 43 ppm), and therefore, only a first- shows the fit. As before, Sin was allowed to float,
order constant is given in Table 1. This shows resulting in a regressed value of 511 ppm (instead of
comparable performance to the Manville packing. 520).
Note that the glucose concentration in the feed was The difference in the value of V~ from the packed-
reduced, since cellulose was available as a carbon bed results could be attributed to different bio-
source. Therefore, it is expected that the life of the mass concentrations. However, the value of Km was
packing would be limited. This methodology (with expected to be the same as before, since the enzyme
500
2°° I
~ 400
~=
~120 c
g 300
._g ,
c ©
0 )
' [] ° [] 8 200
o. iI
o
_o ~o 100
£ 40 I
&
I I J I [ I I I I I I I I I
0 .2 ,4 .6 .8 1.0 2 4 6 8 10 12 14
Flow Rate (ml/min) Flow Rate (ml/min)
Fig. 4. Performance of a packed-bed reactor (2" dia) with Fig. 5. Performance of a packed-bed reactor (4" dia) with a
silica-based catalyst carrier, silica-based catalyst carrier.
Reactor design using a white rot fungus 81
Table 4. Performance of the well-mixedreactor with of toxic compounds. The reduced fungal degradation
alginate-immobilizedcells
activity against 2-chlorophenol observed in the me-
Flow rate 2-CP eonc. in 2-CP conc. in
(ml/min) inflow (ppm) outflow(ppm) chanically stirred fermentor systems could also result
from the high shear effect experienced by the fungus
0.3 520 18
0.4 520 20 in such systems.
0.5 520 69 (3) In packed-bed and immobilized cell systems, the
0.6 520 78
0.7 520 130
fungus was able to degrade 2-chlorophenol at appre-
0.8 520 146 ciable rates even in the absence of certain compounds
0.9 520 176 (such as Tween 80 and veratryl alcohol) which pre-
1.0 520 215
vious investigators (Faison et al., 1986; Linko and
vm= 46 ppm/h.
Km= 16ppm. Zhong, 1987; Tonon and Odier, 1988; Jager et al.,
1985; Asther et al., 1988) found to enhance degrada-
tion activity. This indicates that such compounds
system is presumed to be the same. Furthermore,
may not be required in wastewater treatment, which
mathematical analysis of the mass transfer and reac-
would make the practical application of this technol-
tion rates in the beads indicated that the effectiveness
ogy more attractive.
factor was high (about 0.7-0.9). This would imply
(4) Wood chips can serve as both carrier and
that the internal mass transfer resistance is not very
co-substrate in a packed-bed reactor.
large for the beads. Nevertheless, the rate constants
(5) A packed-bed reactor employing a silica-based
were only apparent constants, and different mass
porous support, and a well-mixed reactor employing
transfer resistances in the two types of reactor could
alginate beads, were designed and operated to
affect the apparent values of 1I, and Kin-
degrade 2-chlorophenol at feed concentrations up
Once again, there was also an attempt to use an
to 460 and 520 ppm, respectively. The upper limit
inhibition model. However, as with the packed-bed
for the 2-chlorophenol concentration before system
reactor, the value of K~ was negative, and therefore
failure was not determined.
the model was inappropriate.
(6) For the packed-bed reactor, the rate constants
obtained with a 2" column were consistant with those
CONCLUSIONS obtained with a 4" column. However, the apparent
rate constants obtained with the well-mixed reactor
(1) Phanerochaete chrysosporium immobilized on
were appreciably different, implying a difference in
alginate beads can degrade 2-chlorophenol at concen-
the mass transfer resistances.
trations up to at least 520 ppm.
(7) The Michaelis-Menten equation can represent
(2) Under the conditions employed in this work,
2-chlorophenol degradation data for both the
degradation of 2-chlorophenol was much more
packed-bed and well-mixed reactors in continuous
efficient in reactor systems in which the fungus was
mode. A substrate inhibition model appeared to be
immobilized. It could be speculated that the fungus
inappropriate, since regression of the data yielded
is less active in a suspended growth reactor, and
negative values for the inhibition constant. To our
needs to be attached to a surface to produce the
knowledge, the Michaelis-Menten rate constants
ligninolytic system responsible for the degradation
obtained in the present work are the only reactor
design parameters available in the open literature for
240
treating a hazardous waste.
f
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~
& 4o
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