Enhanced cometabolic transformation of 4-chlorophenol in the presence of phenol by granular activated carbon adsorption.
Loh, Kai-Chee ; Wang, Ye
Substrate inhibitions that manifest within the cometabolism system
of 4-chlorophenol (4-cp) and phenol were alleviated through the
application of granular activated carbon (GAC) in batch biodegradation.
It was found that 4-cp was preferentially adsorbed over phenol by the
GAC and that 50% to 70% of the adsorption was achieved within the first
two hours of contact. The kinetics of 4-cp adsorption was also much
faster than that of phenol, even when the co-existing phenol was of a
significantly higher initial concentration. As a result, competitive
inhibition between the two compounds was minimized. Adsorption also
caused a lowering of the phenol concentration in solution with a
concomitant reduction in the substrate inhibition effect on cell growth.
The addition of GAC benefited the biotransformation process through
shortening the total degradation time for 600 mg [L.sup.-1] phenol and
100 mg [L.sup.-1] 4-cp from 42 h to 12 h; and it also made it possible
for cells to survive and transform 600 mg [L.sup.-1] phenol and as high
as 400 mg [L.sup.-1] 4-cp in free suspension cultures. Repeated
operations in which GAC was reused showed that GAC could be regenerated
by the cells, thus rendering the GAC incorporated process amenable to
long term operations.
Les inhibitions de substrat qui se manifestent dans le systeme de
cometabolisme du chlorophenol-4 (cp-4) et du phenol ont ete reduites par
l'application de charbon actif granulaire (GAC) lors d'une
biodegradation discontinue. On a trouve que le cp-4 etait le premier a
etre adsorbe sur le phenol par le GAC et que de 50 a 70 % de
l'adsorption avait lieu dans les deux premieres heures du contact.
La cinetique d'adsorption du cp-4 est egalement beaucoup plus
rapide que celle du phenol, meme lorsque le phenol coexistant est
d'une concentration initiale substantiellement plus elevee. En
consequence, l'inhibition competitive entre les deux composes est
minimisee. L'adsorption cause egalement une baisse de la
concentration de phenol dans la solution avec une reduction concomitante
de l'effet d'inhibition du substrat sur la croissance des
cellules. L'ajout de GAC a un effet benefi que sur le processus de
biotransformation en raccourcissant le temps de degradation total pour
600 mg [L.sup.-1] de phenol et 100 mg [L.sup.-1] de cp-4 de 42 h a 12 h;
et cela permet egalement aux cellules de survivre et de transformer 600
mg [L.sup.-1] de phenol et jusqu'a 400 mg [L.sup.-1] de cp-4 en
cultures de suspensions libres. Des operations repetees dans lesquelles
le GAC est reutilise montrent que le GAC pourrait etre regenerees par
les cellules, rendant ainsi le procede d'incorporation du GAS
propice pour des operations a long terme.
Keywords: cometabolism, adsorption, phenol, 4-chlorophenol,
Pseudomonas putida, granular activated carbon
Phenolic compounds, many of which are known to be toxic to many
living organisms, are commonly found in the environment as a result of
the rapid pace of industrialization. The oil refining, textile,
pesticide, as well as the pulp and paper industries have always produced
high concentrations of phenol and chlorinated phenolic compounds in
either their primary products, or their waste effluents. These compounds
do not only taste and smell unpleasant, but they are also known to be
toxic and carcinogenic. Biological treatment of such waste water and
industrial effl uent usually involves utilization and transformation of
mixed substrates. In some cases, the phenolic compounds cannot support
cell growth (non-growth substrates), and cometabolism is encountered.
In cometabolism, biotransformation of the non-growth substrate is
achieved either by growing cells in the presence of a growth substrate,
by resting cells in the absence of a growth substrate, or by resting
cells in the presence of an energy source (Criddle, 1993). In order for
a biological treatment process involving cometabolism to be sustainable,
growth substrates, which provide energy for cell growth and maintenance,
must inevitably be present. One well-known cometabolism system that has
been extensively studied is the biodegradation of phenol (growth
substrate) and 4-chlorophenol (4-cp) (non-growth substrate) by
Pseudomonas putida (Saez and Rittmann, 1991; Loh and Wang, 1998). Phenol
is converted by monooxygenase to catechol and then by catechol 2,
3-dioxygenase to 2-hydroxy muconic semialdehyde (HMSA), after which HMSA
is further oxidized serially to ultimately yield C[O.sub.2], energy for
biosynthesis and maintenance and biomass (Yang and Humphrey, 1975). For
the biotransformation of 4-cp, Saez and Rittmann (1991) postulated that
the fi rst two enzymes for the degradation of phenol can also catalyze 4-cp to 4-chlorocatechol and then to 2-hydroxy-5-chloro-muconic
semialdehyde (HCMSA), which remains in solution as a dead-end
metabolite. In this case where both phenol and 4-cp share the same key
enzymes, competitive inhibition exists and cometabolic transformation of
4-cp is strongly affected by the presence of phenol. On the other hand,
4-cp also inhibits metabolism of phenol because of its toxicity and
recalcitrance, thereby decreasing cell growth and retarding
biodegradation (Criddle, 1993; Saez and Rittmann, 1993). Furthermore,
cell growth on phenol has been observed to display substrate inhibition
effect at high phenol concentrations (Yang and Humphrey, 1975).
To avoid competitive inhibition two-stage or sequencing reactor
systems have been proposed to decouple cell growth and transformation of
the non-growth substrate. For example, Alvarez-Cohen and McCarty (1991)
have proposed the use of a two-stage bioreactor that utilizes
cometabolic biotransformation of trichloroethylene (TCE) by
methanotropic cells in the presence of methane (a growth substrate)
while Hecht et al. (1995) have investigated the feasibility of a
bioscrubber to cometabolically degrade TCE. However, from a practical
viewpoint, such multistage processes are not applicable when treating
co-existing pollutants, like phenol and 4-cp.
Activated carbon adsorption has been extensively studied and
applied as a physical technique for the removal of aromatic hydrocarbons
from waste water (Flora et al., 1994; Moreno-Castilla et al., 1995;
Chatzopoulos and Varma, 1995; Furuya et al., 1997; Haghseresht et al.,
2002; to name a few). In the realm of biological treatment, activated
carbon has also been used as a cell immobilization matrix (Ehrhardt and
Rehm, 1985; Kindzierski et al., 1992; to name a couple). Ehrhardt and
Rehm (1985) noted the survival of the immobilized bacteria in spite of
the addition of normally toxic phenol concentrations and the subsequent
utilization of most of the adsorbed phenol. They concluded that quick
adsorption of phenol on the activated carbon enabled the bacteria to be
exposed for only a short time to the toxic phenol concentrations, and
this seemed to be applicable for the treatment of waste water containing
temporarily high phenol concentrations. According to Kindzierski et al.
(1992), the activated carbon served as an immobilization buffer and
protected the immobilized micro-organisms by adsorbing toxic phenol
concentrations and gradually set free low quantities of the adsorbed
phenol for biodegradation.
In a recent study, Furuya et al. (1997) reported that the
adsorption isotherms of phenolic compounds on activated carbon could be
signifi cantly different depending on the electron cloud density of the
compounds, and their consequential interactions with the surfaces of the
carbons. Based on this, we anticipated that there might be a possibility
of separating phenol and 4-cp utilizing activated carbon adsorption so
as to reduce the extent of competitive inhibition between the two
compounds, and at the same time, lower the phenol liquid phase
concentration, thereby decreasing the substrate inhibition effect.
The objective of this study was to exploit the feasibility of using
granular activated carbon (GAC) in a batch reactor to simultaneously
biodegrade a mixture of phenol and 4-cp. To this end, equilibrium
adsorption isotherms of phenol and 4-cp on a coconut-shell-based GAC,
both singly and in combination, were obtained. In addition, the kinetics
of adsorption was also investigated. Subsequently, the bio-availability
of adsorbed phenol and 4-cp was investigated to evaluate the use of GAC
for phenol biodegradation and consequently cometabolic transformation of
4-cp in the presence of phenol in a sustained treatment operation.
EXPERIMENTAL
Micro-Organisms
Throughout this study, Pseudomonas putida ATCC 49451 was used, and
stock cultures were maintained on nutrient agar slants (Oxoid,
Hampshire, U.K.) and stored at 4[degrees]C. For preparation of inocula,
cells from the nutrient agar slant were induced with the basal medium
(described below) supplemented with 200 mg [L.sup.-1] phenol as the sole
carbon source. All batch cultures were grown with agitation at 200 rpm
and at 30[degrees]C. The working volume in each run was 250 mL, in
cotton plug fitted 500 mL Erlenmeyer flask. All cell culture experiments
were conducted in triplicates.
Culture Medium
The chemically defined culture medium used in this study consisted
of a mineral salt medium and a trace mineral solution, the compositions
of which have been reported by Loh and Wang (1998). 10 mL of the trace
mineral solution and 30 mL of the mineral salt medium were added to each
litre of the medium. The sole carbon source, phenol, was added
accordingly, to obtain the desired concentrations for each experiment.
Chemicals
All chemicals used in this study were of analytical grade. Phenol
and 4-cp, obtained from Merck (Darmstadt, Germany), were each dissolved
in 0.02M NaOH solution to make 10 000 mg [L.sup.-1] stock solution. The
granular activated carbon (GAC) used throughout this study was a
coconut-shell-based type (1~1.5 mm in diameter) and obtained from
Casitan (Petaling Jaya, Malaysia). The GAC was washed with Milli Q water
to remove all the fine particles, and then heated to 70[degrees]C for 24
h, and finally stored in a desiccator for subsequent use.
Adsorption Kinetics and Isotherms
Kinetics of the adsorption was obtained by soaking 0.5 g GAC in 250
mL of phenol or 4-cp at two initial concentrations, 300 mg [L.sup.-1]
and 600 mg [L.sup.-1]. At predetermined time intervals, the
concentrations of phenol or 4-cp in solution were measured from 1 mL
sample taken from the fl ask. All experiments were performed in
triplicates.
To obtain the equilibrium adsorption isotherms of phenol and 4-cp
on the GAC, 0.5 g of GAC were immersed in 250 mL of varying initial
concentrations of phenol (100-1600 mg [L.sup.-1]) and 4-cp (100-1000 mg
[L.sup.-1]), either alone or in combinations. The phenol and 4-cp
solutions were prepared in culture mineral solution. The suspension was
left for 72 h for equilibrium to be established. The amount of phenol or
4-cp adsorbed was determined from material balances after measuring the
equilibrium concentration of the components in solution.
Cell Density Measurement
Cell concentration was determined by measuring the optical density
(OD) at 600 nm using a Shimadzu UV-Visible Spectrophotometer UV-1601 and
1-cm-path length quartz cuvettes with deionized water as reference.
Optical Density (OD) obtained was converted to cell mass based on the
correlation established earlier: X(mg [L.sup.-1])=314.5*O[D.sub.600]
(Wang and Loh, 1999).
Phenol and 4-cp Measurement
Phenol and 4-cp concentrations were determined in accordance with
the protocol reported by Loh and Wang (1998). The detection limit of the
gas chromatography method was within 1 mg [L.sup.-1].
RESULTS AND DISCUSSION
Single Component Adsorption The kinetic profi les of phenol and
4-cp adsorption respectively, on GAC during the fi rst two hours of the
adsorption were fi rst investigated. Previous studies on the rate of
adsorption of phenols on GAC have indicated that the adsorption rate was
rather fast at the beginning. In their study, Zogorski et al. (1975)
reported that the amount adsorbed in the initial stage of adsorption was
linearly related to the square root of time ([t.sup.1/2]), and that more
than 60% to 80% of the adsorption occurred within the first hour.
Nevskaia et al. (1999) reduced kinetic expressions derived from models
based on interaction between the surface and the adsorbate, to the same
parabolic relationship as that shown in Equation (1):
q (t) = [alpha] x [t.sup.1/2] (1)
where [alpha] is an initial concentration dependent coefficient.
Equation (1) was used to fit the experimental kinetic data obtained
(data not shown) with excellent agreement ([r.sup.2] > 0.98).
The top half of Table 1 summarizes the values of [alpha]for
different initial concentrations of phenol and 4-cp. We found that
[alpha] for 4-cp was much larger than that for phenol at both initial
concentrations studied, suggesting that the uptake rate of 4-cp was
faster than that of phenol. During the 2 h period in which the kinetics
experiments was conducted, the adsorbed phenol levels were 51% and 53%
of the equilibrium capacity for initial concentrations of 300 mg
[L.sup.-1] and 600 mg [L.sup.-1], respectively, while the corresponding
percentages for 4-cp, were 71% and 57%. In general, equilibrium
adsorption was reached after 24 h.
The experimental adsorption data for phenol and 4-cp on GAC were
fitted to the Langmuir-Freundlich isotherm (Equation (2)), also with
excellent agreement ([r.sup.2] > 0.97; data not shown).
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (2)
In Equation (2), [q.sub.e], [q.sub.s], and b are defined as the
equilibrium solid phase concentration of the adsorbate, the saturated
solid phase concentration representing the adsorption capacity and the
Langmuir isotherm constant, respectively; v is a parameter related to
the heterogeneity of the active sites on the adsorbent, and when v is
unity, the Langmuir-Frendlich model is reduced to the Langmuir isotherm
for an energetically homogeneous adsorbent (Jaroniec and Madey, 1988).
The Langmuir-Freundlich isotherm was chosen because liquid-solid
adsorption data could be better modelled especially when interactive
effects of multiple substrates were involved.
The second half of Table 1 tabulates the model parameters for
phenol and 4-cp. The obvious difference between the value of [q.sub.s]
for phenol and 4-cp indicates the different adsorption capacities: that
GAC has higher adsorption capacity for 4-cp over phenol. Moreover, the
affinity constant, b, for 4-cp was also larger than that for phenol,
indicating that the GAC used has a greater adsorption affinity for 4-cp
over phenol. The adsorption phenomena of phenolic compounds on GAC has
yet to be thoroughly understood due to the great variety of activated
carbons available and the complexity of the adsorption process itself
(Arafat et al., 1999). It has long been assumed that the adsorption of
phenolic compounds on activated carbon is due to the interaction of the
[pi] electrons of the aromatic ring and the surface of the activated
carbon. Radovic et al. (1997) has reported that the adsorption of
phenolic compounds on activated carbon also depends on the number and
types of substituents on the aromatic ring although the carbon atoms
attached to the chlorine atoms in chlorophenols did not seem to interact
with the active site on the adsorbent.
The fitting results also gave a lower v for 4-cp compared with
phenol, which indicates that the active sites were more heterogeneous
for phenol adsorption than for 4-cp adsorption.
Binary Component Adsorption
The kinetics of adsorption in the binary component system was first
examined. Results from these experiments are shown in Figure 1. These
indicate that the kinetics of adsorption for both phenol and 4-cp were
little affected by their mutual presence in the mixture. Furthermore, it
can be seen again that a significant portion of the adsorption was
accomplished within the first two hours of contact with the GAC.
[FIGURE 1 OMITTED]
In considering the use of the adsorption process to facilitate
cometabolism of phenol and 4-cp, the adsorption rates and the
differences in the rates are very important characteristics. This is
because the overall biodegradation rate could very likely be dominated
by the lag phase, which could vary greatly with different concentrations
of both the non-growth substrate and the growth substrate, due to their
toxicity effect on the degrading cells. Therefore, a faster adsorption
rate of the organic compounds is required to help the cells in
overcoming the toxicity of the substrate through shortening the contact
time for direct contact with the highly concentrated and toxic solution.
The competitive effects that exist between phenol and 4-cp
adsorption on the GAC were investigated by performing adsorption
experiments on mixtures of phenol and 4-cp. Figure 2 features the
resulting isotherms.
[FIGURE 2 OMITTED]
It can be seen that the inhibition of 4-cp on the equilibrium
adsorption of phenol was greater than the converse; the equilibrium
adsorption of 4-cp was not affected to as great an extent by the
presence of the equal initial concentration of phenol.
The adsorption data were fitted to modified interactive isotherms
(Ruthven, 1984) given by Equation 3:
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (3)
where i, j = 1, 2 for phenol and 4-cp as the case may be, and all
other notations have their usual meanings. Specifically, k provides an
indication of the relative inhibition effect between the two substrates.
Figure 2 also shows the corroboration of the model and the
experimental data. The only adjustable parameters were obtained as
[k.sub.2] (inhibition due to 4-cp) = 6.3 (dimensionless) and [k.sub.1]
(inhibition due to phenol) = 1.8 (dimensionless). The fits were very
good with [r.sub.2] in excess of 0.97. For comparison, the single
component adsorption isotherm was also plotted. It can be seen that
phenol adsorption was relatively more significantly decreased in the
presence of 4-cp than the converse. The stronger relative inhibition
exerted by 4-cp on phenol adsorption can be quantitatively assessed by
the ratio:
[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]
where subscript 1 is for phenol, and 2 is for 4-cp. For all the
concentrations studied, this ratio ranged from 1.2 to 1.5 (data not
shown), confirming the stronger inhibition of 4-cp to phenol adsorption.
What was more significant in these results was that for the same
starting concentrations of each of phenol and 4-cp, due to the higher
adsorption capacity of GAC for 4-cp, the equilibrium concentration of
4-cp in the solution mixture was significantly lower than that of
phenol. For example, for starting concentrations of 1000 mg [L.sup.-1]
each of phenol and 4-cp, the equilibrium concentration of phenol was 600
mg [L.sup.-1] while that of 4-cp was only 300 mg [L.sup.-1]. If the
starting concentrations of phenol were much higher than that of 4-cp
which is typically the case in industrial waste water (e.g. 1000 mg
[L.sup.-1] phenol and 100 mg [L.sup.-1] 4-cp; Loh and Wang, 1998), this
difference in equilibrium concentrations would be more dramatic. When
phenol and 4-cp co-exist in a mixture and the 4-cp concentration is
lower than the phenol concentration, GAC can adsorb most of the 4-cp,
leaving a significant amount of phenol in the solution. This result
implies an effective separation of phenol and 4-cp in the waste water,
and the release of the competitive inhibitory effect of 4-cp on phenol
degradation in the phenol-4-cp cometabolism system.
Bioavailablity of Adsorbed Phenol
Before investigating the feasibility of using GAC in the
cometabolism system, studies were conducted to analyze the
bioavailability of the adsorbed phenol for cell growth. Typically,
suspension cells could not grow in phenol concentrations exceeding 800
mg [L.sup.-1] due to substrate inhibition effects (Wang and Loh, 1999).
Figure 3 shows the results of a representative experiment for 600 mg
[L.sup.-1] initial phenol concentration. At this phenol concentration,
in the absence of GAC, the suspension cells experienced a short lag
phase of about 5 h before phenol was gradually degraded, concomitant
with cell growth. The specific growth rate in this case was 0.29
[h.sup.-1], and the maximum cell density attained was 314 mg [L.sup.-1].
Total degradation of phenol was achieved in 21 h.
[FIGURE 3 OMITTED]
With GAC, the phenol concentration profile was very different.
There was a sharp decrease in the first hour due to rapid adsorption of
the phenol on the GAC. This was followed by a gradual removal of phenol
to an undetectable limit. Cell growth did not show significant lag, and
exponential growth immediately ensued. The specific growth rate, in this
case, was 0.4 [h.sup.-1]. Cells grew faster because of the lower (less
toxic) phenol concentration in the solution. Disappearance of phenol
from the solution was achieved in a much shorter time, only 15 h. It is
important to note that in contradistinction to the control experiment,
in which the cell density immediately declined upon depletion of phenol,
cells continued to grow, albeit slowly even though phenol was undetected
in the solution in the presence of GAC. During this period, there was
still a small amount of adsorbed phenol, which slowly desorbed from the
GAC to support cell growth.
All the experiments conducted in this section gave similar results,
and it can therefore be concluded that the adsorbed phenol indeed did
desorb from the GAC, and was therefore available for cell growth.
However, there remained a doubt on whether all of the adsorbed phenol
could reversibly desorb from the GAC. To investigate this, the cell
yield on phenol, based on the suspension cell density obtained and
assuming complete consumption of phenol, was calculated for all the
experiments.
Figure 4 compares the cell yield on phenol in the presence and
absence of GAC. In the absence of GAC, cell yield noted a decrease with
increase in phenol concentrations, while in the presence of GAC, we
observed an increase in cell yield with increase in phenol
concentrations. Notwithstanding, the cell yield obtained with GAC
present was in all cases lower than that without GAC. Superficially, it
seemed that some of the adsorbed phenol had been irreversibly bound on
GAC, and hence not available for cell growth, hence resulting in lower
final cell density.
[FIGURE 4 OMITTED]
However, it has been reported that GAC is a good immobilization
matrix for cells (Kindzierski et al., 1992), and therefore the cells
could have grown on the adsorbed phenol on the GAC, but not registered
in the cell density measurements of solution turbidity. Moreover, it has
also been observed that cells continued to grow for a further duration
beyond phenol depletion in solution, implying that there was continued
desorption of phenol. In the absence of reliable analytical technique to
determine the total cell density present in each of the experiment, it
was difficult to ascertain if all of the adsorbed phenol was presented
to the cells during the biodegradation process. In subsequent
sustainability studies, however, it was confirmed that a small but
constant amount of phenol (and 4-cp) remained irreversibly bound to the
GAC after the first exposure. This will be discussed in a later section.
Regardless, the presence of GAC has allowed for cell
immobilization, hence increasing phenol tolerance (Keweloh et al.,
1989), a lowering of the phenol concentration in solution (at high
initial phenol concentrations) with decreased substrate inhibition on
cell growth, and consequently a much shorter time taken for phenol
removal.
Impact of 4-cp Toxicity on Degradation Time
The inhibitory effect of 4-cp on phenol degradation has been
studied extensively (Saez and Rittmann, 1993; Wang and Loh, 1999). It
has been proven that 4-cp could severely retard phenol degradation and
cell growth. The presence of 4-cp caused a long lag phase for phenol
degradation resulting in a longer overall degradation time. In our
study, we found that the lag phase for phenol degradation of 300 mg
[L.sup.-1] phenol was extended from 6 h to 35 h when 4-cp concentration
increased from 100 mg [L.sup.-1] to 200 mg [L.sup.-1]. Of course, the
level of the phenol concentrations in the mixture also influenced the
lag phase significantly since phenol exerts substrate inhibition
effects. Due to competitive inhibition between phenol and 4-cp,
typically 4-cp would only start to transform upon complete removal of
phenol (Wang and Loh, 1999). Therefore, the total degradation time would
inadvertently be longer than the time taken for complete phenol
depletion. The concentration of 4-cp, nevertheless, has a more
dominating effect on the total degradation time due to its toxicity on
the cells. For example, we have found that complete removal of 600 mg
[L.sup.-1] phenol with 100 mg [L.sup.-1] 4-cp took 41 h, which was still
10 h shorter than the time taken to remove 300 mg [L.sup.-1] phenol and
200 mg [L.sup.-1] 4-cp. Although pure cultures of P. putida could
provide a rather high specific growth rate, the lag phase--as long as 35
h (in presence of 200 mg [L.sup.-1] 4-cp)--makes the total degradation
period longer than 50 h, thus causing the potential application to
deteriorate.
To alleviate the competitive inhibition of the growth substrate
(phenol in our study) and the non-growth substrate (4-cp in our study)
as well as the extension of the lag phase for degradation due to
non-growth substrate toxicity in simultaneous degradation of the two
compounds, many researchers have suggested the sequential degradation of
the growth substrate, followed by the non-growth substrate by resting
cells (Alvarez-Cohen and McCarty, 1991; Hecht et al., 1995).
Considering the special adsorption features of GAC that have been
demonstrated earlier, it is possible that simultaneous degradation could
be improved by simply adding GAC, which could result in the considerable
lowering of the 4-cp concentrations in solution. Before studying the
impact of GAC on phenol and 4-cp, a study on sequential degradation of
phenol and 4-cp was conducted, to assess the effectiveness of using GAC
later. P. putida was grown in different concentrations of phenol, to
cell confluence through phenol depletion and 100 mg [L.sup.-1] 4-cp was
added to the solution. At that stage, no growth substrate was present in
the medium and 4-cp was transformed at the expense of the resting cells.
Table 2 tabulates the experiments conducted and the results of the
degradation times for complete removal/ transformation of the two
compounds.
The complete removal of 100 mg [L.sup.-1] 4-cp (in reasonable time
durations) required cells to be grown in initial phenol concentrations
of as high as 300 mg [L.sup.-1] in order that sufficient resting cells
were available for transforming 4-cp. The times taken for the complete
removal of phenol and 4-cp in these experiments, however, were much
lower than that for simultaneous degradation. For example, the time
taken for removing 500 mg [L.sup.-1] phenol and 100 mg [L.sup.-1] 4-cp
in sequential degradation was 17 h and this was much shorter than that
for simultaneous degradation, which took 27 h (data not shown). However,
when the 4-cp concentration was as high as 300 mg [L.sup.-1], even cells
raised in 500 mg [L.sup.-1] could not transform it. It can be inferred
that the activity of resting cells could be inhibited when they were
suddenly exposed to such a high 4-cp concentration.
Application of GAC in the Phenol-4-cp Cometabolism System
The feasibility of using GAC for the simultaneous degradation of
phenol and 4-cp was examined by conducting the biodegradation
experiments in the presence of 2 g [L.sup.-1] of GAC. Figure 5 shows the
phenol and 4-cp concentration profiles for all the experiments. For easy
reference, indexes for these experiments are P for phenol only, PC for
phenol + 4-cp mixtures, G for experiments with GAC and the number at the
end represents the concentration of 4-cp in hundreds mg [L.sup.-1]. In
all of these, there was a sharp drop in concentration during the first
couple of hours of fast adsorption. Following that, depending on the
initial concentration of 4-cp, there was either no lag in degradation
(at low 4-cp concentrations) or a short lag in degradation (at high 4-cp
concentration). GAC was effective in selectively removing 4-cp from the
solutions rendering improved phenol degradation rates.
[FIGURE 5 OMITTED]
From Figure 5, comparing PC1 and GPC1, in the presence of GAC, the
lag phase for degradation was significantly lowered from 33 h to 6 h and
consequently total degradation time decreased from 42 h to 12 h, or
almost 4 times reduction in time taken. With increasing 4-cp
concentration, the lag phase also increased despite the presence of GAC,
but the overall effectiveness was definitely enhanced. In particular,
when cells were provided with 4-cp concentrations above 300 mg
[L.sup.-1] in the absence of GAC, no biotransformation occurred
regardless of phenol concentration; in the presence of GAC, even 400 mg
[L.sup.-1] 4-cp could be removed within a reasonable duration.
It can be concluded from the above results that the separation of
4-cp and phenol by GAC adsorption could effectively relieve the
inhibitory effect of 4-cp on phenol degradation and maintained phenol at
a sufficient level to ensure a reasonable cell growth rate.
Consequently, the total time needed for removal of phenol decreased from
42 h (PC1) to 12 h (GPC1) for the same phenol and 4-cp concentrations.
The question, however, remained whether 4-cp was merely adsorbed by the
GAC or that it was bioavailable to the cells for transformation. In the
final section of this research, the GAC used in the experiments was
reused to seek an answer to this question.
Reusability of GAC in the Cometabolism System
Multiple degradation runs were conducted to evaluate the
reusability of the GAC. In this reusability test, the same GAC was
repeatedly used in multiple operations of the
adsorption-degradation-desorption cycle. During the test, 800 mg
[L.sup.-1] of phenol mixed with 200 mg [L.sup.-1] of 4-cp, was used
together with 2 g [L.sup.-1] GAC. The degradation and cell growth
profiles of cycles 1, 3, 4, 5 and 6 were recorded. It is important to
mention that each of the experimental runs was maintained for 10-15 h
beyond the complete exhaustion of both phenol and 4-cp to ensure that
all reversibly bound substrates had been released before starting the
next cycle.
According to Figure 6, it can be seen that part of the GAC
adsorption capacity for both phenol and 4-cp was irreversibly lost. The
lost capacity was around 100 mg [g.sup.-1] GAC for phenol and 40 mg
[g.sup.-1] GAC for 4-cp, accounting for 57% and 58% of the GAC
adsorption capacities of phenol and 4-cp, respectively, in the first 4
to 6 h. However, it was found that once GAC had lost this part of its
adsorption capacity, there was no more irreversible adsorption. It was
found that during the third to the sixth run, the same phenol and 4-cp
removal profiles were recorded, despite an extension of the lag phase by
about 10 h after the first run.
[FIGURE 6 OMITTED]
The lost capacity of phenol and 4-cp could be due to several
reasons, including adsorbate oxidation, and hysteresis. Our results,
nevertheless, show that the reversible capacity of GAC could be
maintained at a certain level in a sustained application.
This implies a potential for application in industrial waste water
treatment involving cometabolic transformations. In this study, cell
growth also occurred in quite a similar manner in the third to sixth
runs. After approximately a 35 h lag phase, exponential growth occurred
and ended with consistently about 283 mg [L.sup.-1] cell mass. The
similarity of the results in the last four runs suggests that the
process could reach a steady state operation when the GAC adsorption
capacity has been stabilized.
CONCLUSIONS
In order to sustain cometabolic biodegradations in waste water
treatment, a number of substrate inhibitions have to be taken care of:
(a) substrate inhibition of the growth substrate phenol; (b) toxicity of
the non-growth substrate 4-cp; and (c) competitive inhibition between
the two substrates. Granular activated carbon (GAC) has been exploited
in batch biodegradation for minimizing these inhibitions so that removal
efficiency could be enhanced. GAC has been found to be effective for
selectively removing 4-cp over phenol in synthetic waste water
containing the two substrates. Furthermore, adsorption of the substrates
reduced the solution concentration of both substrates. The feasibility
of using GAC has been demonstrated to not only reduce the substrate
inhibitions involved in the cometabolism system, but the overall
degradation effi ciency has also been improved, with a consequential
reduction in the overall degradation time. Long term operation using
this technique has also been ascertained through repeated operations in
which the GAC was reused.
ACKNOWLEDGEMENTS
The authors wish to acknowledge the graduate scholarship to Ye Wang
provided by The National University of Singapore.
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December 5, 2005; accepted for publication December 6, 2006.
Kai-Chee Loh * and Ye Wang
Department of Chemical and Biomolecular Engineering, National
University of Singapore, 4 Engineering Dr. 4, Singapore 117576,
Singapore
* Author to whom correspondence may be addressed.
E-mail address: chelohkc@nus.edu.sg
Table 1. Kinetics and Langmuir-Freundlich parameters for phenol
and 4-cp adsorption on GAC in single component system
Component Kinetics ([alpha])
(mg[L.sup.-1][h.sup.-1])
Initial concentration
300 mg[L.sup.-1] 600 mg[L.sup.-1]
Phenol 69 108
4-cp 104 167
Langmuir-Freundlich parameters
[q.sub.s]([mgg.sup.-1]) b v
Phenol 1430 0.04 0.30
4-cp 2150 0.10 0.15
Table 2. Effect of 4-cp on degradation time in sequential degradation
of phenol and 4-cp
Phenol 4-cp Time for phenol
(mg[L.sup.-1]) (mg[L.sup.-1]) degradation (h)
50 100 7
75 100 7
100 100 8
200 100 11
300 100 9
500 100 12
500 300 20
600 200 24
Phenol Time for 4-cp Total time of
(mg[L.sup.-1]) removal (h) processes (h)
50 N/A -
75 N/A -
100 78 86
200 66 77
300 9 18
500 5 17
500 N/A -
600 5 29
N/A: 4-cp was not appreciably transformed.
