Effects of cross-substrate interaction on biotrickling
filtration for the control of VOC emissions
Walter Den
a,*, Chihpin Huang
b, Chi-Han Li
ca
Department of Environmental Science, Tunghai University, No. 181, Sec. 3, Taichung-Kan Road, 407 Taiwan, Republic of China bInstitute of Environmental Engineering, National Chiao Tung University, 75 Po-Ai St.,
Hsinchu 30050, Taiwan, Republic of China
cDepartment of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai St., Hsinchu 30050, Taiwan, Republic of China
Received 16 July 2003; received in revised form 7 June 2004; accepted 10 June 2004
Abstract
The effects of cross-substrate interaction to the performance of a gas-phase biotrickling filter for treating a mixture of volatile organic compounds (VOCs), including three structural heterologous in acetone, toluene, and trichloroethyl-ene, were investigated. The biotrickling filter was inoculated with microbial consortium containing at least seven bac-terial species utilizing either acetone or toluene, or both, as their carbon sources. In the performance study, the column operating under variable conditions typifying the waste gas emission from the microelectronics fabrication processes achieved a total hydrocarbon (THC) removal efficiency in excess of 85% and a mineralization capacity over 50% for
THC mass loading as high as 36.2 g-CH4m3h1. The cross-substrate effects were examined by correlating the relative
changes in the mass removal of each substrate with the biodegradative capability of the microbial consortium. The deg-radation of trichloroethylene was primarily due to co-metabolism by the toluene-oxidizing enzymes, but the results also indicated that trichloroethylene partially induced its own degradation. Concentration increases in acetone appeared to cause a diauxie effect that suppressed degradation of toluene and trichloroethylene, and shifted the microbial popula-tion toward the selective acetone-degraders. No irrecoverable toxicity or inhibitory effects were observed throughout the experiments. These results suggest that the relative VOC concentration in the waste gas mixture is a factor as impor-tant as the biodegradative function of the microbial consortium, and thus should be carefully evaluated to satisfy the treatment objectives.
2004 Elsevier Ltd. All rights reserved.
Keywords: Biotrickling filtration; Cross-substrate; Co-metabolism; Industrial waste gas; Volatile organic compound; Bioremediation
1. Introduction
In gas-phase biological processes for the control of industrial waste air emissions, the potential effects of interaction among the mixtures of volatile organic com-pounds (VOCs) are important issues to the process per-formance. Both stimulatory and antagonistic effects 0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2004.06.018 *
Corresponding author. Tel.: +886 4 23590121x3050; fax: +886 4 23594276.
E-mail address:wden@mail.thu.edu.tw(W. Den).
have been documented when multiple substrates (con-taminants) are exposed to mono or mixed microbial
cul-tures (Rittman and Saez, 1993). The stimulatory effects
are primarily attributed to co-metabolism, which re-quires induction of non-specific enzymes capable of cat-alyzing degradation of recalcitrant compounds. These non-specific enzymes, often induced by compounds without chemically reactive functional groups such as alkanes and aromatics, bind to both the growth sub-strate and the co-metabolite to undergo biotransforma-tion. In contrast, antagonistic effects generally arise from substrate competition effects that elicit a variety of re-sponses from diauxie to concurrent utilization of
multi-ple substrates (Maier, 1989). In diauxie the preferred
substrate is utilized first, followed by a lag phase before other substrates are consumed. In general, the relative concentrations of substrates are critical to the microbial responses with respect to the substrate degradation pat-tern. For instance, high concentrations of target sub-strate normally favors diauxie pattern, provided that the concentration levels do not cause toxic inhibition. On the contrary, low substrate concentrations appear to favor concurrent utilization of several substrates. This type of competitive substrate inhibition is known to occur between structural homologous substrates binding to the same active site of an enzyme. A typical example
was delineated by Burbeck and Perry (1993) in their
studies for degradation of alkylbenzene mixture (tolu-ene, benz(tolu-ene, ethylbenz(tolu-ene, xyl(tolu-ene, and styrene) using Mycobacterium vaccae as a dominant species. They observed an enhanced styrene removal due to co-metab-olism induced by toluene. However, the preferential deg-radation of toluene also delayed the benzene utilization when the two compounds were present in equimolar amounts.
Researchers have also proposed mathematical models to relate the substrate consumption and the microbial
growth kinetics for homologous binary mixtures (Klecˇka
and Maier, 1988;Bielefeldt and Stensel, 1999) or tertiary
mixtures (Reardon et al., 2000). These growth kinetic
models proved to be accurate by incorporating various inhibitory parameters to delineate the cross-substrate effects among the homologous substrates. However, for a body of mixture consisting of three or more structurally unrelated substrates (i.e., heterologous substrates), the metabolic role of each substrate for microorganisms in an engineering system is mostly unknown, particularly for a gas-phase biological treatment process.
To evaluate the effects of complex substrate interac-tion in the presence of an easily biodegradable ketonic compound in acetone, a moderately biodegradable aro-matic compound in toluene, and a recalcitrant chlorin-ated alkene compound in trichloroethylene (TCE), a series of biotrickling filter experiments were conducted in the present study. The synthetic VOC mixture and their concentration ranges used in the study was targeted
to mimic the realistic waste air emission from the micro-electronics industry, such as the wafer fabrication and
the printed circuit board facilities (US EPA, 1995;
Coo-gan and Jassal, 1997; Den et al., 1999). The waste emission was typically characterized by substantial fluc-tuation due to the cyclical nature of the batch fabrica-tion processes. In this type of mixture, the metabolic pathway of each VOC all appeared to be vastly different. In particular, the presence of a heavily chlorinated com-pound in TCE among the VOC mixtures forms a chal-lenge to the biological system since biodegradation of the xenobiotic compound requires pathways normally
not found in heterotrophic bacteria (Fetzner, 1998).
Consequently, it is believed that TCE by itself cannot be utilized by heterotrophic bacteria under aerobic con-ditions, but may be co-metabolized in the presence of another substrate to serve as the primary carbon and
en-ergy source (Nelson et al., 1987;Shields et al., 1989;
Ass-inder and Williams, 1990). A number of bacterial cells possessing enzymes with low substrate specificity have been demonstrated to be capable of transforming TCE
via co-metabolism (Wackett and Gibson, 1988;Winter
et al., 1989;Folsom et al., 1990;Hanson and Brusseau,
1994). However, the phenomena of mutual inhibition
and induction of the active enzymes between toluene
and TCE has also been documented (Heald and Jenkins,
1994;McClay et al., 1995). The presence of a third sub-strate (i.e., acetone) further complicates the question of cross-substrate interactions, because acetone is not known as an inducer for TCE-degrading enzymes but could conceivably serve as a growth substrate for the bacteria. The main objective of this study, therefore, is to examine the cross-substrate effects by correlating the biodegradative capability of the microbial consortium with the mass removal of each VOC in the gas mixtures.
2. Materials and methods
2.1. Protocol for microbial cultivation, enrichment, and biodegradation tests
The microbial seeds were originally obtained from an industrial wastewater treatment facility (Hsinchu, Tai-wan) and were initially acclimated by feeding a vapor mixture of filtered air with acetone (600 ppmv), toluene (50 ppmv), and TCE (5 ppmv) into a cell fermentor con-taining 300-ml suspended culture in mineral salt medium
of 8.5 mg l1KH2PO4, 33.4 mg l1K2HPO4, 17.4 mg l1
Na2HPO4, 1.7 mg l1 NH4Cl, 11 mg l1 MgSO4,
aug-mented with 5.0 g l1tryptone and 2.5 g l1yeast extract.
The pH of the mineral medium was neutralized before use. After 60 h of acclimation, an aliquot (1 ml) of culture suspension at three different dilution levels was trans-ferred separately onto plate-count agars and
36 h. After a series of culture enrichment and isolation procedures, the microbial cultures were purified and
stored at 4C until further use. The bacterial
identifica-tion was primarily determined by the standard Biolog procedure interfaced with the MicroLog 1 database
(Biolog Inc., USA).
The growth and biodegradability studies were con-ducted in duplicate by shake-flask experiments. In the growth study, 150 ml of purified cultural suspensions
di-luted to a cell density of approximately 100 CFU ml1
were separately transferred to a series of 250-ml Erlenmeyer flasks sealed with a septum device (for head-space sampling) and placed in a constant-temperature
(30C) rotating incubator. Reagent-grade acetone (15
mM) and toluene (10 lM) were added to separate groups of cultural suspensions containing the same min-eral salt medium without tryptone and yeast extract, such that the pure bacterial cultures could use acetone or toluene as their sole carbon source. A blank group containing only the cultural suspensions in the mineral medium served as the control. The headspace gas and aqueous mass concentration of the substrates can be re-lated using HenryÕs law for a closed equilibrium system:
mtotal¼ mLþ mG and mG mL ¼ Hc RcT Vf VL 1 ð1Þ
where mLand mGdenote the mass of substrate in
aque-ous and gas phases, Vfand VLrefer to the volumes of
flask and liquid suspension, respectively, Hc is the
HenryÕs law constant (atm m3mol1), Rcis the gas
con-stant (8.21· 105atm m3mol1K1), and T is the
tem-perature (K). The aqueous concentrations of acetone and toluene in the suspensions provided headspace con-centrations similar to those to be used in the ensuing biotrickling filter column studies. The same procedure was also followed for the biodegradability studies, ex-cept that the substrates were added as a mixture to the cultural suspensions (15 mM acetone, 60 lM toluene, 10 lM TCE). Blank controls containing sterilized solu-tions were used to account for the abiotic loss of the VOCs. Headspace samples were taken with a 1-ml glass syringe every 30 min for the first 4 h, and every 4 h after-ward. Cell growth was determined by optical density at
600 nm (OD600) on a Hach DR/4000 spectrophotometer
(Hach Co., USA). The OD600 values were correlated
with cell numbers determined by the plate-count method.
2.2. Biotrickling filter system
A bench-scale biotrickling filter (Fig. 1) comprising
of two stages of acrylic column (internal diameter: 7 cm; height: 30 cm each stage) was used to evaluate the effects of VOC mixture to the overall system perform-ance. The column was packed to a depth of 25 cm in each stage with sieved granular activated carbon
(GAC, type BLP, Calgon Carbon Corps., USA) of
6· 12 mesh size. Activated carbon was chosen due to
a combination of favorable properties such as the une-ven surface texture for microbial attachment and growth, the microporous nature for adsorption, and the structural integrity for high permeability. A total of 0.8 kg of mixed bacterial consortium (equal wt.% of each purified strain) was inoculated onto 1.4 kg of car-bon for the column start-up. The pH-controlled (7.0 ± 0.5) recirculating liquid was nozzle-sprayed from the top of the filter bed at a rate of 1.5 l/min. Fresh
min-eral solution (500 mg l1KH2PO4, 500 mg l1K2HPO4,
50 mg l1NH4Cl, pH 7.0) was added daily to the
recir-culating liquid for replenishment of inorganic nutrients to the microorganisms. The VOC-contaminated air stream then entered from the top of the column, forming a co-current mode with the liquid flow. Gaseous samples
were collected daily using 1-l Teflon sampling bags
(Alltech Inc., USA) from each of the four equidistant sampling ports for analysis.
2.3. Experimental methods
As indicated inTable 1, the biotrickling filter column
experiments were broadly divided into ‘‘performance evaluation’’ (phases 1–6) and ‘‘cross-substrate evalua-tion’’ (phases 6–17). Prior to inoculation of microorga-nisms, an independent adsorption breakthrough study was conducted under the ‘‘wetted’’ condition (i.e., with liquid spraying) to serve as a control test for the bio-trickling filter studies. For the column performance evaluation, the influent total hydrocarbon (THC) con-centration, which served as a surrogate concentration for all VOCs in the mixture, was varied from 850 to
2400 ppmv (standardized as CH4). To simulate the
ac-tual emission pattern from microelectronics
manufactur-ing facilities (Den et al., 2003) while minimizing
variation in substrate concentration ratio during the experimental phases, the composition of the three VOCs contributing to the THC was maintained such that the following conditions were satisfied: (i) the volumetric ratio (i.e., ppmv/ppmv) of acetone-to-THC was always within the range of 0.2–0.3, and (ii) the volumetric ratio of toluene-to-TCE remained close to 5. Additionally, the empty-bed-contact-time (EBCT) was maintained at 155 s except for the operation during phase 5.
The cross-substrate effects were evaluated by system-atically varying the concentration of the target VOC (re-ferred to as the ‘‘variable VOC’’ hereinafter) in the sequence of acetone (phases 6–9), toluene (phases 10– 13), and TCE (phases 14–17). A ‘‘baseline’’ condition, chosen as the lower end of the VOC emission spectrum, was established as the reference to which the change in mass removal of each target VOC under the variable conditions was compared. This condition provided an influent acetone concentration of 220 ppmv, toluene of
50 ppmv, and TCE of 10 ppmv, resulting in an equiva-lent THC concentration of approximately 850 ppmv as
CH4. When a step-change of the variable VOC was
made, the mass removal corresponding to the change in the VOC was determined from the steady-state condi-tion. The mass removal for each VOC was calculated using the following equation:
mj¼
QðCin CoutÞjfc
VB
ð2Þ
where mj represents the average steady-state mass
re-moval rate (g m3h1) for VOC species j, Q is the
volu-metric gas flowrate (m3h1), VBis the packing volume
(m3), C
inand Cout denote to the inlet and outlet VOC
volumetric concentrations (ppmv), and fcis a conversion
factor from volume-base to mass-base concentration. The relative change of mass removal at a particular mass loading condition for the non-variable VOCs deviating from those at the baseline condition was quantified using the following formula:
Relative removal¼mj;ref mj
mj;ref
100% ð3Þ
where mref,jis the mass removal of species j at the
base-line condition.
2.4. Analytical methods
The gas-phase VOC concentrations (acetone, tolu-ene, and TCE) were determined by a thermal
desorp-tion/gas chromatograph unit (TDS-GC, GC-14B,
Shimadzu Instrument Co., Japan) equipped with a flame ionization detector and a 30-m, AT-1 fused silica capil-lary column (Alltech Associates, Inc., USA). The THC concentration was analyzed by the same instrument, ex-cept that the analytical column was replaced by a 15-m deactivated fused silica capillary column because no compound separation was needed. The THC concentra-tion was routinely calibrated by the standard 100 and
1000 ppmv methane gases (CH4in N2, Scott Specialty
Gases, USA), and therefore was defined as the CH4
-equivalent concentration. Carbon dioxide was moni-tored at the inlet and outlet ends of the column using
a portable non-dispersive infrared analyzer (TelaireTM
1050, Telaire System Inc., USA) capable of measuring
up to 2000 ppmv of CO2 (±5%). The residual TOC in
the recirculating liquid was periodically analyzed using a TOC 5000A analyzer (Shimadzu Instrument Co., Japan) with automatic injection (200 ll). Potassium hydrogen phthalate and sodium carbonate were used for the cali-bration of total carbon and inorganic carbon, respec-tively. Before analysis, 50-ml aqueous samples were directly withdrawn from the recirculating tank and trea-ted with 0.45 lm cellulose nitrate membrane (Whatman, Inc., USA).
3. Results and discussion
3.1. Microbial characterization and biodegradability After a series of isolation and cultivation cycles over a one-week period, a total of seven bacterial strains capable of aerobically degrading acetone, toluene, or TCE were isolated and identified, including three Pseu-domonas strains (designated as B1–B3) possessing dis-tinct morphological and/or biochemical characteristics
as determined by the Biolog method. As shown inTable
2, all seven strains were capable of growing on acetone
as the sole carbon source, and five of the seven strains also grew on toluene. These bacteria generally exhibited an exponential growth phase lasting 20–24 h before entering their stationary phases, and demonstrated rapid cell growth over eight orders of magnitude during the exponential phase. The observed lag phase typically did not extend beyond 10 h except for the Acetobacteri-aceae species (18 h).
The rapid microbial growth on acetone and toluene indicated that these bacterial strains were efficient degraders of the target VOCs. The specific growth rate (l) of each bacterial culture was calculated for the growth on acetone (15 mM) or toluene (10 lM) as the sole carbon source. Data were interpreted only during their exponential growth phases. The numerical values of the specific growth rate can be also be estimated from
the doubling time of cell number (k = ln 2/t1/2, where t1/2
is the doubling time), as it was done in this study as a
confirmatory procedure (Bustard et al., 2000). As shown
inTable 2, all seven species efficiently utilized acetone as their carbon and energy source, exhibiting a broad range
of l values between 0.49 and 1.03 h1. In contrast, the
five species (B1–B5) that also grew on toluene showed relatively narrow range of l values between 0.34 and
0.46 h1. As a general observation, the three
Pseudo-monas strains (B1–B3) were not as efficient to grow on acetone as the other species, whereas species B4 and B5 were relatively efficient to grow on both acetone and toluene.
The biodegradability experiments were conducted in
the shake-flasks at 30C to correlate the specific growth
rates with the actual substrate consumption rates. The bacterial strains from the growth experiments were indi-vidually transferred to a set of fresh mineral medium
solutions and adjusted to a cell density (from OD600
measurement) of approximately 108cells ml1. The
cul-tures were then exposed to a mixture of acetone, tolu-ene, and TCE. The substrate degradation rate was calculated based on the maximum rate of concentration Table 1
Loading conditions for the biotrickling filter experiments Experimental
objective
Phase Days Acetone
concentration (ppmv) Toluene concentration (ppmv) TCE concentration (ppmv) Corresponding THC concentration (ppmv as CH4) Column performance studies 1 0–31 220 50 10 850 2 32–43 325 75 15 1250 3 43–53 470 200 40 2400 4 53–67 345 145 30 1750 5 67–77 470 200 40 2400 6 77–84 220 50 10 850 Cross-substrate effect studies 7 84–90 365 50 10 1250 8 90–96 560 50 10 1750 9 96–107 815 50 10 2400 10 107–113 220 50 10 850 11 113–120 220 120 10 1250 12 120–129 220 215 10 1750 13 129–137 220 330 10 2400 14 137–142 220 50 10 850 15 142–148 220 50 15 1930 16 148–156 220 50 30 1960 17 156–163 220 50 60 2010 18 163–168 220 50 10 850
reduction in the headspace. Since the maximum sub-strate degradation rates occurred in the first 4 h of the experiments, the headspace air and the initial dissolved oxygen (DO) in the microbial suspensions was presum-ably sufficient to prevent DO depletion during the
study. As shown in Table 2, the acetone degradation
rate displayed a similar tendency as previously seen in the specific growth rates, with the exception for B5 hav-ing high value of specific growth rate but relatively low degradation rate. Contrarily, the degradation rates of toluene did not correlate well with the corresponding specific growth rates for the tested concentration, possi-bly due to the metabolic interference from the presence of acetone in the mixture. All three Pseudomonas species showed significant TCE degrading capability, and the Sphingomonas species also degraded TCE to a much lower extent. It has been demonstrated that Sphingo-monas species share physiological similarities with the
Pseudomonads (Fredrickson et al., 1995), although the
enzymatic mechanism associated to its TCE degrada-tion has not been clarified. In principle, for the four TCE-degrading species (B1–B4), the TCE degradation rates seemed to follow closely to their respective toluene degradation rates, implying a strong biodegradative correlation between the two substrates.
3.2. Removal of VOC mixture under variable loading conditions
As described earlier in Section 2.3, the biotrickling fil-ter column experiments were arranged into two parts; namely the ‘‘performance evaluation’’ (phases 1–6) and the ‘‘cross-substrate evaluation’’ (phases 6–17). The transient column response obtained from the former part has already been extensively presented elsewhere (Den et al., 2003). However, since the latter part was a continuation of the column operation, it would be
perti-nent to reiterate some of the important findings in order to substantiate the discussion on the occurrence of cross-substrate effects due to the presence of the three hetero-logous VOCs.
The step-increase pattern of influent THC mass
load-ing, along with the effluent THC and CO2 production
profiles, are jointly presented inFig. 2for experimental
phases 1–6. During the start-up period (phase 1) with an
influent mass loading of 12 g-CH4m3h1(equivalent
to a THC concentration of 850 ppmv as CH4), the low
production of carbon dioxide suggested that the high THC removal efficiency was primarily due to carbon adsorption. As the microorganisms gradually acclimated to the loading conditions, biodegradation became the predominant removal mechanism, as manifested by the
steady increase in the CO2evolution profile. The carbon
balance calculation showed that approximately 70% of
the organic carbon was recovered as CO2at the end of
phase 1, when an apparent steady state was reached.
Subsequent increases in mass loading to 18 g-CH4
m3h1 (1250 ppmv, phase 2) and then to 36 g-CH4
m3h1(2400 ppmv, phase 3) appeared to deteriorate
the column performance. As a result, the THC removal
efficiency at the peak loading of 36 g-CH4m3h1not
only reduced the removal efficiency to as low as 85%, but also diminished the carbon mineralization (into
CO2) ratio to the vicinity of 50%. Furthermore, for each
of the step-increases, a distinct ‘‘hump’’ was observed in the effluent profile, indicating the occurrence of a lag
phase between THC mass removal and CO2 evolution
in response to the changes of influent mass loading. This phase delay was most probably due to the time needed for microbial adaptation to the new loading conditions. The duration of these phase delays appeared to lengthen at higher mass loading, extending from roughly two
days at 18 g-CH4 m3h1 to nearly four days at 36
g-CH4m3h1.
Table 2
Characteristics and substrate biodegradability of the microbial consortium
No. Identificationa Gram staining Specific growth rate
(l)b, h1
Substrate degradation rate (R)c,
mM h1108cell
Acetone Toluene Acetone Toluene TCE
B1 Pseudomonas G 0.49 0.35 0.12 1.0· 103 1.1· 104 B2 Pseudomonas G 0.59 0.36 0.09 7.1· 104 5.4· 105 B3 Pseudomonas G 0.51 0.34 0.14 1.2· 103 1.0· 104 B4 Sphingomonas G 0.62 0.36 0.08 5.0· 104 2.8· 105 B5 Bacillus sp. G+ 0.73 0.46 0.02 1.3· 104 NA B6 Acetobacteriaceae G 1.0 NA 0.21 NA NA B7 Mycobacterium sp. G+ 0.79 NA 0.24 NA NA
‘‘NA’’ indicates ‘‘no significant biodegradation’’.
a Similarity index > 75% of taxonomic pattern using the Biolog procedure and database. bSpecific growth rate on either acetone (15 mM) or toluene (10 mM) at 30
C. c
Substrate consumption rate normalized with the starting cell number in the batch study. Each cultural suspension was exposed to a mixture of acetone, toluene, and TCE at initial aqueous concentrations of 15, 60, and 10 mM, respectively.
Due to the observed column performance
deteriora-tion at a THC loading of 36 g-CH4m3h1, two
differ-ent procedures were attempted to decrease the infludiffer-ent mass loading; namely, by lowering the influent THC concentration (phase 4), and by reducing the gas
flow-rate in half (phase 5). As shown inFig. 2, reduction of
influent loading from 36 to 27 g-CH4 m3h1 (phase
4) immediately led to improved removal efficiency as well as mineralization capability. At this juncture, the gas flow rate was reduced in half, whereas the influent THC concentration was raised to the same level as in phase 3 to provide an equivalent mass loading of only
18 g-CH4m3h1(phase 5). This change did not result
in significant improvement of THC removal from phase
4. However, a noticeable increase in CO2 production
was observed during this period, indicating that the THC concentration was not inhibitory to biodegrada-tion. This result showed that the reduction of THC re-moval efficiency and mineralization ratio experienced in phase 3 was likely caused by the large mass loading (i.e., combination of high influent concentration and high flow rate), rather than by the high THC influent concentration alone.
As noted, approximately 70–80% of the organic
car-bon was mineralized into CO2when the THC loading
was below 27 g-CH4 m3h1, with no consistently
detectable intermediate products based on GC analyses. Since biomass assimilation typically accounts for 5–10% of the organic carbon depending on the biomass yield
efficiency of the carbon sources (Weber and Hartmans,
1996;Zhu et al., 1996), as much as 20% of the organic carbon was still unaccounted for in the carbon balance. It was therefore assumed that the VOCs––particularly
for acetone, a highly soluble compound––were partially absorbed into the liquid stream in the biotrickling filter and carried into the circulation tank. TOC analyses peri-odically performed on the liquid phase in the circulating
tank were always rather low (<5 mg l1), indicating that
biodegradation occurring in the liquid phase was highly possible in this study. Nevertheless, because the tank liq-uid was either partly or fully replaced by fresh mineral medium solution, no further investigations were made on the extent of aqueous phase biodegradation.
Considering that the system was operated under
nutrient-rich conditions (C/N 4) as compared to the
theoretical nutrient requirement (C/N 6) for cell
syn-thesis, depletion of DO in the excessively grown biofilm could conceivably occur. However, cell enumeration regularly measured from the top, middle, and bottom of the filter demonstrated a sharp reduction in the cell counts by more than three orders of magnitude (from
start-up cell count of 109g1GAC) after 15 days of
operation. For operation of a biotrickling filter, the wash-out of excess cells by the shear force of the contin-uous liquid flow has been reported in a number of
stu-dies (Diks et al., 1994; Weber and Hartmans, 1996).
This mechanism helps stabilize the thickness of biofilm, which can be reflected by measuring the number of via-ble cells. In the present study, relatively steady cell
counts in the order of magnitude of 107cells g1GAC
were consistent obtained after 30 days of operation. The results of the controlled biofilm thickness, coupled with the sufficiently high ratio of conversion from organic
carbon to CO2, suggest that oxygen depletion was not a
significant factor within the VOC concentration range tested in the study. Furthermore, although the pressure Time (day) 0 20 40 60 80 T H C m ass lo ading and rem ov al (g -CH 4 m -3 h -1) 0 10 20 30 40 CO 2 p ro duc ed (g -C O2 m -3 h -1 ) 0 10 20 30 40
Influent mass loading
CO2-C mass produced
Effluent mass remained Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6
Fig. 2. Total hydrocarbon (THC) mass loading and removal profiles in the biotrickling filter. The corresponding carbon dioxide production is also shown in the figure.
drop across the filter bed was not measured, no direct evidence (e.g., liquid flooding) associating with micro-bial clogging was observed throughout the study. 3.3. Effects of variation of influent VOC concentration
In contrast to the experimental strategy employed for the column performance study, the subsequent experi-ments (phases 6–17) were designed to investigate the re-sponse in the removal of each VOC by step-changing the concentration of the variable VOC, while maintaining the others constant. Specifically, the influent loading for each of the VOCs was varied in the order of acetone (220–850 ppmv), toluene (50–330 ppmv), and TCE (10– 60 ppmv). It was noted that the removal data under each ‘‘base condition’’ (phases 6, 10, 14, 18) were somewhat different due to the varying conditions prior to returning to these base levels. Nevertheless, they were comparable enough to give a fair reference to the extent of VOC removals under the ensuing experimental conditions. Typically, it took more than four days to reach a stea-dy-state removal after returning to the base levels.
Fig. 3 shows the mass removal profiles of all three VOCs in response to the change of influent acetone con-centration from 220 to 850 ppmv (phases 6–9, corre-sponding to the acetone mass loading of 12 g-acetone
m3h1up to 44 g-acetone m3h1) as denoted by the
dotted lines in the figure. It can be observed that, with each step-increase in the acetone mass loading, the mass removal efficiency of acetone itself progressively de-creased, reaching a valley of approximately 90% at the
peak loading of 44 g-acetone m3h1. The extents of
mass removal for toluene and TCE also decreased
mark-edly from 4.2 g-toluene m3h1and 1.3 g-TCE m3h1
(at the baseline loading) to 3.8 g-toluene m3h1and 1.1
g-TCE m3h1 (at the peak acetone loading),
respec-tively. The rate of reduction was particularly noticeable when the acetone loading exceeded 30 g-acetone
m3h1.
The removal profiles of the VOCs in response to the step-increase in the toluene loading (phases 10–13) are
compiled inFig. 4. In contrast to the removal patterns
observed when acetone was the variable VOC, the extent of toluene mass removal only marginally lessened when
the influent loading of 4.5 g-toluene m3h1was
step-in-creased to 29 g-toluene m3h1. It was also noted that
TCE removal was not significantly influenced by the influent toluene loading, as its steady-state mass removal
remained at the proximity of 1.2 g-TCE m3h1. The
re-moval of acetone, however, did show a gradual decline
from 12 to 10 g-acetone m3h1. For both TCE and
acetone profiles, noticeable ‘‘dips’’ were again observed immediately after each step-increase in toluene concen-tration, apparently due to the lag-phase phenomenon
discussed earlier. In the similar manner, Fig. 5 shows
the removal profiles of the VOCs when TCE was the variable VOC, whose influent loading was increased
from 1.2 up to 7.5 g-TCE m3h1(phases 14–17). In
general, the removal patterns somewhat resembled those
observed inFig. 3 because all three VOCs exhibited a
declining manner with respect to their removal capabi-lities. The extent of the reductions in their removals, however, appeared to be less substantial than that when acetone was the variable VOC.
The different removal patterns in response to each variable VOC clearly suggest a number of possible cross-substrate interactions involved in the biotrickling filtration of the VOCs. It is noted, however, that the ex-tents of removal reduction (or enhancement) were only moderate, suggesting that the biodegradation of the
Time (days) 0 5 10 15 20 25 30 A ceto ne m a s s lo ad ing a n d rem o va l (g m -3 h -1 ) 10 20 30 40 50 T C E m as s re m ov a l (g m -3 h -1 ) 1.10 1.15 1.20 1.25 1.30 1.35 1.40
Toluene mass removal (g m
-3 h -1) 3.8 4.0 4.2 4.4 4.6 acetone loading acetone removal TCE removal toluene removal
Fig. 3. Mass removal profiles for acetone, toluene, and TCE with step-increases in influent acetone loading (dotted stairlines). The mass removal of acetone (d) is indicated by the single arrow corresponding to the left scale, and mass removal of TCE (}) and toluene (
M
) are indicated by the single and double arrows corresponding to the right-inner and right-outer scale, respectively.variable VOCs were still efficient. These results effectively eliminate the occurrence of self-toxicity that generally leads to irreversible or chronic recovery of microorgan-isms when they were exposed to excessively high concen-trations. Therefore, the observed reductions in the removal efficiency of the variable VOCs were primarily
due to overloading rather than toxic effect (Chou and
Wu, 1999). To present a clearer picture on the potential cross-substrate effects, the relative removal defined in
Eq.(3)was calculated and graphically shown inFig. 6.
The variable VOC and its loading increases (in concen-tration) were indicated in the categorical vertical scale,
and the relative changes of mass removal (in %) of the other VOC species deviating from the baseline condition were indicated in the horizontal scale. As seen in the fig-ure, when the influent toluene concentration was in-creased from 50 to 330 ppmv, the mass removal of TCE neither showed much improvement nor significant deterioration. Since TCE is known to be biodegraded via co-metabolism by toluene-induced oxygenases, the results of the sustained TCE removal clearly suggested the occurrence of toluene-induced co-metabolism. For
a direct comparison,Cox et al. (1998)reported that
con-tinuous supply of toluene at 25 ppmv led to sustained Time (days) 0 5 10 15 20 25 30 0 1 2 3 4 5 6 7 8
Toluene mass removal (g m
-3 h -1) 3.8 4.0 4.2 4.4 4.6
Acetone mass removal (g m
-3 h -1) 9 10 11 12 13 14
TCE mass loading and removal (g m
-3 h -1) TCE loading TCE removal toluene removal acetone removal
Fig. 5. Mass removal profiles for acetone, toluene, and TCE with step-increases in influent TCE loading (dotted stairlines). The mass removal of TCE (}) is indicated by the single arrow corresponding to the left scale, and mass removal of toluene (
M
) and acetone (d) are indicated by the single and double arrows corresponding to the right-inner and right-outer scale, respectively.Time (days)
0 5 10 15 20 25 30
Toluene mass loading and removal (g m
-3 h -1) 0 5 10 15 20 25 30
TCE mass removal (g m
-3 h -1) 1.0 1.1 1.2 1.3 1.4
Acetone mass removal (g m
-3 h -1) 9 10 11 12 13 14 toluene loading toluene removal TCE removal acetone removal
Fig. 4. Mass removal profiles for acetone, toluene, and TCE with step-increases in influent toluene loading (dotted stairlines). The mass removal of toluene (
M
) is indicated by the single arrow corresponding to the left scale, and mass removal of TCE (}) and acetone (d) are indicated by the single and double arrows corresponding to the right-inner and right-outer scale, respectively.TCE degradation (influent concentration of 11 ppmv) when Pseudomonas putida F1 was inoculated as a pure culture in their gas-phase, binary-component biofilter studies. However, increase in the TCE concentration caused temporary toxicity effect that reduced the toluene degradation efficiency, and increase in the toluene con-centration led to a competitive advantage of toluene that inhibited TCE degradation by the same system of active enzymes. Their findings presented an ambiguity between toxicity (by TCE) and competitive inhibition (by toluene) when both substrates co-existed in the same waste stream. In the present study, the fact that no deteriora-tion of TCE removal was observed even at high toluene loading implied that the competitive inhibition was not a factor within the tested range of toluene-to-TCE centration ratios. Moreover, the possibility that the con-sorts in the mixed culture were responsible for the sustained TCE degradation should not be overlooked. The presence of toluene-degrading (but without co-met-abolic function) bacteria can alleviate the effect of com-petitive inhibition on the bacteria co-metabolizing TCE. In that scenario, however, the microbial popula-tion carries a risk of shifting towards toluene degraders that do not degrade TCE over a long period of operating
time (Mars et al., 1998).
When TCE concentration was increased from 10 ppmv (baseline level) to 15 ppmv, the removal of toluene was slightly enhanced. Further increase to 30 and 60 ppmv resulted in progressive decline of toluene removal to a limited extent (less than 4%). The toxicity inhibition of TCE or its intermediate products apparently was not
in effect, as opposed to the observation by Cox et al.
(1998) using the mono-culture of Pseudomonas putida F1. The lack of toxicity effect could also be ascribed to the use of microbial consortium, which generally mani-fests greater efficiency and stability for biodegradation of recalcitrant compounds through sequential degrada-tion of the partial oxidadegrada-tion products.
A more interesting result, however, was that the deg-radation of TCE also increased with its own influent
concentration (shown inFig. 5), even with the fixed
con-centration of toluene. This result is contrary to the pre-sumption that toluene-oxidizing oxygenases cannot be induced by TCE, and that TCE transformation cannot be enhanced without inducing more toluene-oxidizing enzymes. To verify whether the TCE removal was due to biotic or non-biotic causes, the biofilm-coated carbon granules directly taken from the top-quarter of the col-umn packing were washed and re-suspended in the min-eral solution augmented with yeast extract. Without re-separating the microorganisms, the microbial suspen-sion was prepared by following the same protocol used for the batch biodegradation test, with TCE (5 lM) as the sole substrate. The headspace analysis showed that TCE removal was significant (30%) over a 24-h period, after which the rate of removal appeared to slow down (<10% removal for the next 24-h period). This result provided evidence that TCE degradation was possible without toluene as an inducer for the microbial consor-tium, and is partially consistent with the previous
find-ing ofLeahy et al. (1996). These researchers reported
that TCE or its oxidation product not only can act as an inducer of toluene-oxidation activity with several Pseudomonas strains previously grown on toluene and Change in mass removal from base condition (%)
-12 -10 -8 -6 -4 -2 0 2 4 6 S te p -i ncrea se o f VO C con cen trat io n (pp mv ) ac etone 560 365 815 120 215 330 15 30 60 TC E tol u e n e toluene acetone TCE Legend
Fig. 6. The relative changes (in %) in the steady-state mass removal of the non-variable VOCs in response to the step-increases in the influent mass loadings (concentration) of the variable VOC. The relative change was calculated using Eq.(3)in the text.
TCE, but can also enhance its own degradation in the absence of an aromatic inducer. Although the mecha-nism activating the pre-induced toluene-oxidizing oxy-genases for TCE degradation without the continuous presence of toluene has not been clarified, it is hypothe-sized that the mechanism requires toluene-oxidizing cells to be previously grown in the presence of TCE.
Increase in acetone influent concentration from 220 up to 815 ppmv progressively reduced the removal of tol-uene and TCE. Comparing with the baseline condition, the mass removal of toluene was reduced by nearly 5% as the influent acetone concentration was raised to a level at 365 ppmv, and by over 11% at 815 ppmv. The TCE mass removal also deteriorated with the similar pattern. Acetone is oxidized by either acetone monooxygenase or methylhydroxylase into products of acetol and methyl acetate, and then into pyruvate via dehydrogenation (Taylor et al., 1980). Subsequently, these intermediate products enter the common tricarboxylic acid (TCA) cycle that cleaves the acetone skeleton into C1 or C2
frag-ments for further mineralization (Mendz et al., 1994).
Toluene metabolism, on the other hand, is initiated by the insertion of oxygen atoms by toluene mono- or di-oxygenases into the aromatic ring to form catechols. Enzymatic ring fission of catechols then leads to major intermediate products such as pyruvate and acetaldehyde
that also enter the TCA cycle (Ellis and Wackett, 1995).
Therefore, acetone and toluene degradations do not share common enzymatic reaction for their initial decomposition, but their oxidation products may merge into common degradation pathways. The apparent dif-ferences in the active enzymatic reaction effectively elim-inated the possibility of competitive inhibition between acetone and toluene (or TCE) for the same active site of enzyme. A more plausible explanation to this antago-nistic effect is the occurrence of diauxie, in which five of the acetone-degraders also capable of oxidizing toluene (B1–B5) preferably utilized acetone over toluene as the primary substrate. Moreover, when the acetone concen-tration was high enough to become the predominant sub-strate (as in the case where acetone concentration was increased up to 815 ppmv), the competitive balance may be lost in the structure of the microbial consortium. This result can be supported by the observation that strains B6 and B7, two strains that are highly selective for acetone degradation over other toluene- and TCE-degrading bacteria, prevailed over other bacteria in the microbial population at the end of the experiments. Con-sequently, the acetone removal remained highly efficient, whereas the removal efficiencies of toluene and TCE were significantly suppressed.
The use of activated carbon as the packing medium inevitably contributed to a certain extent of VOC re-moval by adsorption. Consequently, there exists an experimental difficulty to completely discriminate the contribution of adsorption from the biodegradation of
the VOCs. However, a number of studies have also shown that, while adsorption plays a prominent role in the initial stage of bioactive carbon systems, its adsorp-tive property would gradually diminish due to the
growth and maturation of biofilm (Schwarz et al.,
1999; Kim et al., 2000). In support of these findings, one can observe that the adsorption breakthrough
even-tually occurred after 20 days of operation (phase 1,Fig.
2), revealing the gradual exhaustion of the adsorption
capacity of the bioactive carbon. Furthermore, in a water-saturated system, water molecules tend to gain competitive advantage for the adsorption sites over or-ganic molecules, leaving lesser chances of direct adsorp-tion of VOCs after an extended period. Therefore, while adsorption may contribute to the beneficial ‘‘buffering’’ effects during transient response to a step-increase in mass loading, it would be much less likely to be a factor for direct removal of gas-phase VOCs under a steady-state condition. In view of these notions, the effect of adsorption was neglected from the analysis for the stea-dy-state removal of the VOCs.
4. Conclusions
The transient and steady-state performance of a bio-trickling filter for the control of a synthetic waste gas mimicking the emission from the microelectronics indus-try was studied. The waste gas mixture contained three representative VOCs in acetone, toluene, and TCE over a range of concentrations, allowing the examination of the possible cross-substrate interactions by relating the variation of mass loading with the functional character-istics of the microbial consortium. A threshold capacity for the column appeared to be reached when the THC concentration was 2400 ppmv with a 155-s gas retention time, but no evidence of irrecoverable inhibition was ob-served. Influent mass loading below this threshold load-ing typically resulted in THC removal efficiencies over 95% and carbon mineralization ratios over 70%. Micro-bial characterization revealed that the consortium con-tained at least seven bacterial strains capable of degrading acetone and/or toluene and TCE. The collec-tive analysis using the ‘‘relacollec-tive change’’ in mass removal against a variable VOC manifested some interactions of practical importance to a biological treatment system. Concentration increases in acetone, a readily biodegrad-able compound, appeared to cause a diauxie effect that suppressed degradation of toluene, and a population shift that favored acetone-degraders (strains B6 and B7). Competitive substrate inhibition was unlikely a fac-tor since the degradation of acetone and toluene follows different enzymatic mechanisms. The interactions be-tween toluene and TCE showed a mutual induction of toluene-oxidizing oxygenases that resulted in enhanced TCE removal at fixed concentration of toluene. Taken
together, this study demonstrated that the relative VOC concentration in the waste gas mixture is a factor as important as the biodegradative function of the micro-bial consortium, and much more information
concern-ing the cross-substrate interactions needs to be
gathered before biological treatment of organic mixtures can be truly considered as a matter of design.
Acknowledgment
This study was partially supported by the National Nano Device Laboratories (NDL), Taiwan, Republic of China.
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