Environ. Sci. Techno/. 1995, 29, 2073-2082
Biodegraddon in a Multi-Stage Bioreactor MICHAEL F. TSCHANTZ,tft JOHN P. BOWMAN,t T E R R Y L. D O N A L D S O N , § PAUL R. BIENKOWSKI,+rt JANET M. STRONG-GUNDERSON,' A N T H O N Y V. P A L U M B O , § STEPHEN E. HERBES,§ AND GARY S. S A Y L E R * ~ t ~ " ~ l Center for Environmental Biotechnology, Department of Chemical Engineering, Department of Microbiology, and the Graduate Program in Ecology, The University of Tennessee, 10515 Research Drive, Knoxville, Tennessee 37932, and Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
A two-stage biorector system was continuously fed a solution of TCE (concentrations ranging between 0.2 and 20 mg L-l) at 2 mL min-'; the system utilized a mutant (PP358) of the methane oxidizing bacterium Methylosinus trichosporium OB3b for the fortuitous cooxidation of TCE by the enzyme-soluble methane monooxygenase (sMMO). A methane-free environment was maintained in the TCE treatment portion of the reactor (plug-flow columns), minimizing the effects of competitive inhibition between TCE and methane for the sMMO. The reactor was operated in two separate flow configurations, single-pass and crossflow, with TCE removal percentages exceeding 78% (for a TCE feed concentration of 20 mg L-l) and 93% (for a TCE feed concentration of 10 mg L-'), respectively. A r,, of 109.4 mg of TCE (g of VS)-l d-' for a TCE feed concentration of 20 mg L-I was obtained, suggesting that high rates of degradation occurred within the reactor. TCE-induced toxicity effects occurred at TCE feed concentrations of 10 mg L-l and greater, resulting in declines of the biomass concentrations and the enzyme activities. However, the extent of this decline was alleviated by the addition of 0.2 M sodium formate. A model describing the rate of TCE degradation in the plug-flow columns was proposed by Alvarez-Cohen et al. and was modified to incorporate the suboptimal activities of sMMO. The model was adjusted to the data, and an apparent rate constant, K, of 0.041 (dimensionless) was obtained. The effect of the finite transformation capacity term, T,, in the model was noticeable only at high TCE feed concentrations. The model suggested that cross-flow operation was kinetically favored over single-pass operation due to enhanced TCE to biomass ratios. The model may be used to predict 0013-938x/95/0929-2073$09.00/0
@ 1995 American Chemical Society
the extent of TCE degradation for a system and may serve as a useful tool for the optimization of flow rates. The optimization may include maximizing the rate of TCE degradation or minimizing the necessary residence time in a methane-starved environment.
Introduction The widespread contamination of groundwater supplies by trichloroethylene (TCE) and other chlorinated solvents has necessitated the development of cost-effective and efficientmeans of remediation. The U.S.EPA has identified counter-current air-stripping and granulated-activated carbon (GAC) adsorption as the most economical and effective means of treating groundwater contaminated by synthetic organic chemicals (EPA Publication 600/9/84/ 004 1 , 2 ) . However, neither of these technologies chemically reduces the contaminants to less hannful compounds; each merely recompartmentalizes the contaminants between media. By offering a remediation system with lowcost feedstocksand energyrequirementsand by eliminating the need to dispose of the concentrated contaminant(s) by producing harmless end products, a low-cost treatment sytem for the remediation of contaminated groundwater would find widespread use in public water works and in the cleanup of contaminated sites. Bioremediation has been shown to be a cost-effective potential alternative to traditional remediation processes (3). Methylosinus trichosporiumOB3b is a methane-oxidizing bacterium capable of cometabolic TCE degradation mediated by the enzyme-soluble methane monooxoygenase (sMMO),which is produced under copper-limitingconditions (4, 5) and is thereby suitable for the remediation of TCE-contaminatedgroundwater at low copper concentrations. In the presence of copper, sMMO synthesis is suppressed,while the synthesisof a membrane-associated methane monooxygenase,referred to as particulate methane monooxygenase (pMMO), is promoted. It has been shown previouslythat the sMMO of M. nichosporium OB3b degrades TCE at a much higher rate (Vma = 278 nmol of TCE min-' (mgof cells)-') (6')than pMMO ( V m a < 0.5 m o l of TCE min-' (mg of cells)-') (7). During TCE oxidation, sMMO oxidizes TCE to an epoxide, which spontaneously breaks down to chloroacetate,glyoxylate, formate, or w b o n monoxide. The conversion products of TCE degradation (epoxide or acylchlorides) can react with the hydroxylase component of the enzyme, rendering it inactive for further degradation (8). Furthermore, the byproducts of TCE degradation or TCE appear to be cytotoxic (9). The kinetics of TCE degradation by resting cell methanotrophs (inthe absence of methane) have been proposed * Corresponding author address: Center for Environmental Biotechnology, 10515 ResearchDr., Suite 100,Knoxville, TN 37932-2567; Telephone: 615/974-8080; Fax: 615/974-8086. + Center for Environmental Biotechnology. Department of Chemical Engineering. 5 Oak Ridge National Laboratory. Department of Microbiology. Graduate Program in Ecology.
*
VOL. 29, NO. 8,1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY m 2073
by Alvarez-Cohen et al. (10) to follow the expression:
r, = dS/dt = kXS/(Ks' + s)
(1)
where r, is the rate of degradationof TCE, kis the maximum rate of contaminant transformation,Xis the active biomass concentration, S is the TCE concentration, and K,' is the apparent half-velocity constant for TCE. In order to incorporate the cytotoxic effects of TCE on the cells, the effective active biomass concentration is decreased by a proposed function of consumed TCE (10):
(2) where T, is defined as the transformation capacity of the active cells (mg of TCE degraded (mg of cells)-') (10).The proposed rate equation implies that the rate of TCE degradation is dependent on the TCE concentration and the biomass concentrationand is independent of the sMMO enzyme activity of the cells. However, fluctuations in the enzyme activity are common, and an adjustment in k to account for the variations is necessary. The transformation capacity and the specific activity (or rate of substrate metabolism as shown by the naphthalene oxidation assay (11)) may be increased by the addition ofmethane or formate (10,12,13). This increased activity is due mostly to an increase in the availability of NADH produced through the methane dissimilaratory pathway (6'). Formate dehydrogenase oxidizes formate to carbon dioxide, which reduces NAD+ to NADH. The increased NADH produced by formate metabolism could support an increased rate in protein turnover, including sMMO. NADH is required for the degradation and the production of the protein. As a result, damaged proteins may be replaced more rapidly, lessening the impact of the deactivation of sMMO by the breakdown products of TCE oxidation. This will reduce the detrimental kinetic effects of TCE toxicity on TCE cometabolism (6'). The methanotrophs gain no usable energy from the oxidation of TCE by sMMO, and therefore the cells must be grown on methane. Because the two substratescompete for the same enzyme,competitiveinhibition occurs, making K,' in eq 1 a function of the methane concentration, I, the half-velocity constant for TCE, K,, and the half-velocity constant for methane consumption, K:
K,' = Ks(l+ 1/41
(3)
Therefore, as the dissolved methane concentration increases, the rate of TCE degradation decreases. Fennell et al. (14) noticed dramatic decreases in the rate of TCE degradation (2.5 mg of TCE (gofVS)-l d-' to 0.3 mg of TCE (gofVS)-' d-l) as the dissolvedmethane feed concentration was increased from 0.01 to 2.5 mg L-l. Severalsingle-stage bioreactor systemshave been proposed,including attached& and fluidized systems (15-17),which required operating with methane feed concentrations of ~ 5 methanel % air (v/v)to avoid competitive inhibition. Strand et al. (18) have shown that competitive inhibition was negligible at gas-phase methane concentrations of less than 5% (v/v air). The effects of competitive inhibition can be alleviated by lowering the liquid-phase methane concentration in single-stage systems; however, the rate of potential TCE cooxidation is incidentally decreased from its maximum rate. Assuming that methane consumption, r1, results in 2074
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 8, 1995
biomass production (during cellular growth phase, where the growth yield is 0.65 mg of VS (mg of CH3-l (I@), the rate of biomass production increases with an increase in methane concentration by (10) (4)
where k~is the maximum rate of methane consumption. It is seen from eq 1 that by increasing the active biomass concentration, the rate of TCE degradation increases. To avoid the effects of competitiveinhibition,dual-stage reactor systemshave been proposed (10)and operated (19). These systems feed methane and oxygen in one compartment for cell growth and then deliverthe cells to a methanefree compartment, fed with TCE, to avoid competitive inhibition during the degradation of TCE. Dual-stage systems allow high methanotrophic growth rates to occur in a region with a high methane concentration. The higher achievable biomass concentrations increase the potential rate of TCE cooxidation in a methane-free or methanelimited environment. The objective of this research was to design a bioreactor around the biology of methanotrophic sMMO production to enhance the efficiency and stability of TCE degradation based on batch culture studies. Factors, other than displacingcompetitiveinhibition by operatinga dual-stage reactor, that might influence the rate of TCE degradation are also investigated, including maintenance of high biomass concentrations through the implementation of filtered dewatering, recycling, and operation under aboveatmospheric pressure to increase the growth rate of the methanotrophs;the addition of formate for enzyme activity maintenance; and the effect of also adding cells midway into the plug-flow reactor to alleviate whole-cell toxicity effects on TCE degradation. Furthermore, the model proposed by Alvarez-Cohen et al. (10) was modified and investigated against experimental data and was utilized to perform a sensitivity analysis on biomass concentrations, TCE feed concentrations,reactor configuration,and dilution effects associated with reactor flow rates.
Experimental Section Bioreactor Design. A schematic of the bioreactor system is depicted in Figure 1 (abbreviationsare outlined in text). The first compartment included a 2.4-LCSTR in which cells were continuously cultivated with methane and oxygen. Pressure control (PI for the entire system occurred in the CSTR,via avariable tension back-pressurerelief valve (BPR), allowing undissolved gases to escape from the system to a vent and allowing excess liquid to escape to a waste tank in a self-regulatingmanner. Dissolved oxygen concentration in the CSTR was measured by a stainless steel oxygen probe (Ingold) connected to a dissolved oxygen meter (DOM) (Cole-Parmer,Chicago, IL). Methane and oxygen introduction into the CSTR occurred through two Model 8272 mass flow controllers (MFC)(MathesonGas Products, Montgomeqville, PA) coupled to a Matheson Model 8284 dynablender. The gases were mixed in a manifold and entered the CSTR through a porous metal diffuser located at the bottom of the CSTR. In order to keep cells from escaping with the effluent stream, a tangential flow dewatering column with feedside recycle was utilized. Liquid was recycled to and from the CSTR through a 6.35-cm i.d. stainless steel pipe 30.5cm in length. Liquid was pumped at 1.0-2.0 mL min-l (as the
I
ji / j
.............j
ps
/
effluent
I ....................................
FIGURE 1. Schematic of the dual-stage recirculating reactor system for methanotrophic TCE biooxidation.
reactor effluent stream) from a 3.81-cm 0.d. porous metal pipe covered with a 0.2-pm Teflon filter, 15.2 cm in length, located inside the pipe, to a waste storage tank. Fluid, comprised of media and cells, was pumped from the CSTR to the second section of the bioreactor, which consisted of a series of four plug-flow contacting columns (Cl-C4) (vol0.56, 0.80, 0.88, and 1.04 L, respectively), in which methane-limiting conditions exist and TCE degradation occurs. These TCE contacting columnswere placed in series to allow versatility for future experiments, additions, and modifications and to allow easy manipulation of residence times. In addition, the placement of discrete samplingpoints allowed monitoring throughout the length of the TCE contacting columns for accurate analysis of the degradation kinetics of the system (ClS-C4S, FS, CSTRS). Another dissolved oxygen probe was positioned in the lowestcolumn for measuringthe oxygen concentrationafter contact of the biomass with TCE. The TCE contacting columns emptied into the CSTR. TCE was fed to the top of column 1 from a stainless steel liquid storage tank in which a 5 psig oxygen blanket was maintained, and formate was fed by a syringe pump into the junction between columns 2 and 3. All internal surfaces of the system potentially in contact with TCE were composed of 316 stainless steel, Teflon, or viton rubber, minimizingTCE adsorptionand cell adhesion. The reactor was pressure tested to 15 a m without leakage, but normal operating pressure was in the range of 1-3 atm (20)*
Bioreactor Inoculation and Routine Operation. M. Richosporium OB3b mutant PP358 (unable to produce pMMO) (21)was provided by the Universityof Texas,Austin, TX. This strain was cultivated either in liquid or on agarsolidifiedNMS media (@ supplemented with 0.025% (wlv) yeast extract and vitamins (22)under a 1:4 methane:&
-
headspace at 28 "C for 3-5 d. The CSTR was inoculated with 1L of PP358 culture (A600 0.51, and NMS media was added to fill the CSTR and the remaining sections of the bioreactor. The CSTR agitator was rotated at 138 rpm. Methane and oxygen at 3 atm were initiallyadded at 10 mL min-l each, and the flow rates were adjusted to final flow rates of 60 and 5 mL min-l, respectively, as the biomass concentration in the reactor increased. The oxygen concentration was maintained at 5-15% saturation (1.4-4.2 mg L-l) at the reactor pressure of 3 atm. PP358 was recirculated throughout the bioreactor in the absence of TCE addition or liquid feed until it had reached stationary growth phase. The simulated TCE contaminated feed was prepared with N M S media and TCE saturatedwater solution (8.31 mM at 25 "C) to a specified TCE concentration. Single-Passvs Cross-FlowOperation. The reactor was operated in two separate flow configurations: single-pass and cross-flow. During single-pass operation, cells were delivered from the CSTR to the top of column 1 at a nominal flow rate of 10 mL min-l, resulting in a CSTR hydraulic retention time (t)of 4 h. This stream was mixed with the entering TCE feed stream, which was pumped into the uppermost TCE contacting column at a rate of 2.1 f0.1 mL min-l. The mixed stream passed downward through the plug-flowcolumns (t= 273 min) and reentered the CSTR. During the cross-flowoperation, two separate cell streams were pumped out of the CSTR (t= 4 h), each at a nominal flow rate of 5 mL min-'. One stream entered the top of column 1where it mixed with the enteringTCE feed stream. This mixed stream flowed downward through columns 1 and 2 (t= 194 min) where the second cell stream entered, mixingwith the stream exiting column 2, and the combined stream flowed downward through colunms 3 and 4 (t = 160 min)and reentered the CSTR. VOL. 29, NO. 8, 1995 I ENVIRONMENTAL SCIENCE & TECHNOLOGY
2075
Sample Acquisition. Valved liquid-phase sampling ports were located at the bottom of the CSTR (CSTRS),at the bottom of each of the TCE contacting columns (ClSC4S), along the simulated TCE wastewater feed line (FS), and in the dewateringline prior to depositioninto the waste tank. Liquid bioreactor samples (4-6 mL) were obtained via 16-gauge needles into screw cap septum vials sealed with Teflon-lined silicon seals (32 mL; Pierce, Rockford, IL)* Analytical Procedures. PP358 culture optical density was determined spectrophotometrically by measuring absorbance at 600 nm and comparingwith a standard curve of optical densityvsdryweight (mgL-9. The sMMO activity was determined by the naphthalene oxidation assay (111, whereby naphthalene is oxidized by sMMO to form naphthol (naphthalene is not oxidized by pMMO). The sMMO activity was normalized to cell dry weight. TCE levels were determined by gas chromatographic analysis ofvial headspace samples. Analysiswas performed on a Shimadzu GC 9AM gas chromatograph (Shimadzu Analytical Instruments Co., Kyoto, Japan)equipped with a split injector port operated at 250 "C. A 60 m x 0.53 mm i.d. RIIXvolatilescapillarycolumn (RestekCorp.,Bellefonte, PA) was used isothermallyat 120 "C with electron capture detection at 300 "C. Nitrogen was used as the carrier gas. The peak areas were integrated with a Shimadzu C-R6A Chromatopac. The concentration of methane in samples was determined by analyzing head space samples by gas chromatography using a Shimadzu GC 9 AM chromatograph equipped with a flame ionization detector and a 15 m x 0.53 mm i.d. AT-1capillary column (Alltech,Deerfield, IL) maintained at 60 "C using nitrogen as the carrier and makeup gas (1 mL min-'1. The bioreactor liquid TCE and methane concentration were then determined by Henry's law (23,241with normalization of chromatographic peaks to a uniform ratio of gas-phase and liquid-phase volume. Oxygen concentrations were measured as percentage of oxygen saturation at the operating reactor pressure (25). TCE Degradation Experiments. Severalsingle-pass and cross-flowexperiments were performedwith different feed concentrations of TCE, ranging from 0.2 to 20 mg L-l.The run time of these experiments varied, with the run time for each of the above TCE inlet concentrations being at least 90 hand ranging up to 428 h. Liquid sampleswere obtained from the inlet stream, all four contacting columns, and the CSTR on a regular basis. The following parameters were routinely determined: biomass levels; TCE, methane, and oxygen concentrations; and sMMO activity level.
Results and Discussion Background Bioreactor Conditions and Performance. Recently, mutants of M.trichosporium OB3b, capable of producing sMMO but not pMMO have been developed through the use of chemical mutagenesis (21). Characterization of these mutants showed they were unable to take up or metabolize copper, thus preventing pMMO synthesis (26). By using one of these mutants, PP358, problems associated with copper suppression of sMMO could be avoided. Unlike the wild type, the nutritional requirements of PP358 are significantly more fastidious, probably induced through the chemical mutagenesis. To obtain growth rates comparable to the wild-type strain, the NMS media had to be supplemented with yeast extract and a vitamin solution. With a dilution rate of 0.02 h-l and CSTR liquid methane and oxygen concentrations kept at 2 0 x 1m ENVIRONMENTAL SCIENCE e, TECHNOLOGY/ VOL. 29, NO. 8.1995
3-5 and 1-4 mg L-l, respectively, biomass levels of 200400 mg L-l were maintained in the reactor. Methane made up 80-90% of the off-gas molar composition. Cell growth rates were found to range from 0.074 to 0.125 mg L-l min-I, and the resultant cell yield ranged from 0.26 to 0.32 mg of cells (mg of methane)-'. Though the system was maintained nonascepticallyin all experiments,the background growth of contaminants never exceeded 10% of the total population as determined by plate counts onvarious media, including nutrient agar (Difco) and R2A medium (Difco, Detroit, MI). No contaminants able to grow on methanol or methane were ever observed. The contaminants were mostly growing on the yeast extract being introduced in the feedstock and were almost entirely non-methylotrophic Gram-positive bacteria of airborne origin, i.e., Curtobacterium, Aureobacterium, Arthrobacter, Pimelobacter, etc. These contaminants did not seem to have a significant effect on the bioreactor operation and TCE degradation. The various TCE feedstock experiments were operated using the dewatering/filtration column. However, rapid clogging of the 0.2-pm Teflon filter membrane by a biofilm resulted in the major part of the effluentleaving the system to escape through the back-pressure relief valve, where no filtration occurred. In some experiments, where little filtration occurred,the biomass levels in the system declined due to these dilution effects. Throughout most of the experiments, a higher biomass was sustained in column 1 than in the rest of the reactor. This was probably due to residual methane in solution, causing a biofilm of PP358 to form in the column. In order to achieve maximal levels of sMMO activity, a number of operating variables were examined and optimized. These variables were defined in batch culture optimization studies. The most critical factor implemented was maintaining a high concentration of methane in the CSTR of the bioreactor. This allowed maximal growth of PP358 and synthesis of sMMO, prevented exhaustion of reductant pools (i.e., NADH) needed formethane oxidation and TCE degradation, and would help recovery of sMMO activity if TCE induced any toxic effects on the cells or on sMMO (6). Most of the methane was consumed by the second contactingcolumn, and thus competitive inhibition of TCE degradation was never observed throughout most of the plug-flow columns. Brusseau et al. (13)found that methane at 5-10% of solution saturation (1.6-3.2 mg of methane L-l) was optimal for TCE degradation by sMMO, whereas Broholm et al. (27) found that methane at levels less than 1.5 mg L-' did not significantly affect TCE degradation. In the contacting columns of the bioreactor, in which most TCE degradation takes place, methane levels ranged from 0 to 0.9 mg L-l, A second factor was maintenance of oxygen levels between 2 and 4 mg L-l in the CSTR. This range was optimal for PP358 growth and sMMO activity (6). When oxygen exceeded 5 mg L-l, lower growth rates and a reduction in sMMO activity occurred. All oxygen was consumed by column 4 ( t p p ~= 275 min). The medium input maintained nitrate (2 mM1, phosphate (2 mM), iron (50 pM), and magnesium (150pM) levels well above limitation. Limitation of these nutrients results in a decline in growth and sMMO activity. During the various bioreactor experiments, these nutrients were always present in the bioreactor at levels adequate for high sMMO activity as determined from batch culture studies (a.
response to the fluctuationsduring steady-state conditions.
CROSSFLOW
06 04
h
i
3.5 3.025-
VI
'w
-100 80 - 60 40
-
2.0-
-
0) 1 5 -
g. 0.51.0-
- 20
Q)
6
0.0
0 2
3
2.0 1.o 0.5 0.0
4
6
0
0 0 1 2 3 4 5 6 7 6 9
1
3
4
5
6
7 100
,o 0
6 s
2
80c 608
-a
4 20 0
2
4
6
-8-
MMOActivity
-+
TCEFeed (mglL)
-
40 20
2
12 10 6 6 4 2 0 0
10
5 0
20 0 0
100 80 60 40 20
b s 5
3m
3.0
.e
8
0 z I ~
TCE Effluent(mglL)
U J
During single-pass operation, greater than 78% of fed TCE was removed during steady-state conditions for feed concentrations ranging between 0.2 and 20 mg L-l. Over 91% removal was achieved when TCE was fed at concentrations of 3 mg L-l and less, with 100% removal for the case of a TCE feed concentration of 0.2 mg L-l. Steady states were achieved for all cases except the 3 mg L-l TCE fed experiment and the 20 mg L-' TCE fed experiment.The 3 mg L-l TCE fed experiment was run for an insufficient time to reach steady state (only 90 h), and during the 20 mg L-' TCE fed experiment, biomass declined and TCE toxicity effects caused a continuous decline in rates of TCE degradationover the 13 daysthe experimentwas performed. Cross-flow operation produced higher TCE removal percentages than single-pass operation for respective nominal TCE feed concentrations. One hundred percent of the fed TCE was removed for a TCE feed concentration of 1 mg L-l, and over 93% was removed for a TCE feed concentration of 10 mg L-l.
0 0
2
4
6
6101214
Time (days) FIGURE 2. Comparative single-pass and cross-flow TCE removal and sMMO activity in the dual-stage reactor system for TCE feed concentrations ranging between 1 and 20 mg L-l.
TCE Degradation Experiments. Abiotic control experiments (20)showed no losses of TCE due to stripping or adsorption. Only trace amounts of TCE were measured in the off-gasstream exiting the CSTR. Therefore, differences of TCE concentrations between the TCE feed stream and the effluent stream were due to accumulationof TCE within the reactor and biodegradation. During periods in which the steady-state was approached, the rate of accumulation of TCE within the reactor was negligible, and measured TCE differences were solely due to biodegradation. The experimentalresults showed that the bioreactorsuccessfully degraded a high proportion of TCE fed at concentrations ranging from 0.2 to 20 mg L-' (Figure 2 and Table 1). Analysis of biomass removed from the bioreactor indicated that TCE degradationwasentirelydue to the methanotroph M. trichosporium OB3b (PP358) and was confirmable by the presence of sMMO activityat alltimes during the various experiments. Fluctuations in the TCE feed concentrations occurred due to partitioning of the TCE between the liquid phase and the gas phase in the constant volume feed tank. However, the system appeared to be particularly stable in
Effect of TCE Toxicity and Formate Addition on sMMO Activity. The sMMO activity of the methanotrophs was directly responsible for TCE cometabolism, and the enzyme concentrationwas proportionalizedby the biomass concentration. The sMMO activity was measured at every time point (Figure 2) to determine the absolute ability of the biomass to degrade TCE and is reported as E, a dimensionlesspercentage of maximum activity (Eexp/Emax) shown in Table 1. E remained relatively constant between 0.3 and 0.4 for both the single-pass and cross-flow experiments with TCE feed concentration at or below 3 mg L-l (but E did reach 0.757 during 1 mg L-' TCE fed cross-flow experiment). This trend is equivalent to previously studied batch cultures,which showed that sMMO activity tends to peak in early stationary growth phase and then declines to a stable level,which may be maintained for weeks in batch culture with periodical inputs of methane and nutrients (6).
Obvious toxicity effects, shown by a dramatic decrease in E, were observed only after 4 d of operation in the bioreactorexperimentsin which the TCE feed concentration was at or above 10 mg L-l. A more pronounced toxicity effect was seen during the 10 mg L-' TCE fed cross-flow experiment than in the corresponding single-pass experi-
TABLE 1
Bioreactor Operating Data (mg 1-'1
reactor mode
TCE feed range (mg 1-l)
proportion of max sMMO activity, F
biomass, X(mg L-1)
effluent TCE concn (me 1-l)
(mg (g of VS1-1 d-l)
TCE removal (%)
0.2 1 3 10 20 1 3 10
SP SP SP SP SP CF CF CF
0.14-0.23 0.49-0.93 2.34-2.73 7.28-9.85 14.69-18.55 0.60-0.82 2.70-2.82 7.14-1 1.07
0.312-0.368 0.304-0.355 0.351 -0.386 0.260-0.355 0.165-0.399 0.368-0.757 0.338-0.377 0.178-0.412
240-500 360-600 320 283-376 170-210 110 255 395
0 0.042 0.457 1.29 4.37 0.037 0.227 0.853
0.90 2.17 8.40 73.47 109.4 12.16 10.62 12.84
100 97.6 91 .o 86.0 78.7 100 93.6 93.5
nom. TCE feed concn
h a
VOL. 29, NO. 8, 1995 I ENVIRONMENTAL SCIENCE & TECHNOLOGY
2077
ment (a reduction in E ot 70% as opposed to 40%, respectively). This effect may be due to an increased amount of degradation and byproduct formation, which occurred during the cross-flow experiment. This observation also reinforces the concept of a finite transformation capacity,where one would expect a larger toxicity effect as the amount of degraded TCE increases. An increase in toxicity effects was further seen as TCE was fed at 20 mg L-' in single-pass operation, where a 70% reduction in E occurred. The relative amount of degraded TCE almost doubled in the 20 mg L-I experiment (compared to the 10 mg L-l experiment),leading to the much reduced sMMO levels. When TCE feed concentrationswere 10mg L- or greater (duringboth single-passand cross-flowoperation),formate was added after 4 d of operation to allow recovery and to enhance TCE degradation due to a readily observable TCE toxicity effect. After adding 0.2 M sodium formate to the 10 mg L-I TCE fed single-pass experiment, the sMMO activity returned to its original value after dropping nearly 25%. When TCE was fed at 10 mg L-' during cross-flow operation, 1.0 M sodium formate addition increased the sMMO activity to 67% of its original value. However, a rapid rise in bioreactor pH was also observed after 5 d, resulting in significant decreases in biomass and sMMO activity and accumulation of TCE within the system. After 8 d, the pH had increasedto 8.5 and was obviously becoming inhibitory to the biomass, and the experiment was halted. Additionally, formate levels had begun to accumulate in the reactor, ranging in concentration from 2 to 4 mM, possibly resulting in additional inhibition to growth and sMMO activity. Batch culture studies (Bowman, unpublished) have shown that formate concentrations exceeding 1mM inhibits cell growth and sMMO production. Changes in the formate introduction were implemented to avoid formate accumulation and to maintain a pH below 8.0. In the case of the 20 mg L-' TCE feed single-pass experiment, the addition of 0.2 M sodium formate caused only a modest increase in the sMMO activity, which plateaued at only 40% of its original value. A significant decline in the relative amounts of TCE being degraded continuedto occur, suggestingthatTCE toxicity effects were outcompeting cell recovery rates. Formate and methane appear to promote sMMO recovery (28). Inhibition of protein synthesis using chloroamphenicol completely blocked sMMO recovery in M. trichosporium OB3b batch cultures (Bowman, unpublished). This suggests sMMO recovery involves cells overcoming toxicity by resynthesis of sMMO rather than through the growth of new cells. Mathematical Modeling and Simulation. Equation 1 describes the degradation rate of TCE as a function of biomass concentration, X, and as a function of TCE concentration, S. The rate of potential TCE oxidation by a quantity of cells is related to the sMMO activityassociated with those cells and not solely with the biomass concentration, with the maximum rate of TCE oxidation as a function of maximum sMMO enzyme activity (Emax= 278 nmol of TCE min-I (mg of protein)-') (6).Because E , an indicator of the extent of sMMO activity, varied between experiments,it was necessary to include a term for E in the proportionality factor, k. This standardized the rate equation such that as E 0, r, 0; and as E l, r, maximum.
'
- -
2078
- -
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 8, 1995
0
50
100 150 200 250 PFR residence time, tau (min)
300
2 0
FIGURE 3. Similarity of apparent maximumTCE transformation rate constants, K, for varied TCE feed concentrations (0.2 and 1 mg 1-' feed concentrations, calcualted utilizing eq 6-81,
The proportionality factor, k, may then be described as
k = ICEE,,~
(5)
where K is the rate constant and 4 conserves the units of k. Equation 1 now relates the rate of TCE degradation to TCE concentration and the total enzyme activityassociated with the biomass Concentration. When eqs 1,2, and 5 are combined and integrated, an equation is formed such that when the terms on the left side of the equation are plotted against the plug-flow residence time, 5, a straight line is produced from which Kmay be determined from the slope:
In order to compare K with varying E by the comparison ofline slopesbetween experimentalruns,eq 6 is rearranged
Y = Y/cE,,#
= Kt
(7)
Figure 3 shows the best fit lines obtained for nominal TCE feed concentrations of 0.2 and 1 mg L-I during the singlepass operation. The data showed good agreement for a single K for each run but differed between experiments. This trend was more evident in Figure 4,where there was considerable scatter in the data and in K between all the single-passand cross-flowruns. The model approximated theexperimentaldatawithavalueforKof0.041 mgofcells (mg of TCE)-', obtained by a least-squares fit of the data. At low TCE feed concentrations, K was insensitive to variance in the transformation capacity, T, (Le., as T, m, IC = constant for each run). However, as the TCE feed concentration increased to 10 and 20 mg L-l, Tc had a significant effect on the determination of K . At high TCE feed concentrations,where a large mass of TCE is degraded, SO- %AS), relative to the initial biomass concentration at the top of column 1,&,the associated loss in biomass from eq 2 has a noticeable detrimental effect on r,. Because Tc was significant only at high TCE feed concentrations when AS was large (and formate addition occurred),a value from the literature for Tcwith formate addition (0.080mg of TCE (mg of cells)-' (10))was used for all modeling cases. The
-
.. . W
S.P. 180 mg celldl
31.2-
0
S.P.3wmgcellsn
B
V
S.P. 720 mg wllc/L
s
P
0 C.F. 180 mg wild
1; '
f 0.8:
0
C.F. 380 mgcallrR
V
C.F. 720 m(l c4llak
P8 0.6-
c.
P
I
i
j, -14 ;-l2I
-201 0
'
'
'
,
50
I 100
150
200
250
300
350
plug-flow residence time, tau (min)
FIGURE 4. Determination of the overall apparent maximum TCE transformation rate constant, K, from single-pass and cross-flow experiments (utilizing eqs 6-8).
value for Ks was determined to be 18.13 mg L-' from previous batch studies performed on the organism M. trichosporium OB3b (a. The maximum rate of TCE degradation, r,,, for each experiment was determined by dividing the obtained rate from eq 1, using & and SO,by the biomass concentration, and is shown in Table 1. The r,, increased with increasing TCE feed concentrations to 109.4mg of TCE (gofVS)-' d-l, during single-passoperation. An improvement in the rm, is seen during cross-flowoperation for the 1and 3 mg L-l TCE feed concentrations. However,the rm,is significantly lower for the 10 mg L-' TCE feed concentration during cross-flow operation. This may be attributed to the pH problems experienced with the formate addition and the corresponding drop in E. The r,, obtained for this reactor design is considerably higher than those reported in the literature for other continuously operated TCE degrading reactor systems. Fennell et al. (14) reported a r,, of 2.9 mg of TCE (g of VS1-l d-' for a TCE feed concentration of 42 mg L-I. McFarland et al. (19) reported a r,, of 21.1 mg of TCE (g of VS1-l d-' for an initial reactor TCE concentration of 29.2 mg L-l in a two-stage reactor utilizing a suspended, mixed culture of methanotrophs. Single-Passvs Cross-FlowOperation. Improved TCE removal efficiencieswere seen during cross-flowoperation when the reactor was fed with TCE concentrations of 1and 3 mg L-l. The differences in TCE removal efficiences may be attributed to effects associated with whole-cell toxicity due to the presence of TCE andlor to kinetic effects. While the hydraulic retention time of the plug-flow columns was 81 min greater during cross-flow operation than during single-passoperation, the ratio of the average time the cells spent in the plug-flow column (PFR) to the average time spent in the CSTR was 1.07 h in PFR (h in CSTR)-l during cross-flowoperation and 1.14 hinPFR (hin CSTR)-l during single-passoperation. If cellular toxicity effects associated with TCE exposure is a function of residence time, then cross-flow operation would be slightly advantageous. However, significant declines in sMMO activity along the plug-flow columns were never seen under the conditions examined. This suggests that there was little or no effect of TCE time exposure on the performance of TCE degradation, and the differences in removal efficiencies must be
0
2
4
6 8 10 12 14 16 TCE feed concentration (mg/L)
18
20
FIGURE 5. Comparative model generated effluent concentrations for given TCE feed concentrations and operating modes (solid lines represent single-pass operation and dashed lines represent crossflow operation).
largely due to the kinetic effects of biomass concentrations and TCE concentrations. Because of the variability in operatingconditions during single-pass and cross-flow operation, it was difficult to determine by direct comparison whether or not singlepass or cross-flowoperation was more efficient in removing TCE. The mathematicalmodel has been shown to correlate the kinetics of TCE degradation reasonably well and was utilized in a sensitivityanalysis to compare single-passand cross-flowoperation under ideal conditions to identifykey parameters causing the kinetic differences. Modeling assumptions included the following: no degradation of TCE occurred in the CSTR biomass inactivated by the finite transformation capacity was regenerated in the CSTR; and the bioreactor operated at steady state. The assumptions are valid because the methane concentrations in the CSTR ranged between 5 and 50 mg L-l, and eq 3 shows that Ks becomes very large (makingr, small)for the reported value for Ki of 3 pM (0.05 mg L-I) (29).The system approached a steady state,where TCE concentrations,enzyme activities, and biomass concentrations remained stable for every case except the TCE feed concentration of 20 mg L-l. The biomass concentrations were varied in the model between 180 and 720 mg L-l, and the TCE feed concentrations were varied between 1and 20 mg L-l. Figure 5 shows the effluent concentrations from the model simulation. Cross-flow operation provided lower effluent concentrations over the range of biomass and TCE feed concentrations examinedwhere the magnitude of differencein TCE effluent concentrations decreased with increasing TCE feed concentrations and decreasing biomass concentrations. This trend suggests that limits exist to the advantage of crossflow operation over single-pass operation. The variable factors that influence the rate of TCE degradation (withE = constant) are Xand S. By comparing the r,, determined from the sensitivityanalysis, shown in Figure 6, it is evident that cross-flowoperation is kinetically favored over single-pass operation, where a higher rate of TCE degradation per unit biomass occurs during crossflow operation. The ratio of &,SP to &,CF is independent of Zst, and is due to the ratio of the dilutionary effects of the cell streams entering the top of column 1 from the CSTR, equaling 1.17. However, this is not the case for SO, VOL. 29. NO. 8, 1995 I ENVIRONMENTAL SCIENCE & TECHNOLOGY
2079
0
2
4
6 8 10 12 14 16 TCE feed concentration (mg/L)
18
2
0 10 20 30 40 50 60 70 80 90 100 Flow rate from CSTR (mumin)
FIGURE 6. Influence of biomass concentration, L, and reactor configuration on maximal TCE removal rates, r , , (varying &, between 180 and 720 mg 1-ti dashed lines represent cross-flow conditions, and solid lines represent single-pass conditions).
FIGURE 7. Effect of cell stream flow rate from CSTR on effluent TCE concentration for varying CSTR biomass concentration, &, (6 = 10 mg 1-l and c = 1; dotted line represents minimum cell stream flow rates for given &, ranging between 90 and 720 mg 1-l).
which is both a function of the dilutionaryratio of the feed concentration, Sf,and the concentration of TCE exiting the plug-flow columns (because of recycle). At low Sf(such as 1 mg L-9,So is very small in relation to K,;therefore,
increasing F, the effluent TCE concentrations approach limits. If exposureto veryhigh TCE concentrationsis found to be toxic to the cells, then the exposure time may be minimized by operating at a high flow rate, resulting in a low plug-flow column residence time while maintaining significant amounts of TCE degradation. But in order to reduce effluent TCE concentrations to minimum levels, very high concentrations of biomass are necessary. At high TCE feed concentrations, or at low biomass conditions, TCE associated toxicity effects may become evident in the plug-flow columns. While Tc is a soundly based toxicity term for whole cell effects, the effect of TCE toxicity on e is more directly measurable. Therefore, transformation capacities, Tc,6,associated with both e and Xneed to be developed in order to fully describethe system, such that in addition to eq 2 an equation of the form
The ratio of rates for single-passand cross-flowoperation is proportional to the ratio: rs,sp/rs,cF = 1*17(sO,Sp/sO,CF) at small So
(9)
Thus,when SO,CF is over 17% greater than SO,SP, then the rate of TCE degradation during cross-flow operation is higher than during single-passoperation. The sensitivityanalysis showed that SO,CF exceeded S0,sp by greater than 50% for every case considered. Even as S became significantin the denominator of eq 1, the larger value of SO,CF over Sop maintained a higher rs,CF and a higher TCE removal efficiency. The dilution characteristics of mixing the cell stream entering column 1 with the TCE feed stream entering column 1 appeared to have the most significant influence on the rate of TCE degradation in the plug-flow columns. Therefore,a sensitivityanalysiswas performed on the model to analyze the effects of varying the cell stream flow rate, F,for a constant TCE feed stream flow rate of 2 mL min-', holding Sfconstant at 10 mg L-' and e = 1. It is seen from Figure 7 that a minimum effluent concentration was obtained at a specificflow rate, F,, for eachJCcst,investigated. Between CSTR biomass concentrations of 180 and 360 mg L-l, varying the cell stream flow rate around Fc had a dramatic effect on the effluent TCE concentration, but at zoeo 1 ENVIRONMENTAL SCIENCE e,
TECHNOLOGY i VOL. 2s. NO. 8 , 1 9 9 5
may be utilized in the rate equation.
Conclusions TCE, continuouslyfed into a dual-stagesuspended growth bioreactor, was degraded by M. trichosporium OB3b (PP358). Over 90% of TCE fed at 5 3 mg L-' was removed
in less than 4.5 h of plug-flow residence time during singlepass operation, and over 93% of TCE fed at 10 mg L-' was removed in under 6 h of plug-flow residence time during cross-flowoperation. Pseudo-steady-stateswere achieved for TCE feed concentrations of 5 10 mg L-I,and the system was robust to fluctuations in the TCE feed concentrations. Rates of TCE degradation were dependent not only on biomass and TCE concentrations but also on enzyme
activities,E (ratioof measured sMMO activity to maximum sMMO activity),which remained stable at suboptimallevels between 0.165 and 0.757. The addition of formate for TCE feed concentrations of 10 mg L-' increased and stabilized enzyme activities nearly to their originalvalues,but formate addition was less effective for a TCE feed concentration of 20 mg L-l. A model proposed by Alvarez-Cohen et al. ( l o ) ,necessarily modified to incorporate the variability in E, correlated reasonablywell with the experimentaldata for an apparent rate constant, K, of 0.041 mg of cells (mg of TCE)-l. A rmax of 109.4 mg of TCE (g of VS1-l d-' was obtained for a TCE feed concentration of 20 mg L-l, which was significantly higher thancomparativevalues obtainedfrom the literature. No significant declines in E were detected in the plug-flow columns, indicating that TCE toxicity effects stabilized at a pseudo-steady-state. Increased TCE removal efficiencies during cross-flow operation were due to favored biomass to TCE concentration ratios produced by the lower cell stream flow rate entering column 1. The model may be used to choose the most efficient reactor configuration and to optimize the flow rates of the streams leading into the plug-flow columns for the maximization of the rate of TCE oxidation. By maintaining adequate biomass concentrationsand optimizingflow rates within this reactor system,the model suggeststhat suitable TCE effluent concentrations (55pg/L)may be achieved for TCE feed concentrations of 520 mg/L. Furthermore, the model may be utilized under nonideal conditions when the enzyme activity is at suboptimal levels, as is usually the case during full-scale or field utilities.
Acknowledgments This work was supported by Martin Marietta Energy Systems, Subcontract 11B99732C S-74, from Armstrong Laboratories,Environics Directorate,Tyndall Air Force Base, Florida. We wish to thank G. Georgiou of the University of Texas at Austin for providing strain PP358.
Nomenclature maximum activity of sMMO (nmol TCE min-' (mg of protein)-') cell stream flow rate entering column 1from the CSTR (mL min-l) critical F producing a minimum TCE effluent concentration (mL min-'1 aqueous methane concentration (mg L-l) maximum rate of TCE transformation (mgof TCE (mg of cells)-' min-') apparent maximum rate constant for TCE transformation (dimensionless) maximum rate of methane consumption (mg of methane (mg of cells)-' min-l) half-velocity constant for methane (mg L-l) half-velocity constant for TCE (mg L-') apparent half-velocityconstant for TCE (mgL-l) rate of methane consumption (mg L-1 min-1) maximum rate of TCE transformation (mgof TCE (g of VS)-l d-l) rate of TCE oxidation (mg L-' min-') rate of TCE oxidation during cross-flowoperation (mg L-' min-')
rate of TCE oxidation during single-pass operation (mg L-' min-') aqueous TCE concentration at time t (mg L-') TCE concentration entering the plug-flow column (mg L-1) TCE concentration entering the top of the plugflow column during cross-flowoperation (mg L-1)
TCE concentration entering the plug-flow column during single-pass operation (mg L-') TCE concentration in the TCE feed stream (mg L-1)
resting cell transformation capacity for TCE (mg of TCE (mg of cells)-') enzyme activity transformationcapacity for TCE (nmol of TCE min-l (mg of protein)-' (mg of cells)-') maximum rate of enzymaticTCE oxidation ( m o l of TCE min-' (mg of cells)-') active aqueous biomass concentration (mgL-l) activebiomass concentration entering the plugflow column (mg L-') active biomass concentration in the CSTR (mg L-1)
integrated form of r, [=I kt (dimensionless) normalized function for Y (mgof cells min-' (mg of TCE)-l) dimensionlessproportion of maximum enzyme activity dimensionlessproportion of maximum enzyme activity entering the plug-flow column units conversion factor [=I (0.5 mg of protein (mg of cells)-' x (131.39 mg of TCE (mmol of TCE)-') x mmol nmol-') residence time in plug-flow columns (min)
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Received for review January 10,1995. Accepted April 24,1995.@
ES940170+
I
@Abstractpublished in Advance ACS Abstracts, June 1, 1995.
