Kinetic Characterization of Methanobacterium bryantii M.o.H.

Hydrogen and thiosulfate limits for growth of a thermophilic, autotrophic Desulfurobacterium species from a deep-sea hydrothermal vent. Lucy C. Stewar...
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Environ. Sci. Technol. 2005, 39, 4900-4905

Kinetic Characterization of Methanobacterium bryantii M.o.H. FATIH KARADAGLI† AND BRUCE E. RITTMANN* Department of Civil and Environmental Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3109

H2 is a key electron donor for many anaerobic microorganisms; thus, keen competition for H2 occurs among H2utilizing microbial groups. Monod kinetic parameters provide essential information for kinetic analysis of competition for H2. In this study, we estimated Monod kinetic parameter values for a methanogen that consumes only H2 as its electron donor, Methanobacterium bryantii M.o.H. Utilization of a single electron donor is an advantage in this study, because complications from alternate metabolic pathways are avoided. Using a set of batch experiments designed to provide the best estimates of each parameter, we obtained these values: maximum specific growth rate (µmax) ) 0.77/ day, maximum substrate consumption rate (qmax) ) 2.36 molH2/gcells/day, true yield (Y) ) 0.325 gcell/mol H2, fraction of donor electrons to synthesis (fs°) ) 0.03 e-cell/e-donor, halfmaximum-rate substrate concentration (Ks) ) 18 000 nM ) 18 µM H2, and endogenous decay rate (b) ) 0.088/ day. This self-consistent set of parameters indicates that, when H2 is not limiting, M. bryantii M.o.H. is a slow grower (low µmax) compared to other H2-oxidizing methanogens and sulfate reducers, and this is mainly due to its low true Y, not a low qmax. The relatively high Ks and b values suggest that M. bryantii also may not be a strong competitor when H2 is limiting.

Introduction Methanogens are unique obligate anaerobes that produce methane and are grouped based on what compound they convert to methane: acetate fermenters, obligate methylotrophs, and autotrophic hydrogen (H2) oxidizers (1). The third group includes Methanothermobacter thermautotrophicus, Methanobacterium bryantii, and Methanobrevibacter arboriphilus (2, 3). H2 is a key end product of organic fermentations and can be used as an electron donor by microorganisms that utilize all terminal electron acceptors (4). Therefore, competition for H2 is a common reality for the H2-oxidizing methanogens. For example, hydrogenotrophic methanogens may compete for available H2 with sulfate reducers and dehalogenators in contaminated subsurface sites (5). Thermodynamic and kinetic factors determine whether the methanogens survive well. * Corresponding author phone: (480)727-0434; fax: (480)965-2765; e-mail: [email protected]. Present address: Center for Environmental Biotechnology, Biodesign Institute at Arizona State University, 1001 South McAllister Avenue, P.O. Box 875701, Tempe, AZ 852875701. † Present address: Department of Environmental Engineering, School of Engineering, Sakarya University, Esentepe Kampusu, Sakarya, Turkey. 4900

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Thermodynamically, the standard Gibbs free energies of reductive dehalogenation (-135 kJ/mol-H2 for transformation of tetrachloroethene to ethane) and sulfate reduction (-75 kJ/mol-H2) are greater than that of methane production with H2 (-57 kJ/mol-H2); therefore, dehalogenators and sulfate reducers gain more energy, synthesize more biomass, and grow faster than methanogens (1, 6). In terms of kinetics, Table 1 summarizes available Monod kinetic values for sulfate reducers, methanogens, and dehalogenators that oxidize H2: µmax, the maximum specific growth rate (1/day); qmax, the maximum specific rate of substrate (H2) utilization (mol H2/gcells-day); and Ks, the half-maximum-rate concentration (µmol H2/L). Equations 1 and 2 show how these parameters describe the growth of active biomass (Xa in g-cells/L) and consumption of H2 (S in µmol H2/L) for a batch reaction.

[ (

) ] [ ( ) ] ( )

dXa S - b Xa ) ) µmax* dt KsH2 + S

Y*qmax*

S - b Xa (1) KsH2 + S

S dS ) -qmax* *X dt KsH2 + S a

(2)

Also included in the mass balances are Y, the true yield (gcells/mol-H2), and b, the endogenous-decay coefficient (1/ day). On one hand, the parameter values in Table 1 overlap enough to suggest that the kinetic competition for H2 could be keen among these microbial groups, with the outcome not always obvious. On the other hand, the available values are limited and do not provide a clear view of how the different microorganisms compete for H2. In this study, we experimentally estimate Monod parameter values for a methanogen that uses only H2 as its electron donor: Methanobacterium bryantii M.o.H. We chose M. bryantii M.o.H. because it is a representative methanogen of anaerobic environments, e.g., sediments, anaerobic digesters, rumen, landfills, and subsurfaces (7, 8). Furthermore, M. bryantii M.o.H. utilizes H2 as the sole electron donor and does not produce or consume acetate and formate (9, 10). This avoids all complications from an alternate metabolic pathway. Our systematically obtained Monod kinetic parameter values for M. bryantii M.o.H. contribute to understanding the kinetics of H2-oxidizing methanogens and help define kinetic competition for H2 among the H2 oxidizers. Past studies on competition for H2 observed stable H2 concentrations, known as H2 thresholds, when a terminal reaction was the predominant process, e.g., methanogenesis, in that environment (11, 12). It has been suggested that H2 thresholds are controlled by microbial thermodynamics or kinetics (13). The microbial kinetics view on H2 thresholds is closely related to our work, because a kinetic threshold can be derived by solving eq 1 for S (H2 in this case) at steadystate, dXa/dt ) 0, and the result is Smin ) b*Ks/(Y*qmax-b) (13-15). At S ) Smin, biomass production due to H2 consumption, which is represented by [µmax*(S/(Ks+S))*Xa] in eq 1, equals biomass decay, b*Xa in eq 1. Therefore, one type of H2 threshold can be Smin.

Materials and Methods We obtained approximately 10 mL of living pure culture of M. bryantii M.o.H. from Deutsche Sammlung von Mikroorganismen und ZellKulturen GmbH (DSMZ #863). After 10.1021/es047993b CCC: $30.25

 2005 American Chemical Society Published on Web 06/01/2005

TABLE 1. Comparison of Kinetic Parameter Values for H2-Oxidizing Sulfate Reducers, Dehalogenators, and Methanogensa microorganism

Desulfovibrio G11 Desulfovibrio G11 Desulfovibrio vulgaris strain Marburg D. vulgaris (Marburg) D. vulgaris (Marburg) Methanospirillum hungatei JF-1 Methanobrevibacter. arboriphilus strain AZ Methanosarcina barkeri Methanogens (mixed culture) Methanogens (range) Methanogens (mix) Dehalobacter restrictus Desulfitobacterium sp. StrainPCE1 Dehalosprillum multivorans Dehalococcoides ethenogenes 195 isolate TT4B dehalogenators dehalogenators (PCE to TCE) dehalogenators (PCE reduction)

qmax

µmax

Sulfate Reducing Bacteria 1.4 1.6 3.6 21.1

Ks 3.3 4.2

(23) (23) (24)

1.3

(25) (26)

5.8-7.3 6.6

(23) (25)

1 ( 0.18 0.08-13 5.8-13

(27) (28) (28) (23)

5.5 Methanogens 1.2-1.3 1.4

0.12

Dehalogenators 0.888 0.413 9.6 1.26 0.25-0.5

ref

(29) (30)

0.23 ( 0.13 0.11 ( 0.04 0.054 ( 0.024

(31) (32) (33) (28) (34) (35)

a The definitions and units for each parameter are as follows: q max, specific substrate utilization rate [mole-H2/gcells/day]; µmax, specific maximum growth rate [1/day]; and Ks, substrate concentration at half of the maximum growth rate [µmol-H2/L].

checking its purity microscopically, we grew M. bryantii in DSMZ medium#119, which is described in detail at their Web site (www.dsmz.de/media/med119.htm). We carried out batch kinetic experiments with M. bryantii in 28-mL anaerobic tubes filled with 5 or 10 mL of liquid media. Medium pH, 7.3, was fixed with NaHCO3 buffer and did not change throughout the experiments. We inoculated growth tubes with approximately 1 mL of living culture, and we aseptically added 80% H2 + 20% CO2 gas mixture to give a total pressure of 0.1-1.0 atm. We then adjusted the total pressure inside the tube to 1 atm with 100% N2 or 80% N2 + 20% CO2, as necessary (16). We incubated the tubes in the dark at 37 °C on a horizontally rotating table that had a rotation speed of 200 rpm. We took gas samples from the headspace of tubes using a gastight syringe and analyzed them for CH4 and H2 with gas chromatography (GC) and a reduction gas analyzer (RGA), respectively. The GC was a Hewlett-Packard 5890 series II, equipped with Porapak Q column (80/100, 6’ × 1/8’’ SS) and operated with temperatures of 70, 60, and 80 °C for injector, column, and detector, respectively. For the GC, the carrier gas, fuel, and air were research-grade N2 at a flow rate of 35 mL/min, H2 at 50 mL/min, and air at flow rate of 470 mL/ min, respectively. We operated the RGA III (Trace Analytical, Inc., Palo Alto, CA) at a 104 °C column temperature and a 265 °C detector temperature. Although the detection limit for RGA was around 0.01 ppmv, interferences from the carrier gas, which had 0.05 ppmv H2, allowed us to measure H2 reliably down to 0.1 ppmv (corresponding to 0.08 nM in the aqueous phase). Based on standards, H2 had a linear response above 0.1 ppmv. The reproducibility of H2 measurements was 2-3%, while it was 4-5% for methane. We computed the liquid-phase H2 and CH4 concentrations from the gas-phase measurements using Henry’s law, which assumes equilibrium between the gas and liquid phases. The Henry’s law constants at 25 °C, 7.8 × 10-4 mol/L-atm for H2 and 1.3 × 10-3 mol/L-atm for CH4, were corrected for 37 °C using the van’t Hoff equation, giving 7.3 × 10-4 and 1.04 × 10-3 mol/L-atm for H2 and CH4, respectively (17, 18).

We measured the biomass concentrations using optical density (OD) with a spectrophotometer at wavelength of 600 nm and via a modified version of Bradford protein assay (19). The correlation between OD and protein was linear: (mg-protein/L) ) 71.344*(OD) + 1.494 (R2 ) 1.00). The reproducibility of OD values was 2-3%. We assumed that protein was 50% of the cell dry weight (20). Control experiments carried out with no biomass inoculation showed no change in H2 concentration; an example is presented later in Figure 3.

Mass Balances for a Batch Reactor Because H2 and CH4 are gases, we accounted for mass in the gas and liquid phases using Henry’s law. Reaction rates, represented by Monod kinetics, were based only on aqueousphase concentrations, since biomass does not exist in the gas phase. The total mass (MT) of a gaseous solute is equal to the mass in the gas phase (MG) plus the mass in the liquid phase (ML), and mass is the product of concentration times volume

MT ) MG + ML

(3)

MT ) CG*VG + CL*VL

(4)

where M is mass (moles), C is concentration in mol/L, V is the volume in liters, and subscripts G and L are for gas and liquid phases, respectively. As long as gas-liquid mass transfer is rapid compared to reaction rates, the gas-phase concentration is in equilibrium with liquid-phase concentration through Henry’s law

PG ) CL/KH

(5)

CG ) CL/(KH*R*T)

(6)

or

where KH is the Henry’s Law constant in mol/L-atm, PG is the VOL. 39, NO. 13, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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partial pressure in atm, R is the universal gas constant (0.082 L-atm/mol-K), and T is temperature in Kelvin. In our experimental system, we established equilibrium between gas and liquid phases by vigorous shaking at 200 rpm rotation speed of the incubator. We observed slow growth at rotation speeds less than 50 rpm. By substituting CG from eq 6 into eq 4, we relate the total mass to the liquid-phase concentration:

MT )

(

)

CL *V + CL*VL R*T*KH G

(7)

Because biomass is not present in the gas phase, eq 1 remains valid as the mass balance for Xa in the batch reactor. Because the change in the total mass of H2 in the system is only through microbial consumption, eq 2 must be adapted to be a total-mass balance using eq 7

dMT,H2 dt

)

(

)

VG d[H2]L + VL ) dt R*T*KH

( ( -qmax *

[H2]L

KsH2 + [H2]L

) )

*Xa *VL (8)

dt

)

(

)

VG d[CH4]L +1 ) dt R*T*KH*VL d[H2]L 0.25 (1 - fs°) + bXa(0.0222) dt

[ ( )

]

(9)

where d[H2]L/dt is the rate of change in H2 concentration in the liquid phase (eq 8), 0.25 is the stoichiometric molar ratio of CH4-produced to amount of H2-consumed for methanogenesis from H2 oxidation, (1 - fs°) is the fraction of donor electrons respired to generate CH4, fs° is the fraction of donor electrons synthesized into new active biomass, and 0.0222 is the stoichiometric ratio of moles CH4 produced per g of biomass (represented as C5H7O2N with formula weight ) 113 g-cells/mol-cells) oxidized by endogenous respiration (21). fs° is proportional to Y according to fs° (e-cells/e-H2) ) Y (g-cells/mol-H2)*(1 mol-cells/113 g-cells)*(20 e-cells/molcells)*(1 mol-H2/2 e-H2) (21).

Results We performed a series of experiments that allowed us to systematically evaluate each of the parameters in an experiment best suited for estimating that parameter. This approach involved selecting optimal values for the H2 and biomass concentrations. Estimating µmax. To estimate the maximum growth rate, we used eq 1 and biomass concentrations from exponential growth with nonlimiting H2 concentrations. When S . Ks, the term S/(Ks+S) becomes 1, and eq 1 simplifies to

( ) dXa dt

growth

) µmax*Xa

(10)

To estimate µmax, we pressurized each tube with 80% H2 + 20% CO2 mix gas to 1 atm. In addition, we repressurized the tubes to 1 atm at every 12-24 h to replace the H2 consumed through microbial activity, and the remaining pressure was 4902

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usually 0.6 atm after 12 h in a thick culture; thus, H2 was always nonlimiting in the system. Figure 1 presents the biomass (OD) data for the two batch growth experiments used to estimate µmax. After a lag period of 40-50 h, M. bryantii M.o.H. grew exponentially for about 30 h. We computed µmax from the integrated form of eq 10

ln (Xt/Xo) ) µmax*t

where [..]L represents liquid-phase H2 concentrations (mol H2/L). Methane is produced in proportion to the oxidation of H2 in respiration and also for endogenous respiration of biomass (21). Equation 9 is the total-mass balance for CH4 and reflects both routes for forming CH4

dMT,CH4

FIGURE 1. Biomass (OD) results for M. bryantii at 37 °C in two batch growth experiments to estimate µmax.

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 13, 2005

(11)

where Xo is the OD at the start of the exponential growth, and Xt is the OD at time t within the period of exponential growth. For the results shown as diamonds in Figure 1 (t1 ) 49.5 h, X1 ) 0.11 OD; t2 ) 68.5 h, X2 ) 0.315 OD), µmax ) 0.79/day. For the results indicated by squares (t1 ) 49.5 h, X1 ) 0.074; t2 ) 68.5 h, X2 ) 0.24), µmax ) 0.75/day. We set µmax to the average of 0.77/day with standard deviation, 0.028/ day. Our µmax value gives a doubling time of 21.5 h. Estimating qmax. The second parameter is the maximum specific substrate utilization rate - qmax. To obtain a reliable qmax value directly, we used eq 8 with experimental data collected under two conditions that satisfy two criteria. First, the H2 concentration in the system must be significantly greater than the Ks value so that the (S/(Ks+S)) term can be simplified to 1. Second, net biomass growth should be small for the duration of the experiment so that biomass term, Xa, can be set to a constant value. With these criteria satisfied, the rate of H2 utilization in a batch nongrowth experiment is represented by

dMT,H2 dt

)

(

)

VG d[H2]L + VL ) (-qmax *Xa)*VL (12) dt R*T*KH

or qmax is

( )( )(

qmax ) -

d[H2]L VG 1 + VL Xa*VL dt R*T*KH

)

(13)

We conducted the qmax experiments with 10 mL of fresh medium and inoculated with 1 mL of the source culture. Hence, the tubes had 11 mL of liquid volume (VL) and 17 mL of gas phase (VG). The values for the other constants in eq 13 are R ) 0.08205 (L-atm/mol-K), T ) 310.3 K (37 °C), and KH ) 7.3 × 10-4 mol/L-atm at 37 °C. We obtained experimental (d[H2]L/dt) data at various biomass concentrations and then computed qmax according to eq 13. We performed the experiments to estimate qmax after the biomass had reached the exponential growth phase. At the beginning of an experiment, we pressurized the tubes with the 80% H2 + 20% CO2 mix and monitored the H2 consumption over time. The time of each test was on the average of 6 h, during which biomass increased maximum of 27% in all tubes in proportion to a doubling time of 21.5 h. We used

TABLE 2. Data for Estimating qmax for Different Biomass Levels mean mean estimate biomass biomass H2 consumption of qmaxa rate (-dCL/dt) growth concn concn (mol-H2/ tube (OD at 600 nm) (mg-cells/L) (mol-H2/L/h) g-cells-day) a b c d e a

0.094 0.115 0.194 0.368 0.388

16.4 19.4 30.7 55.5 58.4

1.88 × 10-5 1.93 × 10-5 3.68 × 10-5 7.65 × 10-5 5.58 × 10-5

2.35 2.05 2.29 3.18 1.96

Mean ) 2.36 mol-H2/g-cells/day.

FIGURE 3. Biomass and methane concentrations during two endogenous decay experiment. The lines are linear regression fits with the parameters shown. At the start of the decay experiment, we flushed the headspace of each tube with 80% N2 + 20% CO2 gas mixture to remove all H2 and CH4, and we started the experiment once H2 and CH4 concentrations were undetectable. We monitored biomass, CH4, and H2 concentrations. To estimate b from the decay results, we dropped the synthesis terms in eq 1, because no H2 was present in the beginning of the experiment. The mass balance simplifies to

( ) dXa dt

decay

) -b*Xa

(14)

Integrating eq 14 gives FIGURE 2. H2 consumption for different biomass concentrations. H2 concentration is expressed as liquid-phase concentration in 10-4mol/L. Biomass concentrations are 0.094, 0.115, 0.194, 0.368, and 0.388 as OD(600) for panels a, b, c, d, and e, respectively. Panel f shows the H2 concentration in a medium-only control tube. eq 13 and the mean biomass concentrations in Table 2 to compute qmax. Figure 2 shows the results for H2 concentrations in five experiments having different initial biomass concentrations. The H2 concentration declined steadily in each experiment. We performed linear regression to obtain the slopes and R2 values shown in Figure 2 and tabulate the relevant values in Table 2. We computed the normalized substrate-consumption rates using eq 13, in which Xa values are the mean biomass concentrations listed in Table 2, (d[H2]L/dt) values are the slopes in Figure 2, and the value of constant (VG/ R*T*Kh)+VL)/VL) is 85.53. The qmax values in the last column of Table 2 are consistent with each other and approximately 2 mol-H2/gcells-day for all biomass concentrations. The mean and standard deviation are 2.36 and 0.24 mol-H2/gcells-day, and we accept the mean value as the best estimate of qmax. Estimating Y and fs°. Using experimental µmax and qmax values0.77/day and 2.36 (mol-H2/g-cells-day), respectivelys we can compute the yield for M. bryantii according to Y ) µmax/qmax, or Y ) 0.77 (1/day)/2.36 (mol-H2/gcells-day) ) 0.325 g-cells/mol-H2. Furthermore, we can compute fs° from Y as (0.325 g-cells/mol-H2)*(1 mol-cells/113 g-cells(C5H7O2N))* (20 e--eq-cells/mol-cells)*(1 mol-H2/2 e--eq) ) 0.03 e- cells/ e- H2. Estimating b. Endogenous decay includes the oxidation of biomass to support cell maintenance. To obtain the endogenous decay constant experimentally, we grew M. bryantii M.o.H. to biomass concentrations of 0.18 and 0.24 (OD units) under nonlimiting H2 conditions. These OD values represent biomass taken from the exponential growth phase.

ln(Xo/Xt) ) b*t

(15)

where Xo is the initial Xa concentration, and Xt is the Xa value at time t of the decay experiment. Because H2 was absent, all CH4 production was due to endogenous decay, which is represented by

dMT,CH4 dt

)

(

)

VG d[CH4]L + 1 ) [bXa(0.0222)] (16) dt R*T*KH*VL

Solving eq 16 for b gives

b)

(

)(

)

d[CH4]L VG + 1 /[Xa(0.0222)] dt R*T*KH*VL

(17)

Figure 3 shows the experimental results from the two decay experiments. When no H2 was present in the growth tubes, exponentially grown M. bryantii cells decayed over time. As the cells oxidized themselves to provide energy for maintenance, they also produced methane. We conducted linear-regression analyses on the biomass data to find the slope for biomass decay (i.e., bXa ) -dXa/dt). We then divided these slope values by the average biomass concentration to compute the endogenous-decay constant. Table 3 summarizes b values computed from experimental biomass data in Figure 3, and the average b value is 0.085/ day. Using eq 16, we conducted a similar linear regression analyses on the methane data of the two experiments in Figure 3 and computed the slopes (d[CH4]L/dt). We then computed the b values using eq 17. The b values are summarized in Table 4, and the average b value is 0.09/day. Estimating Ks. To find Ks, we conducted batch growth experiments under conditions in which H2 was rate limiting. Because we already had µmax, qmax, Y, and b estimates from VOL. 39, NO. 13, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Endogenous Decay Constant, b, Values Based on Biomass Data from Experiments 1 and 2 in Figure 3a expt no.

-dXa/dt (OD/day)

mean biomass concn (OD at 600 nm)

endogenous decay constant (b) (1/day)

1 2

0.0176 0.0128

0.198 0.15

0.089 0.08

a

Mean ) 0.085/day.

TABLE 4. Endogenous Decay Constant, b, Values Based on Methane Data from Experiments 1 and 2 in Figure 3a expt no.

d[CH4]L/dt (µM/day)

mean biomass concn (OD) at 600 nm

endogenous decay constant (b) (1/day)

1 2

1.16 0.94

0.198 0.15

0.08 0.1

a

Mean ) 0.09/day.

FIGURE 4. Model simulations (lines) and experimental data (symbols) for H2 consumption (+), CH4 production (o) (panels a, c, and e), and biomass growth (triangles) (panels b, d, and f) in three Ks experiment. A Ks value of 18 000 nM provides a good match between model predictions and experimental data. Initial biomass concentration is the same for all three experiments (∼0.01 OD600 or 4-5 mg-DW/L). The initial H2 concentrations for parts a, c, and e are 52 000, 32 400, 16 400 nM, respectively. the preceding experiments, we found Ks by fitting the experimental data from the Ks experiments. The two critical conditions for good Ks experiments are the initial H2 and biomass concentrations. If initial [H2]L . Ks, the Monod term, [H2]L/(Ks + [H2]L) equals 1; thus, finding a unique Ks value is not possible. However, if initial [H2]L ) ∼Ks, then the data emphasize the smooth transition from zero order to first order in substrate concentration. The second important issue is the initial biomass concentration, which should be large enough so that we can monitor H2 utilization in a practical time frame, which is a few days. We used an initial biomass concentration of 4.2-4.5 mg-cells/L (0.01 as OD), and we could complete the experiment in 3 days. We performed three experiments having initial H2 concentrations of 52 000, 32 400, and 16 400 nM. Using eqs 1 and 8, we simulated substrate consumption and biomass growth in each tube. Figure 4 compares the experimental results with model simulations for H2 consumption, CH4 production, and biomass growth with a Ks value of 18 µM. Using a common Ks value of 18 µM fit all experiments 4904

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satisfactorily for H2, CH4, and biomass. The good fit in the last experiment is especially important, because the initial H2 concentration was close to the Ks value (18 µM) but was also high enough so that the changes in H2, CH4, and biomass could be captured by the experimental data.

Discussion Based on the maximum specific growth rate (µmax), M. bryantii M.o.H. is at a relative disadvantage when the H2 concentration is high. Table 1 shows that the maximum specific growth rates for other methanogens (in 1/day) are 1.4 for Methanosarcina barkeri, 1.24 for Methanosprillum hungatei JF-1, 4.0 for Methanobrevibacter smithii, and 0.49-6.5 for a range for methanogens. In comparison, M. bryantii M.o.H. is slow growing, because its experimental µmax (0.77/day) is at the lower end of the µmax range. Comparing to sulfate reducers, M. bryantii’s µmax is almost half the lowest µmax (1.4/day) reported for sulfate reducers, which indicates that sulfate reducers have a clear growth-rate advantage over M. bryantii M.o.H. as well as other methanogens in general. In contrast, the limited number of µmax values for dehalogenators bracket 0.77/day, with one exception being Dehalosprillum multivorans (µmax ) 9.6/day). This comparison of maximum specific growth rates indicates that M. bryantii and other methanogens may not compete well with sulfate reducers and dehalogenators, when none of the three groups is limited by its electron acceptor. Considering that concentrations of sulfate, except for marine environment, and halogenated compounds are much lower than bicarbonate in natural environments and treatment reactors, growth of sulfate reducers and dehalogenators is controlled by availability of their electron acceptor, making it possible for methanogens to compete with H2 is amply available. Although few qmax values are reported (Table 1), the range (0.6-3.25 mole-H2/gcells-day) brackets M. bryantii’s experimental qmax value (2.36 mol-[H2]/gcells/day). This finding means that M. bryantii has a normal capability to oxidize H2. Therefore, its relatively low µmax is due to its low Y and fs° values (0.325 gcells/mol-H2 and 0.03 e-cells/e-H2). The literature Ks values for methanogens (Table 1) range from 0.08 to 13 µM, and the experimental Ks value for M. bryantii M.o.H. (18 µM) is close to the upper end of these values. A wide range of literature Ks values can be attributed, at least in part, to varying mass-transfer limitations in experimental setups. However, mass-transfer limitations were at a minimum in our experiments, because we grew M. bryantii as completely dispersed cells, and the tubes were well-shaken during the experiments. Therefore, the relatively large Ks value of M. bryantii means that this microorganism has a relatively low affinity for H2 uptake and dehydrogenation. Compared to sulfate-reducing bacteria, the Ks value for M. bryantii M.o.H. is large; therefore, sulfate reducers have a kinetic advantage over M. bryantii when the H2 concentration is low. It is difficult to compare our Ks value to those of dehalogenators, because only several Ks values are available for dehalogenators, and they are well below 18 µM. A typical endogenous-decay coefficient for anaerobic microorganisms is b ) 0.02/day (21, 22). This value is almost 5 times smaller than our experimental b value, 0.087/day. The typical endogenous-decay coefficient for heterotrophic aerobic microorganisms has been reported as 0.1-0.3/day (21). Although the endogenous decay rate for exponentially grown M. bryantii is smaller than typical for aerobic heterotrophs, it appears to be much larger than for many anaerobes. The high b value makes M. bryantii poorly suited for survival in H2-limited environments. It is possible that M. bryantii exposed to a low H2 concentration would adapt by reducing its endogenous-decay rate, but we did not evaluate this possibility here.

Perhaps the most valuable contribution of our work is having accurate and self-consistent values of µmax, qmax, Y, b, and Ks for the strict H2-oxidizing methanogen, M. bryantii M.o.H. On one hand, these values can be used to evaluate how well M. bryantii can compete with other methanogens, sulfate reducers, dehalogenators, or other H2-oxidizing microorganisms. For example, M. bryantii M.o.H. should not be well suited to compete for low H2 concentrations due to its high Ks and b values. For nonlimiting H2, M. bryantii might be outgrown by H2 oxidizers having larger µmax values. On the other hand, these parameter values make it possible to evaluate kinetic effects on M. bryantii’s growth, H2 uptake, endogenous decay, and CH4 production as well as on a kinetic H2 threshold. In particular, we compute the kinetic threshold for M. bryantii M.o.H. as Smin ) 2.4 µm. We expect that the net growth rate of M. bryantii is positive only for H2 concentrations greater than approximately 2.4 µM.

Literature Cited (1) Zinder, S. H. Physiological ecology of methanogens. In Methanogenesis: Ecology, Physiology, Biochemistry and Genetics; Ferry, J. G., Ed.; Chapman and Hall, Inc.: New York, 1993. (2) Oremland, R. S. Biogeochemistry of methanogenic bacteria. In Biology of Anaerobic Organisms; Zehnder, A. J. B., Ed.; JohnWiley and Sons: New York, 1988. (3) Boone, D. R.; Mah, R. Methanogenic archeobacteria. In Bergey’s Manual of Determinative Bacteriology; Holt, J. G., Ed.; Williams and Wilkins: Baltimore, 1989. (4) Dolfing, J. Acetogenesis. In Biology of Anaerobic Microorganisms; Zehnder, A. J. B., Ed.; John-Wiley and Sons: New York, 1988. (5) National Research Council (NRC). In Situ Bioremediation: When does it work? National Academy Press: Washington, DC, 2000. (6) Yang, Y.; McCarty, P. L. Competition for hydrogen within a chlorinated solvent dehalogenating anaerobic mixed culture. Environ. Sci. Technol. 1998, 32, 3591-3597. (7) Bryant, M. P.; Wolin, E. A.; Wolin, M. J.; Wolfe, R. S. Methanobacillus omelianskii, a symbiotic association of two species of bacteria. Archiv. Mikrobiol. 1967, 59, 20-31. (8) Whitman, W. B.; Bowen, T. L.; Boone, D. R. The methanogenic bacteria. In The Prokaryote: a Handbook of the Biology of Bacteria; Balows, A., Ed.; Springer-Verlag: New York, 1992. (9) Peters, V.; Janssen, P. H.; Conrad, R. Transient production of formate during chemolithotrophic growth of anaerobic microorganisms on hydrogen. Curr. Microbiol. 1999, 38, 285289. (10) Benstead, J.; Archer, D. B.; Lloyd, D. Formate utilization by members of the genus Methanobacterium. Arch. Microbiol. 1991, 156, 34-37. (11) Lovley, D. R.; Dwyer, D. F.; Klug, M. J. Sulfate reducers can out-compete methanogens at freshwater sulfate concentrations. Appl. Environ. Microbiol. 1982, 43, 1373-1379. (12) Lovley, D. R.; Klug, M. J. Kinetic analysis of competition between sulfate reducers and methanogens for hydrogen in sediments. Appl. Environ. Microbiol. 1983, 45, 187-192. (13) Lovley, D. R.; Goodwin, S. T. Hydrogen concentration as an indicator of the predominant terminal electron-accepting reactions in aquatic sediments. Geochim. Cosmochim. Acta 1988, 52, 2993-3003. (14) Rittmann, B. E.; McCarty, P. L. Model of steady-state-biofilm kinetics. Biotech. Bioeng. 1980a, 22, 2343-2357. (15) Rittmann, B. E.; McCarty, P. L. Evaluation of the model of steadystate-biofilm kinetics. Biotech. Bioeng. 1980b, 22, 2359-2373. (16) Ljungdahl, L. G.; Wiegel, J. Working with anaerobic bacteria. In Manual of Industrial Microbiology and Biotechnology; Demain, A. L., Solomon, N. A., Eds.; American Society of Microbiology: Washington, DC, 1986.

(17) Stumm, W.; Morgan, J. J. Aquatic Chemistry: Chemical Equilibria and Rates in Natural Waters, 3rd ed.; John-Wiley and Sons: New York, 1996. (18) Manahan, S. E. Environmental Chemistry; Lewis Publishers: Boca Raton, Florida, 1994. (19) Ma¨gli, A.; Martingo, F. A.; Leisinger, T. Acetogenesis from dichloromethane by a two-component mixed culture comprising a novel bacterium. Appl. Environ. Microbiol. 1995, 61, 29432949. (20) Heine-Dobbernack, E.; Schobert, S. M.; Sahm, H. Relationship of intracellular coenzyme F420 content to growth and metabolic activity of Methanobacterium bryantii and Methanosarcina barkeri. Appl. Environ. Microbiol. 1988, 54 (2), 454-459. (21) Rittmann, B. E.; McCarty, P. L. Environmental Biotechnology: Principles and Applications; McGraw-Hill: New York. 2001. (22) Lawrence, A. W.; McCarty, P. L. A unified basis for biological treatment design and operation. J. Sanitary Eng. ASCE 1970, 757-785. (23) Robinson, J. A.; Tiedje, J. M. Competition between sulfatereducing and methanogenic bacteria for H2 under resting and growing conditions. Arch. Microbiol. 1984, 137, 26-32. (24) Badziong, W.; Thauer, R. K. Growth yields and growth rates of Desulfovibrio vulgaris (Marburg), growing on hydrogen plus sulfate and hydrogen plus thiosulfate as the sole energy sources. Arch. Microbiol. 1978, 117, 209-214. (25) Kristjansson, J. K.; Scho¨nheit, P.; Thauer, R. K. Different Ks values for hydrogen of methanogenic bacteria and sulfate reducing bacteria: An explanation for apparent inhibition of methanogenesis by sulfate. Arch. Microbiol. 1982, 131, 278-282. (26) Nethe-Jaenchen, R.; Thauer, R. K. Growth yields and saturation constant of Desulfovibrio in chemostat culture. Arch. Microbiol. 1984, 137, 236-269. (27) Weimer, P. J.; Zeikus, J. G. One carbon metabolism in methanogenic bacteria: cellular characterization and growth of methanosarcina barkeri. Arch. Microbiol. 1978, 119, 49-57. (28) Smatlak, C. R.; Gossett, J. R.; Zinder, S. H. Comparative kinetics of hydrogen utilization for reductive dechlorination of tetrachloroethene and methanogenesis in an anaerobic enrichment culture. Environ. Sci. Technol. 1996, 30, 2850-2858. (29) Holliger, C.; Hahn, D.; Harmsen, H.; Ludwig, W.; Schumacher, W.; Tindall, B.; Vazquez, F.; Weiss, N.; Zehnder, A. J. Dehalobacter restrictus gen. nov. and sp. nov., a strictly anaerobic bacterium that reductively dechlorinates tetra- and tricholoroethene in an anaerobic respiration. Arch. Microbiol. 1998, 169, 313-321. (30) Gerritse, J.; Renard, V.; Pedro Gomes, T. M.; Lawson, P. A.; Collins, M. D.; Gottschalk, J. C. Desulfitobacterium sp. strain PCE1, an anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene or ortho-chlorinated phenols. Arch. Microbiol. 1996, 165, 132-140. (31) Scholz-Muramatsu, H.; Neumann, A.; Messmer, M.; Moore, E.; Diekert, G. Isolation and characterization of Dehalosprillum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing, strictly anaerobe bacterium. Arch. Microbiol. 1995, 163, 48-56. (32) Maymo-Gatell, X.; Chien, Y. T.; Gossett, J. M.; Zinder, S. H. Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethane. Science 1997, 61, 3928-3933. (33) Krumholz, L. R.; Sharp, R.; Fishbain, S. S. A freshwater anaerobe coupling acetate oxidation to tetrachloroethylene dehalogenation. Appl. Environ. Microbiol. 1996, 62, 4108-4113. (34) Haston, Z. C.; McCarty, P. L. Chlorinated Ethene Half-velocity coefficients (Ks) for reductive dehalogenation. Environ. Sci. Technol. 1999, 33, 223-226. (35) Carter, S. R.; Jewell, W. J. Biotransformation of tetrachloroethylene by anaerobic attached-films at low temperatures. Water Res. 1993, 27, 607-615.

Received for review December 19, 2004. Revised manuscript received May 3, 2005. Accepted May 6, 2005. ES047993B

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