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Applied and Environmental Microbiology, November 1999, p. 5059-5065, Vol. 65, No. 11
Department of Microbiology, University of
Illinois at Urbana-Champaign, Urbana, Illinois 61801
Received 16 July 1999/Accepted 25 August 1999
For the hyperthermophilic and barophilic methanarchaeon
Methanococcus jannaschii, we have developed a medium and
protocols for reactor-scale cultivation that improved the final cell
yield per liter from ~0.5 to ~7.5 g of packed wet cells (~1.8 g
dry cell mass) under autotrophic growth conditions and to ~8.5 g of packed wet cells (~2 g dry cell mass) with yeast extract (2 g liter Methanococcus jannaschii
(13) is a hyperthermophilic (optimal growth temperature,
85°C) and barophilic (18, 19) methanarchaeon isolated from
the surface material collected from the base of a "white smoker"
submarine hydrothermal vent (13). Although this organism was
the first archaeon for which the sequence of the entire genome was
determined (6), the biochemical studies have been limited
due to the inability of investigators to successfully mass culture the
organism to high cell densities. The entire genome sequence for another
methanarchaeon, Methanobacterium thermoautotrophicum Previous to the present study, the typical maximum cell yield for
M. jannaschii in reactor-scale cultures was ~0.5 g of
packed wet cells per liter of culture (University of Illinois Fermentor Facility records) (27, 32). The published information on the growth of M. jannaschii is very limited and almost entirely
pertains to small-scale (5- to 200-ml) cultures (13, 18, 19,
24). In addition, none of these studies sought to optimize the
cell yield. A rare study that involved the cultivation of M. jannaschii in a reactor offers no details of the reactor and
reports a very slowly growing culture under limited gas supply and at
65°C that reaches an optical density at 660 nm (OD660) of
0.8 to 1.0 in 5 days (9). We describe here the media recipes
and protocols for mass culture of this organism in a 16-liter
constantly stirred tank reactor to high cell densities and provide
guidelines for scaling up such cultures to higher volumes.
Organism and media.
M. jannaschii JAL-1
(13) was obtained from David R. Boone (Oregon Collection of
Methanogens, Portland, Oreg.) and maintained in 25 ml of medium 1 in a
160-ml serum bottle as described below.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Reactor-Scale Cultivation of the Hyperthermophilic Methanarchaeon
Methanococcus jannaschii to High Cell Densities
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
1) and tryptone (2 g liter1) as medium
supplements. For growth in a sealed bottle it was necessary to add Se
to the medium, and a level of 2 µM for added Se gave the highest
final cell yield. In a reactor M. jannaschii grew without
added Se in the medium; it is plausible that the cells received Se as a
contaminant from the reactor vessel and the H2S supply.
But, for the optimal performance of a reactor culture, an addition of
Se to a final concentration of 50 to 100 µM was needed. Also, cell
growth in a reactor culture was inhibited at much higher Se
concentrations. These observations and the data from previous work with
methanogen cell extracts (B. C. McBride and R. S. Wolfe,
Biochemistry 10:4312-4317, 1971) suggested that from a
continuously sparged reactor culture Se was lost in the exhaust gas as
volatile selenides, and this loss raised the apparent required level of
and tolerance for Se. In spite of having a proteinaceous cell wall,
M. jannaschii withstood an impeller tip speed of 235.5 cms1, which was optimal for achieving high cell density
and also was the higher limit for the tolerated shear rate. The
organism secreted one or more acidic compounds, which lowered pH in
cultures without pH control; this secretion continued even after
cessation of growth.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
H,
has also been determined (26, 36). The availability of whole
genome sequences provides excellent opportunities for thorough comparative biochemical studies. The sequencing of the genome of
M. jannaschii has generated interest in studying this
organism; the requests for cultures, cell pastes, and protocols for
growing this organisms have greatly increased (our own experience)
(4, 11).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
Small-scale cultivation protocol.
To study growth of
M. jannaschii in tubes or bottles and to prepare inocula for
reactors, the techniques of Balch and Wolfe (2) were used,
with the following modifications. Two types of culture bottles were
used: one was a 160-ml serum bottle (catalog no. 223748; Wheaton
Science Products, Millville, N.J.) which contained 25 ml of medium and
which was sealed with a solid rubber stopper (catalog no. 2048-11800;
Bellco Glass Inc., Vineland, N.J.) (2) and the other was a
530-ml serum bottle (catalog no. 223952; Wheaton Science Products)
(7) which contained 150 ml of medium and which was sealed
with a no. 1 black rubber stopper that had one-third of its bottom cut
off (7); the rubber stoppers were crimped in place. Unless
otherwise mentioned (see below), for the preparation of medium, all
components (including vitamins, yeast extract, and tryptone, wherever
indicated) except MgCl2 · 6H2O,
CaCl2 · 2H2O, and NH4Cl (and
also excluding
Fe(NH4)2(SO4)2 · 6H2O and Na3-nitrilotriacetate for medium 1)
were dissolved in distilled deionized water. This solution was made
anaerobic by boiling (2) under a stream of
N2-CO2 (80:20 [vol/vol]). After the anaerobic solution was cooled under a N2-CO2 atmosphere
and transferred inside an anaerobic bag (2),
MgCl2 · 6H2O, CaCl2 · 2H2O, and NH4Cl were added as solids to it to
desired final concentrations. This procedure prevented precipitation of
Mg2+ and loss of NH3 during boiling due to a
rise in pH. Such a precaution was not needed for a medium with MES as
the buffer, since here the pH did not rise during boiling
(pKa/°C for MES, 
0.009) (19), and
therefore MgCl2 · 6H2O,
CaCl2 · 2H2O, and NH4Cl were
included in the medium before boiling. Further steps were carried out
as detailed previously (2), except the headspace gas of the
bottles with anaerobic medium was exchanged with
H2-CO2 (80:20 [vol/vol]; 0.15 to 0.2 × 105 Pa before the bottles were autoclaved. Before
inoculation of the sterile medium, sodium sulfide was added from an
anaerobic sterile stock to a final concentration of 2 mM. Also, for
medium 1, at this stage
Fe(NH4)2(SO4)2 · 6H2O and Na3-nitrilotriacetate were added from
a sterile anaerobic stock solution. For growth experiments the cultures
were initiated with an inoculum (2 ml of mid-logarithmic phase culture
per 100 ml of medium) that had been grown by two sequential transfers
in the intended test medium. The inoculated cultures were pressurized
to 1.7 × 105 Pa with H2-CO2
(80:20 [vol/vol] and shaken at 85°C and 200 rpm in a gyratory
shaker (model 3527X Orbit Environ-Shaker; Lab-Line Instruments, Inc.,
Melrose Park, Ill.). For growth of inocula each bottle was removed from
the shaker every 2 h and the headspace was repressurized with
H2-CO2. For growth experiments, the atmosphere in each bottle was repressurized every hour, except every third hour
the atmosphere was flushed out for 30 s by allowing 2 liters of
gas to escape through a 22-gauge needle prior to pressurization. To
minimize the temperature drop in a culture during this rejuvenation process, an insulating sleeve made out of soft cellulose was put on the
bottle; even then the culture temperature often dropped to as low as
78°C. For studying the effect of Se concentration on growth, the
serum bottles were soaked overnight in 1 M
H2SO4 and then washed extensively with
distilled deionized water before use; the rubber stoppers were washed
with distilled deionized water. Here, medium 1 was prepared without
Na2SeO4, but it was added to the sterile medium
to a desired final concentration from a sterile anaerobic stock.
Reactor and accessories.
The reactor-scale experiments were
carried out in a 16-liter (12-liter working volume) stainless steel
constantly stirred tank reactor (model Microgen; New Brunswick
Scientific Company, New Brunswick, N.J.) with four vertical baffles and
three six-bladed Rushton type turbine impellers (diameter, 7.5 cm) (see
Fig. 1 of reference 8). The bottommost impeller was
situated 5 cm above the vessel bottom and 1.3 cm above the single-hole
sparger. The distance between two consecutive impellers was 10.2 cm.
The top impeller was 7 cm below the liquid surface (when the vessel content was not being stirred). The vessel was fitted with a gel-filled sterilizable pH probe and the same type of redox probe (Broadly James
Corp., Irvine, Calif.) for the measurements of culture pH and redox
potential, respectively, in situ. For making anaerobic and sterile
additions (manual or automatic) to the culture two of the addition
ports on the head plate of the vessel were fitted with rubber stoppers
(no. 5 1/2 black rubber stopper) as detailed in Fig.
1 (20). All manual additions
of sterile and anaerobic solutions to the culture were performed
through one of these two rubber stoppers by using either a sterile
syringe fitted with a 22-gauge needle (Fig. 1) or a double-needle
(22-gauge) system of Baresi and Wolfe (3) (Fig. 1). Each
automatic addition (NaOH solution or water) was via a 20-gauge needle
inserted through the other rubber stopper and connected to a supply
line by using a 1.6-mm polypropylene male Luer fitting (catalog no.
E-30504-00; Cole Palmer Instrument Co., Vernon Hills, Ill.). For
automatic control of pH, a pH controller (model M1055-1000; New
Brunswick Scientific Co.), a reservoir (a 530-ml sealed serum bottle
fitted with a 21-gauge needle through its stopper) of sterile and
anaerobic solution of 5 N NaOH and a peristaltic pump (a Masterflex LS
variable-speed modular drive and an LS size 13 pump head; Barnant Co.,
Barrington, Ill.) were used. The flow path for the NaOH solution,
including the pumping section, was made up of a Masterflex size 13 Norprene A60G tube (internal diameter, 0.8 mm; Norton Performance
Plastic Loop, Akron, Ohio). A similar system was used for the
continuous addition of sterile anaerobic water to the reactor, and here
a 1-liter culture bottle, described by Balch and Wolfe (2),
fitted with a 21-gauge needle through its rubber stopper served as the reservoir. The inoculum was transferred to the sterile medium from a
160-ml serum bottle by use of the double-needle system shown in Fig. 1
(3). To obtain consistent results we recommend that the
detailed procedures presented in the legend for Fig. 1 be used. Unless
otherwise mentioned, all gases were supplied to the culture at the
bottom of the vessel through a single-hole sparger situated directly
below the agitator shaft. The N2, CO2, and
H2 streams were made oxygen free by passage through a
common heated bed of copper turnings (2); the flow of
hydrogen ensured continuous regeneration of oxidized copper. The flow
rates of gases were measured and controlled by using rotameters (Cole
Palmer Instrument Co.), and the reported values correspond to a
pressure of 1 atm or 1.01 × 105 Pa. Heating and
cooling of reactor contents were accomplished by using flows of steam
(2.8 × 105 Pa) and water (22°C and 2.8 × 105 Pa), respectively, through the hollow baffles. The
cells were harvested by use of a Sharples centrifuge (type AS-14),
frozen quickly in liquid nitrogen, and stored at 70°C.
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Reactor-scale cultivation protocol.
For the preparation of
medium 1 or 2, all components except MgCl2 · 6H2O, CaCl2 · 2H2O, and
NH4Cl (and also excluding
Fe(NH4)2(SO4)2 · 6H2O and Na3-nitrilotriacetate for medium 1)
were dissolved in the vessel in water and the vessel content was
sterilized at 121°C for 40 min. The sterilized medium was cooled to
30°C under nitrogen (supplied as an overlay from the top of the
vessel), and the pH controller was reset to the pH for a sample of the sterilized medium, determined outside of the vessel. Then the flows of
hydrogen (2,500 ml min1) and CO2 (700 ml
min1) were initiated and the nitrogen flow was
discontinued. As a result the medium pH dropped rapidly to 6 to 6.2. At
this stage an anaerobic sterile solution of MgCl2 · 6H2O, CaCl2 · 2H2O, and NH4Cl from a sealed 530-ml bottle was added to the medium
by use of the double-needle system (3); this solution had
been made anaerobic by evacuation of the headspace and had been placed
under N2 (0.15 to 0.2 × 105 Pa) before
autoclaving and was pressurized to 1.7 × 105 Pa after
poststerilization cooling. The same protocol was used to add yeast
extract and tryptone. For medium 1, at this time an aerobic solution
(~20 ml) of
Fe(NH4)2(SO4)2 · 6H2O (final concentration, 0.0122 g liter1 or
31 µM) and Na3-nitrilotriacetate (final concentration,
0.083 g liter1 or 0.32 mM) was added to the medium
through a 0.2-µm sterile syringe filter (Fig. 1). After the redox
potential reading for the medium stabilized (typically at 160 mV
after ~30 min of gassing), a continuous flow of a gas mixture of
N2 and H2S (90:10 [vol/vol]; henceforth
referred to as H2S) was initiated at a flow rate of 50 ml
min1. After the medium was gassed with H2S
for 20 min, the redox potential reading stabilized at about 330 mV.
At this stage the agitation speed and gassing rates were adjusted to
desired values (see Results and Discussion), and the medium was
inoculated with 25 ml of a mid-log-phase culture (OD600,
0.6 to 0.8) from a 160-ml culture bottle by use of the double-needle
system (3). Further additions and manipulations of culture
conditions were as indicated in the Results section. Throughout the
cultivation period the vessel was maintained at a positive pressure of
1.25 × 105 Pa and the culture temperature was
maintained at 85°C. Whenever needed, foaming in the culture was
suppressed by addition of a 0.2-ml anaerobic and sterile aqueous
solution (50%) of Sigma Antifoam 289 (Sigma Chemical Co., St. Louis,
Mo.). The protocols for medium with MES as the buffer were essentially
the same as detailed above, except the pH of the medium was adjusted to
6.0 with NaOH before sterilization and, for the reason mentioned above
(see "Small-scale cultivation protocol") MgCl2 · 6H2O, CaCl2 · 2H2O, and
NH4Cl could be included in the medium before sterilization.
Analytical methods.
The OD of a culture was monitored at 600 nm by use of a model DU 640 UV-visible spectrophotometer (Beckman
Instruments, Inc., Fullerton, Calif.) with a cuvette having a 1-cm
light path. The protein content of a cell pellet was determined as
follows. At a desired time a sample of the culture was withdrawn and
centrifuged at 14,000 × g to obtain a cell pellet;
this pellet was resuspended in water (1 ml of water per ml of culture
centrifuged) and stored at 20°C for processing at a later time.
Each sample of resuspended cell pellet was thawed, mixed with 34 µl
of 1.5 M NaOH (final concentration, ~50 mM), and incubated at 80°C
for 20 min to maximize the release of proteins from the cells; these
optimized digestion conditions were established through experimentation
(data not shown). Each digest was then cooled to room temperature,
mixed with 34 µl of 1.5 M HCl, and assayed for protein content as
described by Bradford (5) with the dye reagent from Bio-Rad
Laboratories (Richmond, Calif.); serum albumin served as the standard.
The data from our early experiments showed that each of the growth patterns obtained from the values of OD600 was parallel to
the corresponding profile for the pellet protein content per milliliter of culture. Hence, all growth patterns reported in this work were determined by measuring the values of OD600. For
determining the dry-cell content of a culture, the cell pellet from a
known volume of culture (collected by centrifugation at 14,000 × g) was resuspended in a minimum volume of water and dried
(initially overnight at 95°C and then at 105°C) to constant weight.
The sulfide concentration in the culture liquid was determined by the
methylene blue method of Pachmayr (21) as detailed by
Trüper and Schlegel (28).
1) for the logarithmic growth
phase and the linear growth rate (change in OD600 units per
hour) for the linear growth phase. Each rate or OD600 value
reported in Fig. 3 is an average of data from duplicate experiments.
The data set in Fig. 2 is from one of the two experiments performed
under identical conditions.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Growth in serum bottles.
For a 150-ml culture in a 530-ml
serum bottle, the values for maximum specific growth rates in medium 1 and in other (9, 13, 24) media were in the range of 1 to 1.5 h1 and were comparable to those observed by Jones et al.
(13); here the final concentration of Se in medium 1 was 2 µM. The values for the final culture turbidities and cell yields in
these media were also comparable to each other, except that those for
medium 1 (OD600, ~1.7; ~0.65 g dry cell weight
liter1) were about twofold higher than those for others.
Certain observations at the reactor scale (see below) prompted us to
study the effect of added-Se level in medium on the growth of M. jannaschii at the serum bottle scale, and we chose medium 1 for
this purpose. We found that M. jannaschii was dependent on
added Se for growth. In the 0 to 100 µM range, a Se concentration of
2 µM offered the highest cell yield, and the levels higher than 10 µM were inhibitory. Our data show that a series of 530-ml bottles
each containing 150 ml of medium can be used for generating gram
quantities of cells. This system also allows withdrawal of samples for
monitoring growth and for analysis of culture liquid without
significantly changing the growth conditions and provides sufficient
cell mass at the mid-logarithmic and late logarithmic stages for enzyme and cofactor level measurements.
Growth cycle, medium composition, impeller speed, shear, foaming,
and water loss in a reactor.
To allow close monitoring of the
logarithmic and late logarithmic stages, we inoculated the sterile
medium late in the evening and allowed the cultivation to proceed at
200 rpm overnight with the following gas flow rates (in milliliters per
minute): H2, 2,500; CO2, 700;
N2-H2S (90:10 [vol/vol]), 20. At this
agitation rate, which limited the gas transfer to the liquid, the
organism grew slowly and was ready in the morning (~8 h after
inoculation) at an OD600 of ~0.2 (with each medium
tested) for immediate rapid logarithmic growth when the higher
agitation (600 rpm) and gassing rates (H2, 19,200 ml
min1; CO2, 4,800 ml min1;
H2S, 215 ml min1) were imposed (Fig.
2). All reactor-scale data presented here were collected by following this protocol. It was also possible to
extend the initial slow growth stage up to 12 to 16 h without significantly shortening the vigorous logarithmic growth stage (data
not shown). The option of initiating the agitation and gassing at
optimal rates immediately after inoculation of the medium proved impractical. This was because such a growth cycle often included a lag
period of ~4 h and, as seen in Fig. 2, after the onset of the
logarithmic growth phase a reactor culture of M. jannaschii took ~12 h to reach the late logarithmic or stationary stage. Also,
as the stationary phase began, cells started to lyse.
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on
the physiology of M. jannaschii, MES (pKa at
85°C, 5.6) (23) could be used as the buffer in the medium.
M. jannaschii derives all the needed energy and the reducing
power needed for cell material biosynthesis from the oxidation of
H2. On the other hand H2 is a very sparingly
soluble gas; Henry's constant for H2 (in units of
atmospheres per mole fraction of solute dissolved in solution) at
80°C is 7.55 × 104 and at 90°C is 7.51 × 104 (16). Thus, the rate of H2
dissolution is expected to limit this organism's growth rate. With
medium 1, an increase in impeller rotational speed from 400 to 600 rpm
improved the value of the maximum specific growth rate twofold,
although the value of the final cell density improved only marginally.
The increase in impeller rotational speed from 400 to 600 rpm
corresponded to a change in the value of the impeller Reynolds number
or NRe from 3.75 × 104 to 5.6 × 104 and to a 1.5-fold increase in shear rate. The
NRe values were calculated from the relationship
NRe = (rotations per second) × (impeller
diameter in centimeters)2 × (density of culture
liquid in grams per cubic centimeter)/viscosity of culture liquid in
grams per centimeter per second and by assuming the density and
viscosity of culture liquid to be 1 g cm3 and
0.01 g cm1 s1, respectively. The
relative shear rates were judged from the values of impeller tip speed,
which are given by 
× impeller diameter × impeller
rotational speed. Neither the growth rate nor the final cell density
values at an impeller speed of 800 rpm (NRe, 7.5 × 104) were different from those at 600 rpm. Rather, at 800 rpm the culture, after reaching an OD600 of 0.75, foamed
almost continuously in spite of frequent antifoam additions; this
effect was most likely caused by shear-induced cell lysis. But it
should be noted that, in spite of having only a proteinaceous cell
envelope (15), this organism tolerated a fairly high shear
rate (600 rpm for an impeller with a diameter of 7.5 cm; a tip speed of
235.5 cms1), which is routinely used for reactor
cultivation of M. thermoautotrophicum (8, 20), an
organism with a rather rigid cell wall composed of pseudomurein, a type
of peptidoglycan (15). Our observation is in contrast to the
reported fragility of Methanococcus vannielii (12), which is why this organism has been cultivated in a
400-liter reactor without mechanical mixing. A sensitivity to higher
agitation rates has also been seen with Methanothermus
fervidus (22), even though the organism's cell
envelope compositions (15) suggest a capacity to withstand
high shear; a very likely explanation for this observation is the
inability of the reactor system in use to prevent oxygen contamination
(22). Our observations suggest that other methanogens with
seemingly weaker cell envelopes should be tested for shear sensitivity
while developing a method for mass culture with sparingly soluble gases
as substrates. Each of the values of NRe reported here
corresponded to a turbulent mixing regime (25). Our results
could form the basis for scale-up of M. jannaschii culture
to higher operating volumes. It is customary to scale-up gas transfer
rate-limited microbial cultures based on the optimized value for power
input per unit volume or mass transfer coefficient at the smaller scale
(29). However, the moderate shear sensitivity of M. jannaschii, as observed by us, suggested that a scale-up effort
should use the shear rate as the first basis to determine the mixing
rate at the higher scale. For this purpose an impeller tip speed of
235.5 cm s1 (corresponding to an impeller rotational
speed of 600 rpm in our system) could be used. It is expected that such
a scale-up method would give for the larger scale a H2
transfer rate lower than the one that was in effect for the reactor
used in this study. For this reason, after the suitability of the use
of the tip speed of 235.5 cm s1 at the larger scale is
established, attempts should be made to optimize the power input as
well as the gassing rates.
Occasionally and unpredictably, foaming occurred in reactor cultures
even at an impeller speed of 600 rpm, after the culture turbidity
(OD600) reached 0.5 to 0.8. We used Sigma Antifoam 289 for
suppressing foam formation. For a culture volume of 12 liters, a 0.2-ml
50% aqueous anaerobic and sterile solution of the antifoam was used
for each addition. Such additions at about 2- to 4-h intervals did not
alter the growth rates and final cell yields.
Our reactor was operated without a condenser on the exhaust gas line.
The consequence was a substantial evaporative loss of water in this
thermophilic culture; at a total gas flow rate of 24,215 ml
min1 the water loss rate was ~200 ml h1.
To compensate for this loss, we instituted a continuous addition of
sterile anaerobic water at a rate of ~200 ml h1.
Formation of acids in reactor cultures. It was necessary to add NaOH to maintain the pH at 6 ± 0.5. In the absence of pH control, the culture pH dropped (to a value as low as 3.5 for media 1 and 2) after the OD600 of the culture reached a value of about 0.7 and the growth rate and final cell yield were reduced (data not shown). Intermittent manual addition of base and addition of base via a control system with a set point of 6 ± 0.5 were found to be equally effective. With 10 mM NaHCO3 and a partial pressure of 0.445 × 105 Pa for CO2 (H2/CO2 ratio [vol/vol], 80:20; total pressure, 1.25 × 105 Pa), the experimentally determined value for the pH of medium 1 at 85°C was 6.1. The corresponding calculated value, based on the available solubility and dissociation constant data (16, 30), was 6.9. However, this calculation did not take into account the effect of salts at high concentrations on the values for the constants, as well as the effects of salts and elevated temperatures on the probe. Nevertheless, both the experimentally determined and the calculated values for the pH of uninoculated medium were much higher than those of cultures grown without pH control. It is possible that M. jannaschii excreted certain acidic compounds (with low values for their pKas) in amounts that, in the absence of pH control, overwhelmed the buffering capacity of the medium. In each culture raised with pH control, the NaOH consumption paralleled cell growth; Fig. 2 shows an example where, by the end of growth, the culture consumed as much as 16 meq of base per g of dry cell produced. The base consumption continued even after the cessation of growth. It has been shown that Methanosarcina species excrete acetate when they are grown on H2-CO2, methanol, or trimethylamine (31, 35). At this point neither the nature of the excreted acidic compound(s) nor its metabolic origin nor its effect on the physiology of M. jannaschii is known. We are currently pursuing this topic.
Selenium.
Jones et al. (13) reported that the
addition of selenium in medium significantly stimulates the growth of
M. jannaschii. A previous careful study has established an
absolute requirement of Se for the growth of Methanococcus
voltae on H2-CO2 and has shown that for
this organism Se at a final concentration of 10 µM increases growth
rate by 50% and culture turbidity by 2.7-fold over those obtained with
unsupplemented medium presumably containing Se as a contaminant
(33). For Methanococcus maripaludis an added-Se level of 10 µM enhances the growth rate by 20% over the control (14), and for Methanococcus vannielii a level of
1 µM substantially improves both growth rate and cell yield on
formate at large scale (12). Also, these organisms possess
several Se-containing enzymes that are linked to their energy
metabolism (10). Since our medium development work started
from the medium recipes of Jones et al. (13) and Rajagopal
and Daniels (24), where the added-Se levels were 2.4 and 2 µM, respectively, in our early attempts to cultivate this
methanococcus in a reactor we added Na2SeO4 to
the medium at a level of 2 µM. These experiments offered relatively
high growth rates (~1 h1) and cell densities
(OD600, ~3.5; ~6 g of wet cell paste liter of
culture1; ~1.34 g dry cell mass liter of
culture1) compared to past accomplishments (see
Introduction). However, after several months of success we could no
longer reproduce these observations, especially with respect to the
growth rate (data not shown). This switch coincided with a change of
the H2S supply tank. This problem was alleviated when the
medium was supplemented with additional
Na2SeO4. Although a detailed study using
H2S tanks from several sources is needed to obtain a clear
explanation of these observations, it is plausible that our earlier
success in growing M. jannaschii at high growth rates and to
high cell densities in medium 1 with 2 µM added Se was due to a
continuous supply of H2Se to the culture as a contaminant
in the H2S gas. Whitman et al. (33) have
indicated that most methanogen cultures probably receive Se as a
contaminant in sodium sulfide that is used as a medium reductant
(2). Therefore, we studied the effect of added Se on the
growth rate and final cell yield in bottle- and reactor-scale cultures
of M. jannaschii. The data for the bottle cultures have been
presented above and are discussed below. Figure 2 shows the parameter
profiles in a reactor culture with added Se at a concentration of 50 µM. Figure 3 presents data from an analysis of such cultures at various Se levels. Similar to what is seen
in Fig. 2, each culture showed a transition from a logarithmic to a
linear growth phase at a culture OD600 of 0.7 to 1. The
corresponding specific growth rate (µ), calculated from the culture
turbidity data in the logarithmic growth phase, showed an apparent
hyperbolic saturation kinetics with respect to the added-Se level in
medium (Fig. 3A). An extrapolation of the data in Fig. 3A showed that the calculated value of Se level for µ = 0 was 5.2 µM. In
other words, if the extrapolated section of the plot were to reflect a
real culture property, the contaminating level of Se in a medium without added Se would be 5.2 µM. Since an inspection of the plot in
Fig. 3A showed a sign of inhibition of logarithmic growth at high Se
concentrations, attempts were made to fit the data to the standard
substrate inhibition model µ = µmax[Se]/{Ks + [Se] + ([Se]2/Ki)} where [Se] is the
Se concentration, Ks is the substrate affinity
constant (in micromolar units), and Ki is the
inhibition constant (in micromolar units). For this purpose the [Se]
values were corrected for the contaminating Se by using the
above-calculated concentration value of 5.2 µM. But the simulation
showed that the data did not fit well to the above-mentioned substrate
inhibition model and thus suggested a more complex behavior (see
below). The observed maximum culture density also showed a hyperbolic saturation kinetics with respect to Se concentration (Fig. 3B), and
from this set of data the calculated concentration value for Se
appearing as a contaminant was 7.3 µM. The response of the linear
growth rate to the Se concentration is shown in Fig. 3C. Up to a
concentration of 5 µM, the growth rate was dependent on the Se level,
but further addition of Se did not influence the growth rate. Here the
calculated level of contaminating Se was 4.2 µM. At an added Se
concentration of 100 µM, the autotrophically grown cultures
approached a maximum OD600 of 4.9 and produced up to
7.5 g of wet cell paste or 1.8 g of dry cells per liter.
|
inhibits methane formation
from methylcobalamin in cell extracts of Methanobacterium
and that the extracts can reduce selenate to hydrogen selenide and
produce methylated selenide (17). Thus, in a continuously
sparged reactor culture of M. jannaschii, Se could be lost
in the exhaust gas as volatile selenides, and this loss would raise the
apparent required level of and tolerance for Se; such as an effect
could also account for the fact that the data shown in Fig. 3A did not
fit a standard substrate inhibition model. In that case, the
concentrations presented in the previous paragraph and in Fig. 3 would
correspond to the added amount of SeO42 but not to
the actual concentrations in a developing culture (cells plus fluid).
However, these values would remain useful for the mass culture of the organism.
Other growth parameters.
Jones et al. (13) observed
that organic compounds such as yeast extract, Trypticase, vitamins,
acetate, and formate are neither required for nor stimulate the growth
of M. jannaschii. But, in our hands, supplementation of
medium 1 containing 20 µM Se with yeast extract (2 g
liter1), tryptone (2 g liter1), and
vitamins (34) improved the final cell yield by as much as
1.3-fold and yielded up to 8.5 g of wet cell paste liter of culture1 or 2 g dry cell mass liter of
culture1; the specific and linear growth rates improved
by ~1.5- and ~1.25-fold, respectively. Of these supplements, when
added individually, yeast extract and tryptone, but not the vitamin
mixture, provided substantial enhancement of the final cell yield (data
not shown). It would be interesting to know whether the improvements in
cell yields by the heterotrophic supplements were due to some sort of
limitation on the cell material biosynthesis imposed by the growth
conditions employed. The data from our preliminary experiments at an
added Se concentration of 2 µM and low gas flow rates provided some clues. For most growth studies with methanogens an 80:20 (vol/vol) mixture of H2 and CO2 is used, and this ratio
corresponds to the stoichiometry in the methanogenesis reaction
(4H2 + CO2
CH4 + 2H2O). We observed that for M. jannaschii
cultures raised at H2 and CO2 flow rates of,
respectively, 5,500 and 1,410 ml min1
(H2/CO2 ratio, 79.6:20.4) the cell yield was
approximately threefold higher than that with H2 and
CO2 flow rates of 5,500 and 450 ml min1
(H2/CO2 ratio, 92.4:7.6), respectively. When
cultures were examined at the latter gas flow rates, it was found that
the inclusion of tryptone (2 g liter1)-yeast extract (2 g
liter1)-vitamin into the medium provided up to 1.8-fold
enhancement in cell yield, whereas no such enhancement was observed at
the earlier flow rates. The exact reason for the observed enhancement in cell yield by complex supplements under CO2-limiting
conditions is currently unknown. Neither do we know whether there are
other environmental conditions that would invoke such a dependence on an exogenous source of nutrients for vigorous growth. Experiments on
these aspects are in progress.
is 7.04 (1). Also, it was noted that the growth phase change coincided with a rise in culture redox potential (Fig. 2); the reason
and implication of this change are not known.
It has been reported previously that for M. jannaschii both
growth rate and methanogenesis rate increase with an increase in
pressure up to 750 atm or 7.60 × 107 Pa (18,
19), and this observation reflects the situation in this
organism's natural habitat (13). It is not known whether the final cell density is influenced by the higher pressures; we
carried out our work at the working pressures of 1.7 × 105 Pa at the bottle scale and of 1.25 × 105 Pa at the reactor scale, and most laboratories would be
able to use the protocols optimized at such pressures.
In summary, with a reactor of the type used here, the best values of
the autotrophic growth rate (~1 h1) and final cell
yield (approaching 7.5 g of packed wet cells and 1.8 g of dry
cells per liter of culture) for M. jannaschii were obtained
under the following conditions: growth medium, medium 1 with 100 µM
Se; impeller rotational speed, 600 rpm (impeller tip speed, 235.5 cms1); flow rates for H2, CO2,
and 10% H2S (in N2), respectively, 19,200, 4,800, and 215 ml min1; and a controlled culture pH of
6 ± 0.5. Supplementation of growth medium with yeast extract (2 g
liter1) and tryptone (2 g liter1) improved
the growth rate and cell yield substantially. The described protocols
will also be very useful in designing experiments for studying the
physiology of M. jannaschii as well as in examining other
medium recipes for further improvements in growth rates and cell yield.
| |
ACKNOWLEDGMENTS |
|---|
We thank Vipool J. Patel and Cynthia L. Kreder for excellent technical assistance. We thank Kevin R. Sowers, William B. Whitman, H. Hippe, and D. R. Boone for communicating unpublished observations.
This work was supported by the Department of Energy grant DE-FG02-87ER13651. All reactor culture experiments were conducted in the Department of Microbiology Fermentor Facility, University of Illinois at Urbana-Champaign.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: University of Illinois at Urbana-Champaign, Department of Microbiology, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. Phone: (217) 333-1397. Fax: (217) 244-8485. E-mail: biswarup{at}life.uiuc.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Applied and Environmental Microbiology, November 1999, p. 5059-5065, Vol. 65, No. 11
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