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Nutrient control by the gas evolution in methanogenesis of methanol by Methanosarcina barkeri
JOURNALOF FERMENTATIONAND BIOENGINEERING VOI. 73, No. 6, 481-485. 1992
Nutrient Control by the Gas Evolution in Methanogenesis of Methanol by Methanosarcina barkeri NAOMICHI NISHIO, T O S H I H I D E KAKIZONO, ROBERTO G. SILVEIRA, SUSUMU TAKEMOTO,§ AND SHIRO NAGAI*
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Kagamiyama 1-4-1, Higashi-ttiroshima 724, Japan Received 21 November 1991/Accepted 11 April 1992 A practical fed-batch culture, in which consumed amounts of methanol and other nutrients were supplied in response to a direct signal of the gas production of CH4 and CO2, was carried out for the cell production of methanol-utilizing Methanosarcina barkeri. In this fed-batch culture system equipped with level sensors to detect the gas production, a high cell concentration of 24.4 g/1 was attained in 175-h cultivation maintaining the optimized nutrient concentrations of methanol, NH4+, PO43-, Na ÷, Mg 2+, Ca 2+, Fe 2+, Ni 2+, Co 2+ and cysteine (S source) throughout the culture.
In a previous work, medium optimization in methanogenesis of methanol by Methanosarcina barkeri has been achieved to enhance the methane production rate, or the cell growth rate by applying orthogonal array method based on a statistical multi-variable analysis (1). In fact, the optimized medium increased the methane production rate two-fold compared with the original basal medium. This result suggested that a dense culture of the methanogen cells might be possible within a short period if one can maintain the respective medium components in the range of each optimized concentration throughout the culture. In this work, an attempt to maintain the ten medium components at the optimal levels has been conducted by measuring the volume of gas (CH4 and CO2) production in a fed-batch culture of methanogenesis of methanol by M. barkeri. In this fed-batch culture, the stoichiometric relationships between the gas evolution, the cell growth, the methanol consumption, and the nutrient consumption were analyzed so as to optimally supply the required amount of the respective nutrients based on the volumetric increase of the evolved gas from the fermentor. In this fed-batch culture with the nutrient-feeding controlled by the evolved gas, a dense cell mass of the methanogen of 24.4g// was attained in 175-h culture maintaining the respective nutrient concentration at the optimal level with little variation.
tion (1) used in batch and fed-batch cultures was as follows (per liter of deionized water): K2HPO4, 1.4g; KH2PO4, 0.9g; NH4C1, 0.5 g; MgSO4-7H20, 1.0g; CaC12.2H20, 0.75g; FeSO4-TH20, 5.0mg; NiC12.6H20, 0.17mg; COC12.6H20, 0.17rag; cysteine.HCI-H20, 0.3g; the vitamin mixture (2), 5.0ml; the trace mineral solution (2), 18.0ml, and methanol, 8.0g. Strategy for fed-batch culture After the progress of batch culture, a fed-batch culture was initiated with the addition of the concentrated nutrients, in which the objective nutrients to be maintained were PO4, Na, Mg, Ca, Fe, Ni, Co, cysteine and methanol. The vitamin mixture and trace mineral solution were not taken into account as they were included in the batch culture. Since NH4 + was supplied together with the pH adjustment (see later), it was not included as an objective along with the nutrients. Potassium was also not considered since it was supplied sufficiently from K2HPO4 and KH2PO4 for PO, requirement (see later). In the methanogenesis of methanol, the following mass balance equation can be established.
A&=ASc+ ASx
(1)
where, AST, consumed methanol (mol methanol); ASG, methanol catabolized to CHa and CO2 (mol methanol); hSx, methanol anabolized to the cells (mol methanol). In Eq.1, hSx can be rewritten as follows;
aSx
a~ASx AST _ axaX
MATERIALS AND METHODS
ASx= AST AST= AS~
Microbial strain and culture technique M. barkeri strain Fusaro (DSM804) obtained from Deutsche Sammlung yon Mikroorgasismen (G6ttingen, Germany) was used throughout this study. The culture was maintained by frequent transfer into methanol basal medium (1). All manipulation of media and cultures were carried out under an O2-free atmosphere of N2 (99.999% (v/v) purity, Chugoku Teisan Co., Hiroshima) as reported (1). Culture medium An optimized medium composi-
= ~-c ASTYx/s
a~ -- AST
AST
a~ (2)
where, a~, carbon content of methanol (12 g carbon/mol methanol); hX, cell mass produced (g dry cell mass); ax, carbon content of dry cell mass (0.441 g carbon/g cell, Ref. 4); Yx/s, growth yield from methanol (4.23 g cell/mol methanol, see results). While AS6 in Eq. 1 can be rewritten by
AS~= a~--~G= ~G * Corresponding author. § Present address: Research Center, Suntory Ltd., 1-I-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan.
(3)
where ~G, gas (CH4 and COz} produced from the catabolized methanol (tool gas), i.e., CH3OH=0.75 CH4+0.25 481
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NISHIO ET AL.
J. FERMENT.BIOENG.,
CO2+0.5 H20. In fact, almost the same stoichiometry was obtained from the mass balance o f methanogenesis of methanol (3). Hence, AS6/AG comes to 1. Substitution o f Eqs. 2 and 3 into Eq. 1 yields that zIG AST= 1--ax.ac Yx/s -- 1.18 AG
(4)
Equation 4 means that the consumed methanol is in p r o p o r t i o n to the gas production (CH4 and CO2). Thus, by measuring the gas production for a short period, it is possible to calculate the consumed a m o u n t o f methanol for the same period, and to supplement it based on the amount o f the produced gas (Eq. 4). The amounts o f the other nutrient requirements corresponding to the consumed amount o f methanol, YN/S (rag nutrient/tool methanol) were determined by measuring the consumed amount o f respective nutrients in the batch culture o f M. barkeri. Fed-batch reactor system by gas evolution Figure 1 shows a schematic diagram o f the fed-batch culture system with level sensors for the evolved gas measurement. The cultivation was carried out in a conical flask (3.0/), equipped with a temperature control (37°C) and a magnetic stirrer (150 rpm). The p H was controlled at 5.9 ± 0 . 1 by a pH-controller using NH4OH solution (28%, w/w). Before a fed-batch culture was started, a batch culture was started with 1.5 1 of the optimized medium. When the evolved gas reached to the top of the level sensor, the objective nutrients were fed from the nutrient reservoirs. The detailed procedures are as follows: the evolved gas was passed through a vessel containing saturated NaC1 solution, and the gas volume equivalent to the displaced solution in the vessel was determined by the two level sensors ( 6 I F type, Omron, Kyoto). When the liquid level in the vessel reached the level of the sensor located at the top, the electric circuit between the top and the b o t t o m sen-
sors was closed turning the pump ON. Then, the concentrated nutrients were fed into the fermentor at intervals adjusted by a timer (H5CN type, Omron, Kyoto). Thus, the objective nutrients were supplied in p r o p o r t i o n to the methanol consumption, while the NaCI solution in the cylinder was quickly discharged by a pump (755300 type, Yamato Scientific Co., Tokyo) at a high flow rate (e.g. 1,3 l/min) until the liquid level reached the sensor at the bottom. Then, the circuit between the two b o t t o m sensors was closed turning the pump OFF, and then a new cycle o f the feeding was restarted. Design for objective nutrient concentrations As all the objective nutrients do not dissolve simultaneously in pure methanol, the nutrients were grouped into three (see Fig. 1), i.e., #1: methanol, MgSO4.7H20 and COC12. 6H20, #2: K2HPO4, KH2PO4 and NaC1, and //3: FeSO4. 7H20, NiC12.6H20, CaC12.6H20 and cysteine. HC1. H20. To design the nutrient concentrations in each group, an as small as possible flow rate o f the pump (Fig. lg) for each nutrient group was selected so as to avoid an increase o f culture volume during a fed-batch culture (see Results). Operation time for feeding The evolved gas, AG between the two level sensors in the cylinder which corresponded to 0.87 l ( = 0 . 0 3 9 mol gas at the standard condition) was substituted into Eq. 4 to calculate the consumed methanol, i.e., AST: 1.18AG=0.046 (mol methanol). Then, an operation time o f a timer, At (s) for supplying all the nutrients into the fermentor can be calculated as follows: AST= CMOH"FMoH" At
(5)
where CMOH and FMOH are the methanol concentration, and its feeding rate, respectively. Analytical procedure Methanol concentration was determined by gas chromatography as described previously (3). Cell dry weight was calculated from the whole cell protein content by a dye binding method described previously (2). A m m o n i u m (NH4+), phosphate ( P O 2 - ) and sulfide (S 2-) were determined by the methods o f indophenol, m o l y b d e n u m blue and methylene blue, respectively (5). Cysteine was determined by the Gaitonde method (6). Fe, Co, Ni, Mg and Ca were determined by atomic absorption spectrometry (GA-2B, Hitachi, Tokyo) after passing the supernatant through a XAD-2 column to retain Fe, Co and Ni-tetrapyrroles (7).
RESULTS AND DISCUSSION
FIG. 1. Schematic diagram of the fed-batch culture system controlled by evolved gas. a, Fermentor (3/); b, liquid level sensor; c, pH controller; d, concentrated nutrient reservoirs (nutrient groups I to 3, see Table 2); e, NaCI saturated solution; f, timer for nutrient supplies; g. pump for nutrient feeding; h, pump for NaCI solution discharging; i, pump for NI-I4OH feeding; j, NI-LOH solution (28~ w/w).
Measurements of YN/s TO supply the objective nutrients during the fed-batch culture according to the gas evolution (see Fig. 1), YN/S values were determined experimentally in the batch culture as shown in Table 1. In contrast with these values, YN/s values were also calculated from the elemental composition of M. barkeri cells, and the growth yield, Yx/s. The experimental YN/s was in agreement with the calculated values except for Fe and Ni. Therefore, the fed-batch culture was carried out using the experimentally determined Y•/s. Objective nutrient concentration Based on the flow rates o f the pumps (Fig. 1), one can decide the respective nutrient concentration, Cw (mg/ml) o f the three groups as follows: CN = CMoH'FMoH
FN
where, CMOH
. YN/S
(6)
is methanol concentration (mg/ml), and
VOL. 73, 1992
NUTRIENT CONTROL IN METHANOGENESIS
483
TABLE I. Estimation of the respective nutrient requirement equivalent to the consumed methanol (Yws)
YN/S Nutrient used
Element
K2HPO4 c KH2PO/ NaCI
MgSO4"7H20 CaCl2.2H20 FeSO4.7H20 NiCl2.6H20 CoCl2.6H20 Cysteine•HCI. H20
Ywx (mg nutrient/g cell)
(mg nutrient/mol methanol) Experimentala Calculatedb
K, P K, P Na
201.5c 80.6: 94.2
188.7 c 75.3: 99.0
44.6 ~ 17.8¢ 23.4
Mg Ca Fe Ni Co S
69.2 56.1 4.84 1.21 0.97 202.0
72.8 58.8 45.3 2.33 1.02 255.0
17.2 13.9 10.7 0.55 0.24 60.2
a Experimentally obtained from the consumed amounts of methanol and respective nutrient. b y~/s= YN/x"Yx/swhere YN/x (rag nutrient/g cell), the amount of nutrient required for I g cell formation, can be calculated from the elemental composition ofM. barkeri, e.g. for Na: 9.2 (rag Na/g cell) x 58.5 (mg NaCl)/23 (rag Na), and Yx/sis 4.23 (g cell/tool methanol, experimental value). The elemental compositions (mg/g cell) were referred from (4): P, 12.0; K, 2.5; N, 128; Na, 9.2; Mg, 1.7, Ca, 3.8; Fe, 2.15, Ni, 0.135; Co, 0.06; S, 11.0; C, 441. : Calculated from the ratio of K2HPO4/KH2PO4of 2.5 (not theoretical). Therefore, the amount of K is ca. 10-fold higher than the value required from the elemental analysis of M. barkeri cells (2.5 mg K/g cell) (4). FMOH and FN are flow rates of methanol and other nutrients (ml/min), respectively. Substitution of YN/s (Table 1) and CMO., FMOH, and Fr~ (Table 2) gives the respective CN as shown in Table 2. O p e r a t i o n time f o r f e e d i n g The operation time of a timer, At (s) to supply the concentrated nutrients of the three groups into the fermentor, can be calculated by substituting ASr (0.046 tool, Eq. 4), CMo. (794 m g / m l , Table 2), and FMOH (3.0 m l / m i n , Table 2) into Eq. 5. Then, 37 s was obtained for At. P e r f o r m a n c e o f the fed-batch culture c o n t r o l l e d by evolved gas A fed-batch culture of M. barkeri in which methanol and other essential nutrients were supplied intermittently based on the gas production was carried out to investigate whether methanol and other nutrient concentrations can be controlled at their optimum levels. The results obtained were depicted in Figs. 2 and 3. Methanol concentration could be maintained between 8.0 and 8.8 g/l, and both the cell concentration and the gas production rate increased exponentially up to the final cell concentration of'24.4 g cell/l, and 3.6 1 gas/l/h, respectively in 175-h culture (Fig. 2). As to these results, it took 264-h to attain 8.5 g c e l l / / i n the previous fed-batch culture of M. barkeri, in which the essential nutrient concentrations were not controlled during the culture (2). Also, in a repeated fed-batch culture combined with membrane module (8), a longer culture time (550-h) was required to reach TABLE 2. Nutrient concentrations to supply in a fed-batch culture of M. barkeri controlled by the evolved gas Nutrient group" Nutrient supplied 1 2 3
Pump Flow used rate (ml/min) (mg/ml)
Methanol BIP--1 b MgSO4.7H20 CoCl2.6H20 K2HPO4 SW3--1 c KH2PO4 NaCl FeSO4.7H20 SW3--1 NiCl2.6H20 CaCl~.2H20 Cysteine. HCI. H20
• see Fig. 1.
b,c JASCO HPLC pump.
3.0 0.633 0.840
Nutrient concentration 794 1.718 0.024 23.63 9.45 I 1.05 0.43 0.11 4.98 17.86
the same cell concentration. These results suggest that a precise control of methanol and the other nutrient concentrations might be necessary to give a longer exponential growth of M. barkeri. As for other nutrient concentrations during the culture (Fig. 3), concentrations of all the objective nutrients tested except for NH4 + seemed to be well controlled at the respective optimized levels (see culture t i m e = 0 ) . In the case of NH4 +, which was employed with pH adjustment, it was 50% above the optimum value at the end of the culture. In the methanogenesis of methanol by M. barkeri, it was successful to maintain the ten nutrients at the optimized levels in a fed-batch culture by automatic feeding of the nutrients in response to the gas evolution. Thus, the period of the exponential growth phase was drastically extended to far more than a hundred hours when the medium composition was maintained at the optimized level throughout the cultivation (Fig. 2), while the exponential period was only about twenty hours in the optimized medium without the nutrient control (1). This result also indicated that the M. barkeri cells seemed to utilize methanol for assimilation to the cell mass and for dissimilation to CH4 and CO2 (Eq. 1) at an almost constant ratio, thus utilizing other nutrients in proportion to the cell growth. Although the
11o.o 8"°1 ~I
~
3O
•
°'-"---_14.0 E. 3.0
,~u
2.0
lO
1.0
1
0
50
100 Time
150
,1 0.1
(11)
FIG. 2. A fed-batch culture of M. barlceri by intermittently supplying methanol and other essential nutrients by the evolved gas. Arrow indicates initiation of fed-batch culture. Symbols: o , methanol concentration (g/0; e , gas production rate (I gas/l broth/h); A, cell concentration (g//).
484
NISHIO ET AL.
J. FERMENT.BIOENG.,
P043
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FIG. 3. Time course of nutrient concentrations in the fed-batch culture of M. barkeri with automatic feeding of nutrients in response to the evolved gas control system. vitamin mixture and trace mineral solution were not measured for the nutrient control in this experiment, it would be necessary to take account of these components in a longer cultivation.
ac ax
: c a r b o n content of methanol, 12g c a r b o n / m o l methanol : carbon content of dry cell mass, 0.441 g c a r b o n / g cell
NOMENCLATURE
ACKNOWLEDGMENT
CMOH : m e t h a n o l concentration in the feed reservoir, mg/ml C~ : each nutrient concentration in the feed reservoir, mg/ml FMOH : methanol feeding rate, m l / m i n FN : each nutrient feeding rate, m l / m i n AG :gas (CH4+CO2) produced from the catabolized methanol, mol ASG : methanol catabolized to CH4+CO2, mol AST : methanol consumed, mol ASx : methanol anabolized to the cells, mol At : operation time of a timer, s AX : cell mass produced, g dry cell mass Y~/s : n u t r i e n t requirement equivalent to the consumed methanol, mg nutrient/tool methanol Y~/x : n u t r i e n t requirement equivalent to 1 g cell, mg n u t r i e n t / g cell Yx/s : growth yield from methanol, 4.23 g cell/mol methanol
The authors wish to thank Mr. E. Shoto, K. Ito, and M. Masumoto, in Wastewater Treatment Center, Hiroshima University, for their help for use of atomic absorption spectrometry. REFERENCES 1. Siiveira, R.G., Kakizono, T., Takemoto, S., Nishio, N., and Nagai, S.: Medium optimization by an orthogonal array design for the growth of Methanosarcina barkeri. J. Ferment. Bioeng., 72, 20-25 (1991). 2. Mazumder, T.K., Nishio, N., Hayashi, M., and Nagai, S.: Production of vitamin B12 compounds from methanol by Methanosarcina barkeri. Appl. Microbiol. Biotechnol., 65, 511516 (1987). 3. SHveira,R. G., Nishio, N., and Nagai, S.: Growth characteristics and corrinoid production of Methanosarcina barkeri on methanol-acetate medium. J. Ferment. Bioeng., 71, 28-34 (1991). 4. Scherer, P., Lippert, G., and Wolff, G.: Composition of the major elements and trace elements of 10 methanogenic bacteria determined by inductively coupled plasma emission spectrometry. Biol. Trace Element Res., 5, 149-163 (1983).
VOL. 73, 1992 5. American Public Health Association: Standard methods for the examination of water and wastewater, 17th ed. American Public Health Association, Inc., Washington D.C. 0989). 6. Gaitonde, M. K.: A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem. J., 104, 627-633 (1967). 7. Lin, D., Nishio, N., Mazumder, T. K., and Nagai, S.: Influence
NUTRIENT CONTROL IN METHANOGENESIS
485
of Co 2+, Ni 2+, Fe2+ on the production of tetrapyrroles by Methanosarcina barkeri. Appl. Microbiol. Biotechnol., 30, 196200 (1989). 8. Silveira, R. G., Nishida, Y., Nishio, N., and Nagai, S.: Corrinoid production by Methanosareina barReri in a repeated fed-batch reactor with membrane module. Biotechnol. Lett., 12, 721-726 (1990).
Nutrient Control by the Gas Evolution in Methanogenesis of Methanol by Methanosarcina barkeri NAOMICHI NISHIO, T O S H I H I D E KAKIZONO, ROBERTO G. SILVEIRA, SUSUMU TAKEMOTO,§ AND SHIRO NAGAI*
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Kagamiyama 1-4-1, Higashi-ttiroshima 724, Japan Received 21 November 1991/Accepted 11 April 1992 A practical fed-batch culture, in which consumed amounts of methanol and other nutrients were supplied in response to a direct signal of the gas production of CH4 and CO2, was carried out for the cell production of methanol-utilizing Methanosarcina barkeri. In this fed-batch culture system equipped with level sensors to detect the gas production, a high cell concentration of 24.4 g/1 was attained in 175-h cultivation maintaining the optimized nutrient concentrations of methanol, NH4+, PO43-, Na ÷, Mg 2+, Ca 2+, Fe 2+, Ni 2+, Co 2+ and cysteine (S source) throughout the culture.
In a previous work, medium optimization in methanogenesis of methanol by Methanosarcina barkeri has been achieved to enhance the methane production rate, or the cell growth rate by applying orthogonal array method based on a statistical multi-variable analysis (1). In fact, the optimized medium increased the methane production rate two-fold compared with the original basal medium. This result suggested that a dense culture of the methanogen cells might be possible within a short period if one can maintain the respective medium components in the range of each optimized concentration throughout the culture. In this work, an attempt to maintain the ten medium components at the optimal levels has been conducted by measuring the volume of gas (CH4 and CO2) production in a fed-batch culture of methanogenesis of methanol by M. barkeri. In this fed-batch culture, the stoichiometric relationships between the gas evolution, the cell growth, the methanol consumption, and the nutrient consumption were analyzed so as to optimally supply the required amount of the respective nutrients based on the volumetric increase of the evolved gas from the fermentor. In this fed-batch culture with the nutrient-feeding controlled by the evolved gas, a dense cell mass of the methanogen of 24.4g// was attained in 175-h culture maintaining the respective nutrient concentration at the optimal level with little variation.
tion (1) used in batch and fed-batch cultures was as follows (per liter of deionized water): K2HPO4, 1.4g; KH2PO4, 0.9g; NH4C1, 0.5 g; MgSO4-7H20, 1.0g; CaC12.2H20, 0.75g; FeSO4-TH20, 5.0mg; NiC12.6H20, 0.17mg; COC12.6H20, 0.17rag; cysteine.HCI-H20, 0.3g; the vitamin mixture (2), 5.0ml; the trace mineral solution (2), 18.0ml, and methanol, 8.0g. Strategy for fed-batch culture After the progress of batch culture, a fed-batch culture was initiated with the addition of the concentrated nutrients, in which the objective nutrients to be maintained were PO4, Na, Mg, Ca, Fe, Ni, Co, cysteine and methanol. The vitamin mixture and trace mineral solution were not taken into account as they were included in the batch culture. Since NH4 + was supplied together with the pH adjustment (see later), it was not included as an objective along with the nutrients. Potassium was also not considered since it was supplied sufficiently from K2HPO4 and KH2PO4 for PO, requirement (see later). In the methanogenesis of methanol, the following mass balance equation can be established.
A&=ASc+ ASx
(1)
where, AST, consumed methanol (mol methanol); ASG, methanol catabolized to CHa and CO2 (mol methanol); hSx, methanol anabolized to the cells (mol methanol). In Eq.1, hSx can be rewritten as follows;
aSx
a~ASx AST _ axaX
MATERIALS AND METHODS
ASx= AST AST= AS~
Microbial strain and culture technique M. barkeri strain Fusaro (DSM804) obtained from Deutsche Sammlung yon Mikroorgasismen (G6ttingen, Germany) was used throughout this study. The culture was maintained by frequent transfer into methanol basal medium (1). All manipulation of media and cultures were carried out under an O2-free atmosphere of N2 (99.999% (v/v) purity, Chugoku Teisan Co., Hiroshima) as reported (1). Culture medium An optimized medium composi-
= ~-c ASTYx/s
a~ -- AST
AST
a~ (2)
where, a~, carbon content of methanol (12 g carbon/mol methanol); hX, cell mass produced (g dry cell mass); ax, carbon content of dry cell mass (0.441 g carbon/g cell, Ref. 4); Yx/s, growth yield from methanol (4.23 g cell/mol methanol, see results). While AS6 in Eq. 1 can be rewritten by
AS~= a~--~G= ~G * Corresponding author. § Present address: Research Center, Suntory Ltd., 1-I-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618, Japan.
(3)
where ~G, gas (CH4 and COz} produced from the catabolized methanol (tool gas), i.e., CH3OH=0.75 CH4+0.25 481
482
NISHIO ET AL.
J. FERMENT.BIOENG.,
CO2+0.5 H20. In fact, almost the same stoichiometry was obtained from the mass balance o f methanogenesis of methanol (3). Hence, AS6/AG comes to 1. Substitution o f Eqs. 2 and 3 into Eq. 1 yields that zIG AST= 1--ax.ac Yx/s -- 1.18 AG
(4)
Equation 4 means that the consumed methanol is in p r o p o r t i o n to the gas production (CH4 and CO2). Thus, by measuring the gas production for a short period, it is possible to calculate the consumed a m o u n t o f methanol for the same period, and to supplement it based on the amount o f the produced gas (Eq. 4). The amounts o f the other nutrient requirements corresponding to the consumed amount o f methanol, YN/S (rag nutrient/tool methanol) were determined by measuring the consumed amount o f respective nutrients in the batch culture o f M. barkeri. Fed-batch reactor system by gas evolution Figure 1 shows a schematic diagram o f the fed-batch culture system with level sensors for the evolved gas measurement. The cultivation was carried out in a conical flask (3.0/), equipped with a temperature control (37°C) and a magnetic stirrer (150 rpm). The p H was controlled at 5.9 ± 0 . 1 by a pH-controller using NH4OH solution (28%, w/w). Before a fed-batch culture was started, a batch culture was started with 1.5 1 of the optimized medium. When the evolved gas reached to the top of the level sensor, the objective nutrients were fed from the nutrient reservoirs. The detailed procedures are as follows: the evolved gas was passed through a vessel containing saturated NaC1 solution, and the gas volume equivalent to the displaced solution in the vessel was determined by the two level sensors ( 6 I F type, Omron, Kyoto). When the liquid level in the vessel reached the level of the sensor located at the top, the electric circuit between the top and the b o t t o m sen-
sors was closed turning the pump ON. Then, the concentrated nutrients were fed into the fermentor at intervals adjusted by a timer (H5CN type, Omron, Kyoto). Thus, the objective nutrients were supplied in p r o p o r t i o n to the methanol consumption, while the NaCI solution in the cylinder was quickly discharged by a pump (755300 type, Yamato Scientific Co., Tokyo) at a high flow rate (e.g. 1,3 l/min) until the liquid level reached the sensor at the bottom. Then, the circuit between the two b o t t o m sensors was closed turning the pump OFF, and then a new cycle o f the feeding was restarted. Design for objective nutrient concentrations As all the objective nutrients do not dissolve simultaneously in pure methanol, the nutrients were grouped into three (see Fig. 1), i.e., #1: methanol, MgSO4.7H20 and COC12. 6H20, #2: K2HPO4, KH2PO4 and NaC1, and //3: FeSO4. 7H20, NiC12.6H20, CaC12.6H20 and cysteine. HC1. H20. To design the nutrient concentrations in each group, an as small as possible flow rate o f the pump (Fig. lg) for each nutrient group was selected so as to avoid an increase o f culture volume during a fed-batch culture (see Results). Operation time for feeding The evolved gas, AG between the two level sensors in the cylinder which corresponded to 0.87 l ( = 0 . 0 3 9 mol gas at the standard condition) was substituted into Eq. 4 to calculate the consumed methanol, i.e., AST: 1.18AG=0.046 (mol methanol). Then, an operation time o f a timer, At (s) for supplying all the nutrients into the fermentor can be calculated as follows: AST= CMOH"FMoH" At
(5)
where CMOH and FMOH are the methanol concentration, and its feeding rate, respectively. Analytical procedure Methanol concentration was determined by gas chromatography as described previously (3). Cell dry weight was calculated from the whole cell protein content by a dye binding method described previously (2). A m m o n i u m (NH4+), phosphate ( P O 2 - ) and sulfide (S 2-) were determined by the methods o f indophenol, m o l y b d e n u m blue and methylene blue, respectively (5). Cysteine was determined by the Gaitonde method (6). Fe, Co, Ni, Mg and Ca were determined by atomic absorption spectrometry (GA-2B, Hitachi, Tokyo) after passing the supernatant through a XAD-2 column to retain Fe, Co and Ni-tetrapyrroles (7).
RESULTS AND DISCUSSION
FIG. 1. Schematic diagram of the fed-batch culture system controlled by evolved gas. a, Fermentor (3/); b, liquid level sensor; c, pH controller; d, concentrated nutrient reservoirs (nutrient groups I to 3, see Table 2); e, NaCI saturated solution; f, timer for nutrient supplies; g. pump for nutrient feeding; h, pump for NaCI solution discharging; i, pump for NI-I4OH feeding; j, NI-LOH solution (28~ w/w).
Measurements of YN/s TO supply the objective nutrients during the fed-batch culture according to the gas evolution (see Fig. 1), YN/S values were determined experimentally in the batch culture as shown in Table 1. In contrast with these values, YN/s values were also calculated from the elemental composition of M. barkeri cells, and the growth yield, Yx/s. The experimental YN/s was in agreement with the calculated values except for Fe and Ni. Therefore, the fed-batch culture was carried out using the experimentally determined Y•/s. Objective nutrient concentration Based on the flow rates o f the pumps (Fig. 1), one can decide the respective nutrient concentration, Cw (mg/ml) o f the three groups as follows: CN = CMoH'FMoH
FN
where, CMOH
. YN/S
(6)
is methanol concentration (mg/ml), and
VOL. 73, 1992
NUTRIENT CONTROL IN METHANOGENESIS
483
TABLE I. Estimation of the respective nutrient requirement equivalent to the consumed methanol (Yws)
YN/S Nutrient used
Element
K2HPO4 c KH2PO/ NaCI
MgSO4"7H20 CaCl2.2H20 FeSO4.7H20 NiCl2.6H20 CoCl2.6H20 Cysteine•HCI. H20
Ywx (mg nutrient/g cell)
(mg nutrient/mol methanol) Experimentala Calculatedb
K, P K, P Na
201.5c 80.6: 94.2
188.7 c 75.3: 99.0
44.6 ~ 17.8¢ 23.4
Mg Ca Fe Ni Co S
69.2 56.1 4.84 1.21 0.97 202.0
72.8 58.8 45.3 2.33 1.02 255.0
17.2 13.9 10.7 0.55 0.24 60.2
a Experimentally obtained from the consumed amounts of methanol and respective nutrient. b y~/s= YN/x"Yx/swhere YN/x (rag nutrient/g cell), the amount of nutrient required for I g cell formation, can be calculated from the elemental composition ofM. barkeri, e.g. for Na: 9.2 (rag Na/g cell) x 58.5 (mg NaCl)/23 (rag Na), and Yx/sis 4.23 (g cell/tool methanol, experimental value). The elemental compositions (mg/g cell) were referred from (4): P, 12.0; K, 2.5; N, 128; Na, 9.2; Mg, 1.7, Ca, 3.8; Fe, 2.15, Ni, 0.135; Co, 0.06; S, 11.0; C, 441. : Calculated from the ratio of K2HPO4/KH2PO4of 2.5 (not theoretical). Therefore, the amount of K is ca. 10-fold higher than the value required from the elemental analysis of M. barkeri cells (2.5 mg K/g cell) (4). FMOH and FN are flow rates of methanol and other nutrients (ml/min), respectively. Substitution of YN/s (Table 1) and CMO., FMOH, and Fr~ (Table 2) gives the respective CN as shown in Table 2. O p e r a t i o n time f o r f e e d i n g The operation time of a timer, At (s) to supply the concentrated nutrients of the three groups into the fermentor, can be calculated by substituting ASr (0.046 tool, Eq. 4), CMo. (794 m g / m l , Table 2), and FMOH (3.0 m l / m i n , Table 2) into Eq. 5. Then, 37 s was obtained for At. P e r f o r m a n c e o f the fed-batch culture c o n t r o l l e d by evolved gas A fed-batch culture of M. barkeri in which methanol and other essential nutrients were supplied intermittently based on the gas production was carried out to investigate whether methanol and other nutrient concentrations can be controlled at their optimum levels. The results obtained were depicted in Figs. 2 and 3. Methanol concentration could be maintained between 8.0 and 8.8 g/l, and both the cell concentration and the gas production rate increased exponentially up to the final cell concentration of'24.4 g cell/l, and 3.6 1 gas/l/h, respectively in 175-h culture (Fig. 2). As to these results, it took 264-h to attain 8.5 g c e l l / / i n the previous fed-batch culture of M. barkeri, in which the essential nutrient concentrations were not controlled during the culture (2). Also, in a repeated fed-batch culture combined with membrane module (8), a longer culture time (550-h) was required to reach TABLE 2. Nutrient concentrations to supply in a fed-batch culture of M. barkeri controlled by the evolved gas Nutrient group" Nutrient supplied 1 2 3
Pump Flow used rate (ml/min) (mg/ml)
Methanol BIP--1 b MgSO4.7H20 CoCl2.6H20 K2HPO4 SW3--1 c KH2PO4 NaCl FeSO4.7H20 SW3--1 NiCl2.6H20 CaCl~.2H20 Cysteine. HCI. H20
• see Fig. 1.
b,c JASCO HPLC pump.
3.0 0.633 0.840
Nutrient concentration 794 1.718 0.024 23.63 9.45 I 1.05 0.43 0.11 4.98 17.86
the same cell concentration. These results suggest that a precise control of methanol and the other nutrient concentrations might be necessary to give a longer exponential growth of M. barkeri. As for other nutrient concentrations during the culture (Fig. 3), concentrations of all the objective nutrients tested except for NH4 + seemed to be well controlled at the respective optimized levels (see culture t i m e = 0 ) . In the case of NH4 +, which was employed with pH adjustment, it was 50% above the optimum value at the end of the culture. In the methanogenesis of methanol by M. barkeri, it was successful to maintain the ten nutrients at the optimized levels in a fed-batch culture by automatic feeding of the nutrients in response to the gas evolution. Thus, the period of the exponential growth phase was drastically extended to far more than a hundred hours when the medium composition was maintained at the optimized level throughout the cultivation (Fig. 2), while the exponential period was only about twenty hours in the optimized medium without the nutrient control (1). This result also indicated that the M. barkeri cells seemed to utilize methanol for assimilation to the cell mass and for dissimilation to CH4 and CO2 (Eq. 1) at an almost constant ratio, thus utilizing other nutrients in proportion to the cell growth. Although the
11o.o 8"°1 ~I
~
3O
•
°'-"---_14.0 E. 3.0
,~u
2.0
lO
1.0
1
0
50
100 Time
150
,1 0.1
(11)
FIG. 2. A fed-batch culture of M. barlceri by intermittently supplying methanol and other essential nutrients by the evolved gas. Arrow indicates initiation of fed-batch culture. Symbols: o , methanol concentration (g/0; e , gas production rate (I gas/l broth/h); A, cell concentration (g//).
484
NISHIO ET AL.
J. FERMENT.BIOENG.,
P043
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•
~
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-
,
.
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.
.
,
.
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.
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100
150
200
Tlme (h)
A 1.o 0.5 0
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100
5o
,
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150
,
200
Time (h)
FIG. 3. Time course of nutrient concentrations in the fed-batch culture of M. barkeri with automatic feeding of nutrients in response to the evolved gas control system. vitamin mixture and trace mineral solution were not measured for the nutrient control in this experiment, it would be necessary to take account of these components in a longer cultivation.
ac ax
: c a r b o n content of methanol, 12g c a r b o n / m o l methanol : carbon content of dry cell mass, 0.441 g c a r b o n / g cell
NOMENCLATURE
ACKNOWLEDGMENT
CMOH : m e t h a n o l concentration in the feed reservoir, mg/ml C~ : each nutrient concentration in the feed reservoir, mg/ml FMOH : methanol feeding rate, m l / m i n FN : each nutrient feeding rate, m l / m i n AG :gas (CH4+CO2) produced from the catabolized methanol, mol ASG : methanol catabolized to CH4+CO2, mol AST : methanol consumed, mol ASx : methanol anabolized to the cells, mol At : operation time of a timer, s AX : cell mass produced, g dry cell mass Y~/s : n u t r i e n t requirement equivalent to the consumed methanol, mg nutrient/tool methanol Y~/x : n u t r i e n t requirement equivalent to 1 g cell, mg n u t r i e n t / g cell Yx/s : growth yield from methanol, 4.23 g cell/mol methanol
The authors wish to thank Mr. E. Shoto, K. Ito, and M. Masumoto, in Wastewater Treatment Center, Hiroshima University, for their help for use of atomic absorption spectrometry. REFERENCES 1. Siiveira, R.G., Kakizono, T., Takemoto, S., Nishio, N., and Nagai, S.: Medium optimization by an orthogonal array design for the growth of Methanosarcina barkeri. J. Ferment. Bioeng., 72, 20-25 (1991). 2. Mazumder, T.K., Nishio, N., Hayashi, M., and Nagai, S.: Production of vitamin B12 compounds from methanol by Methanosarcina barkeri. Appl. Microbiol. Biotechnol., 65, 511516 (1987). 3. SHveira,R. G., Nishio, N., and Nagai, S.: Growth characteristics and corrinoid production of Methanosarcina barkeri on methanol-acetate medium. J. Ferment. Bioeng., 71, 28-34 (1991). 4. Scherer, P., Lippert, G., and Wolff, G.: Composition of the major elements and trace elements of 10 methanogenic bacteria determined by inductively coupled plasma emission spectrometry. Biol. Trace Element Res., 5, 149-163 (1983).
VOL. 73, 1992 5. American Public Health Association: Standard methods for the examination of water and wastewater, 17th ed. American Public Health Association, Inc., Washington D.C. 0989). 6. Gaitonde, M. K.: A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem. J., 104, 627-633 (1967). 7. Lin, D., Nishio, N., Mazumder, T. K., and Nagai, S.: Influence
NUTRIENT CONTROL IN METHANOGENESIS
485
of Co 2+, Ni 2+, Fe2+ on the production of tetrapyrroles by Methanosarcina barkeri. Appl. Microbiol. Biotechnol., 30, 196200 (1989). 8. Silveira, R. G., Nishida, Y., Nishio, N., and Nagai, S.: Corrinoid production by Methanosareina barReri in a repeated fed-batch reactor with membrane module. Biotechnol. Lett., 12, 721-726 (1990).