88
4 Application of Biocatalysts for the Production of Methanol …
(3) Carbon dioxide
High concentrations of carbon dioxide partially inhibit MDH activity. However,
the maximum productivity of methanol under carbon dioxide-inhibited conditions is
very low (3 μmol L
−1 h
−1 ) compared with other methanol synthesis methods using
methane-oxidizing bacteria [13]. However, the use of carbon dioxide as an inhibitor
has the advantage of not requiring chemical modification of methane-oxidizing bacteria or the addition of expensive external electron donors or MDH inhibitors to the
reaction mixture.
(4) Combinations of inhibitors
EDTA has not been used alone as an MDH inhibitor. EDTA binds to the
cytochrome-binding area of MDH, blocking electron transfer from MDH to
cytochrome c. In fact, the oxidation of methanol by MDH can be inhibited by 0.05–
1 mM of EDTA. However, EDTA also reduces MMO activity, probably because of
the removal of metal ions from MMO via the chelation effect of EDTA [25, 26].
The combination of EDTA and other inhibitors such as NaCl has been tested as a
method to decrease the concentration of the other inhibitor. The methanol production
observed for a combined EDTA/NaCl system exceeded that of the NaCl-only system
approximately two-fold [15, 18].
MgCl 2 also has not been used as a sole inhibitor, but has been added along with
either phosphate or NaCl [15, 17, 19]. The mechanism by which MgCl 2 suppresses
the oxidation of methanol is still unclear [109], but various studies have reported
that methanol production increases significantly when 5–20 mM MgCl 2 is added
to phosphate buffer [7, 16, 22, 24, 25]. Mg ions promote sMMO activity and the
growth of methane-oxidizing bacteria [16]. The use of MgCl 2 in conjunction with
phosphate resulted in greater productivity than the use of EDTA with phosphate,
but EDTA showed a greater inhibitory effect on MDH activity than MgCl 2 [22,
25]. The use of 400 mM phosphate and 10 mM MgCl 2 resulted in a cell density of
17.3 g L
−1 and the highest methanol concentration of 1.13 g L
−1 , although phosphate
concentrations above 100 mM have been reported to inhibit MMO activity [10, 22,
25]. These results demonstrate that higher methanol production can be achieved by
increasing the inhibitor concentration as well as the cell density [16].
It has been suggested that 50% of the methanol oxidation activity of MDH should
be retained to achieve the highest methanol production [10]. This hypothesis is based
on the assumption that MDH supplies electrons to MMO. 100% MDH inhibition
would thus result in no carbon assimilation and no electron production. Thus, the
methane-to-methanol conversion ratio should be considered along with the methane
uptake rate. For example, NH 4 Cl and NaCl have high methane-to-methanol conversion rates during the initial period of the reaction, but a significant decrease in the
methane conversion rate is observed as the reaction proceeds [17, 19]. This decrease
was attributed to the decreased MMO activity and resulting lack of electrons from
MDH.
4 Application of Biocatalysts for the Production of Methanol …
(3) Carbon dioxide
High concentrations of carbon dioxide partially inhibit MDH activity. However,
the maximum productivity of methanol under carbon dioxide-inhibited conditions is
very low (3 μmol L
−1 h
−1 ) compared with other methanol synthesis methods using
methane-oxidizing bacteria [13]. However, the use of carbon dioxide as an inhibitor
has the advantage of not requiring chemical modification of methane-oxidizing bacteria or the addition of expensive external electron donors or MDH inhibitors to the
reaction mixture.
(4) Combinations of inhibitors
EDTA has not been used alone as an MDH inhibitor. EDTA binds to the
cytochrome-binding area of MDH, blocking electron transfer from MDH to
cytochrome c. In fact, the oxidation of methanol by MDH can be inhibited by 0.05–
1 mM of EDTA. However, EDTA also reduces MMO activity, probably because of
the removal of metal ions from MMO via the chelation effect of EDTA [25, 26].
The combination of EDTA and other inhibitors such as NaCl has been tested as a
method to decrease the concentration of the other inhibitor. The methanol production
observed for a combined EDTA/NaCl system exceeded that of the NaCl-only system
approximately two-fold [15, 18].
MgCl 2 also has not been used as a sole inhibitor, but has been added along with
either phosphate or NaCl [15, 17, 19]. The mechanism by which MgCl 2 suppresses
the oxidation of methanol is still unclear [109], but various studies have reported
that methanol production increases significantly when 5–20 mM MgCl 2 is added
to phosphate buffer [7, 16, 22, 24, 25]. Mg ions promote sMMO activity and the
growth of methane-oxidizing bacteria [16]. The use of MgCl 2 in conjunction with
phosphate resulted in greater productivity than the use of EDTA with phosphate,
but EDTA showed a greater inhibitory effect on MDH activity than MgCl 2 [22,
25]. The use of 400 mM phosphate and 10 mM MgCl 2 resulted in a cell density of
17.3 g L
−1 and the highest methanol concentration of 1.13 g L
−1 , although phosphate
concentrations above 100 mM have been reported to inhibit MMO activity [10, 22,
25]. These results demonstrate that higher methanol production can be achieved by
increasing the inhibitor concentration as well as the cell density [16].
It has been suggested that 50% of the methanol oxidation activity of MDH should
be retained to achieve the highest methanol production [10]. This hypothesis is based
on the assumption that MDH supplies electrons to MMO. 100% MDH inhibition
would thus result in no carbon assimilation and no electron production. Thus, the
methane-to-methanol conversion ratio should be considered along with the methane
uptake rate. For example, NH 4 Cl and NaCl have high methane-to-methanol conversion rates during the initial period of the reaction, but a significant decrease in the
methane conversion rate is observed as the reaction proceeds [17, 19]. This decrease
was attributed to the decreased MMO activity and resulting lack of electrons from
MDH.