4.2 Methane Metabolism in Methane-Oxidizing Bacteria
85
three types of methane-oxidizing bacteria, while the growth rates of Type III methaneoxidizing bacteria were much slower rate than those of the other two types. However,
the growth rates of methanotrophs are much slower than that of industrially utilized
Escherichia coli (~1.3 h
−1 [97]), and slightly slower than that of Saccharomyces
cerevisiae (~0.40 h
−1 ). One of the major reasons for the low growth rate is the
agitation of culture medium that is required to overcome the low solubility (~1 mM
at 30 °C and 1 atm) [98] and very low transfer rate [99] of methane gas to the water
phase.
Achieving high-cell-density cultivation of methane-oxidizing bacteria is expected
to require even more technical improvements than improving their growth rate. Pressurized bioreactors have been used to improve the solubility of methane and its delivery to bacteria, and high biomass densities in the range of 18–65 g L
−1 have been
achieved. However, high-pressure bioreactors consume more energy than cultivation
under atmospheric pressure [105–107]. Alternatively, two-phase partitioned bioreactors using a non-aqueous phase that has a higher affinity for methane than water are
being tested. For example, the addition of 10% v/v silicone oil to the culture medium
led to a 3.3-fold increase in the growth rate of the strain Methylosinus sporium DSMZ
17706 [108]. Using the same concept, Methylosinus trichosporium OB3b was cultured in a medium containing 5% v/v paraffin oil and achieved a biomass density
of 14 g L
−1 after 240 h of cultivation [109]. However, much higher bacterial cell
density is achieved in industry. For example, S. cerevisiae and E. coli have shown
biomass densities of up to 170 g L
−1 and 190 g L
−1 , respectively [110, 111].
In addition to the growth rate and yield, the quality of the biocatalyst is also
important. The content of MMO in methane-oxidizing bacteria determines its quality as a biocatalyst. The content of pMMO can be controlled by the copper concentration in the culture medium, with increasing copper concentration leading to
increased pMMO content. However, the growth rate of methane-oxidizing bacteria
decreases when the copper concentration is increased above 10–20 μM, and copper ions become toxic to bacterial cells at concentrations higher than 50 μM [112,
113]. Chan and co-workers developed a method to produce high-quality methaneoxidizing bacteria using a fermenter adapted with a hollow-fiber bioreactor. The spent
media was filtered through the hollow-fiber membrane, allowing cell waste and toxic
metabolites to be discarded from the reactor, while the same time the reactor was
simultaneously replenished with fresh cell media. Thereby, the copper concentration
of the culture solution could be controlled to avoid toxicity to the cells. Using this
method, M. capsulatus Bath cells with high pMMO-expression (80% of the total
cytoplasmic membrane) were obtained [114].
85
three types of methane-oxidizing bacteria, while the growth rates of Type III methaneoxidizing bacteria were much slower rate than those of the other two types. However,
the growth rates of methanotrophs are much slower than that of industrially utilized
Escherichia coli (~1.3 h
−1 [97]), and slightly slower than that of Saccharomyces
cerevisiae (~0.40 h
−1 ). One of the major reasons for the low growth rate is the
agitation of culture medium that is required to overcome the low solubility (~1 mM
at 30 °C and 1 atm) [98] and very low transfer rate [99] of methane gas to the water
phase.
Achieving high-cell-density cultivation of methane-oxidizing bacteria is expected
to require even more technical improvements than improving their growth rate. Pressurized bioreactors have been used to improve the solubility of methane and its delivery to bacteria, and high biomass densities in the range of 18–65 g L
−1 have been
achieved. However, high-pressure bioreactors consume more energy than cultivation
under atmospheric pressure [105–107]. Alternatively, two-phase partitioned bioreactors using a non-aqueous phase that has a higher affinity for methane than water are
being tested. For example, the addition of 10% v/v silicone oil to the culture medium
led to a 3.3-fold increase in the growth rate of the strain Methylosinus sporium DSMZ
17706 [108]. Using the same concept, Methylosinus trichosporium OB3b was cultured in a medium containing 5% v/v paraffin oil and achieved a biomass density
of 14 g L
−1 after 240 h of cultivation [109]. However, much higher bacterial cell
density is achieved in industry. For example, S. cerevisiae and E. coli have shown
biomass densities of up to 170 g L
−1 and 190 g L
−1 , respectively [110, 111].
In addition to the growth rate and yield, the quality of the biocatalyst is also
important. The content of MMO in methane-oxidizing bacteria determines its quality as a biocatalyst. The content of pMMO can be controlled by the copper concentration in the culture medium, with increasing copper concentration leading to
increased pMMO content. However, the growth rate of methane-oxidizing bacteria
decreases when the copper concentration is increased above 10–20 μM, and copper ions become toxic to bacterial cells at concentrations higher than 50 μM [112,
113]. Chan and co-workers developed a method to produce high-quality methaneoxidizing bacteria using a fermenter adapted with a hollow-fiber bioreactor. The spent
media was filtered through the hollow-fiber membrane, allowing cell waste and toxic
metabolites to be discarded from the reactor, while the same time the reactor was
simultaneously replenished with fresh cell media. Thereby, the copper concentration
of the culture solution could be controlled to avoid toxicity to the cells. Using this
method, M. capsulatus Bath cells with high pMMO-expression (80% of the total
cytoplasmic membrane) were obtained [114].