Bioconversion of methane to methanol in a conventional membrane bioreactor
We constructed a new MBR (Fig. 1a). Its detailed structure and assembly drawing are shown in Fig. 1b and 1c, respectively. A sheet of flat membrane on a support grid separates two chambers for the gas phase and the aqueous phase. First, this reactor was used for the bioconversion of CH4 into CH3OH as a conventional MBR, in which a hydrophobic polyvinylidene difluoride (PVDF) filter was employed to efficiently transport CH4 to the bottom aqueous phase. A biofilm of M. capsulatus (Bath) cells of 12.5 mg-dry cell weight (DCW) on the PVDF filter, which was prepared by filtration, was set in the middle of the reactor, facing the aqueous phase. However, the biofilm that was immersed in the aqueous phase was detached, and the cells were resuspended in the aqueous solution that contained 10 μM cyclopropanol as an MDH inhibitor and 10 mM sodium formate as an electron donor. The released cells were circulated at 10 mL min-1 in the bottom liquid chamber that contained 10 mL of the aqueous solution (Fig. 2a). The mixed gas containing 20% (v/v) CH4 in air was continuously infused into the top gas chamber from the inlet at 3 mL min-1 of the gas flow rate without gas pressurization. The time courses of the CH4 concentration of the exhausted gas and the accumulated concentration of CH3OH in the solution container are shown in Fig. 2b. During the 6-h reaction, the concentration of CH4 at the outlet was maintained at 19.9% (v/v). By calculating the difference between CH4 concentrations at the inlet and the outlet, we estimated that the average consumption rate of CH4 was 7.1 μmol h-1. CH3OH produced by the circulating M. capsulatus cells accumulated in the liquid chamber, and its concentration gradually increased to 1 mM in 6 h. As a result, the conventional MBR without gas pressurizing had a low consumption ratio of CH4 (0.5%) and an average production rate of CH3OH (1.7 μmol h-1).
Bioconversion of methane to methanol in an inverse membrane bioreactor
To simultaneously deliver gaseous CH4 to the methanotrophic cells, supply cyclopropanol and formate from the aqueous phase, and harvest CH3OH from the aqueous phase, we developed a novel MBR, IMBR, which had the same reactor configuration as described above; however, the membrane sheet was placed in an inverse direction to that of the conventional MBRs so that the biofilm faced the gas phase (Fig. 3a). M. capsulatus (Bath) cells were immobilized on a sheet of hydrophilic glass fiber filter, which was employed to efficiently transport water and soluble chemicals through the membrane and was set so that the cells were not immersed in the aqueous solution in the liquid chamber. Thus, gaseous CH4 and O2 were directly delivered to the immobilized whole-cell catalysts in the gas phase, and the produced CH3OH was transported to the aqueous phase via the hydrophilic membrane. In contrast, chemicals in the aqueous phase, including cyclopropanol, sodium formate, and inorganic nutrients, were delivered to the biofilm from the aqueous phase via the membrane. A peristaltic pump was used for liquid circulation and to harvest CH3OH from the solution container. The water level in the solution container remained lower than the position of the membrane sheet in the IMBR, generating negative pressure in the direction from the biofilm to the liquid chamber. Thus, the biofilm was maintained in a semidry condition on the hydrophilic membrane in the gas phase.
Using this new IMBR, bioconversion of CH4 to CH3OH was performed. All the operation parameters were the same as those in the conventional MBR except the direction of the biofilm and the material of the filter. The time courses of the CH4 concentration of the exhausted gas at the outlet and the concentration of CH3OH accumulated in the solution container are shown in Fig. 3b. After infusing CH4 into the IMBR, the concentration of CH4 decreased from 20% (v/v) at the inlet to 19.6% (v/v) at the outlet. CH3OH was rapidly produced and accumulated in the aqueous phase; the concentration of accumulated CH3OH reached 2.0 mM in 2 h and then gradually increased to 3.7 mM in 6 h, which was approximately 4 times higher than that with the conventional MBR. During the 6 h operation, the average consumption rate of CH4 was 25.3 μmol h-1, which was significantly enhanced after using the inverse biofilm in the IMBR, while the overall conversion of CH4 to CH3OH was 24.4% and 23.5% in the IMBR and the conventional MBR, respectively. This indicates that even though the cell activities in these two systems are almost identical, resistance to the mass transfer of CH4 limits the rate of CH4 consumption and dominates the rate of CH3OH production. Thus, the gas-phase bioreaction in the IMBR increased the rate of CH4 consumption compared with that of the aqueous-phase bioreaction and caused an increase in the rate of CH3OH production.
Effects of the operating conditions of the IMBR on the consumption and conversion of methane
In the aforementioned result (Fig. 3b), the consumption ratio of CH4, which directly demonstrates the efficiency of the substrate utilization, was very low (only 2%). The space time, which is defined as the mean residence time of reactants in the reactor, is determined by calculating the ratio between the gas chamber volume and the volumetric flow rate of the inlet gas. In a well-mixed condition in a bioreactor, in which reactants and biocatalysts efficiently collide by agitation or bubbling for the liquid phase (such as an activated sludge process), the space time is close to the actual residence time of reactants, and therefore, a longer space time results in a higher conversion or consumption ratio. In our IMBR, although the gas phase was not agitated, the diffusion rate of gaseous reactants with a small molecular mass, such as CH4, was quick enough in the gas phase that gaseous reactants were expected to contact the biofilm efficiently. Therefore, the increase in the space time might improve the ratio of methane consumption. To confirm this, a new reactor with a gas chamber volume of 25 mL (Fig. 4a and b) was fabricated. In this reactor, the outlet was also repositioned on the opposite side of the inlet, as far away from the inlet as possible, to prevent the gas from passing through without contacting the biofilm. This IMBR with the large gas chamber was compared with the previously mentioned IMBR with the smaller gas chamber in terms of the consumption ratio of CH4. The glass fiber filter with 10 mg-DCW of immobilized M. capsulatus (Bath) cells was set between the gas and liquid chambers of these IMBRs, and the aqueous solution in the absence of cyclopropanol and sodium formate was infused into the liquid chamber and circulated at 10 mL min-1. The volumetric flow rate of the inlet gas containing 20% (v/v) CH4 was fixed at 1 mL min-1.
As shown in Fig. 4c, in the IMBR with the larger gas chamber, the consumption ratio of CH4 increased slightly over time up to 0.51% during the first 1.5 h and then remained constant afterward. When the IMBR with the smaller gas chamber was used, the consumption ratio of CH4 increased sharply over time, reaching up to 11.9% after 1 h, and then became constant at approximately 12.4%. In the gas chambers of 2.5 mL and 25 mL, the space time of the inlet gas was 2.5 min and 25 min, respectively. Although the space time in the gas chamber of 25 mL was 10 times longer than that in the gas chamber of 2.5 mL, the consumption ratio of CH4 was approximately 1/24 lower in the larger gas chamber than that in the smaller one. Therefore, other operating conditions had to be examined to improve the consumption ratio of CH4, and the IMBR with a 2.5 mL gas chamber was used in further experiments.
Next, we examined the effects of the CH4 concentration of the inlet gas on the CH4 consumption ratio and conversion of CH4 into CH3OH in the IMBR. For this purpose, M. capsulatus (Bath) cells of 12.5 mg-DCW were immobilized on the filter in the IMBR, in which the bottom liquid chamber carried 10 mL of the aqueous solution containing 10 μM cyclopropanol and 10 mM sodium formate, with circulation at 10 mL min-1. A gas containing CH4 at concentrations from 2% (v/v) to 30% (v/v) in air was continuously pumped into the gas chamber at a gas flow rate of 1 mL min-1. The consumption rate of CH4 and the production rate of CH3OH were determined by the total amounts of CH4 consumed and CH3OH produced in the first 1 h of the reaction. Thereafter, from the ratio of these two values, the conversion of CH4 into CH3OH as a percentage was calculated. As a result, the CH4 consumption rate increased from 3.1 μmol h-1 to 17.2 μmol h-1, and the consumption ratio of CH4 increased from 0.7% to 3.7% when the CH4 concentration of the inlet gas increased from 2% (v/v) to 20% (v/v), but both of the values remained constant when the CH4 concentration was higher than 20% (v/v) (Fig. 5a). The CH3OH production rate showed the same trend as the CH4 consumption rate; it increased from 1.1 μmol h-1 to 10.2 μmol h-1 on 2-20% CH4 (v/v) and remained constant at >20% CH4 (v/v). This suggested that CH3OH production was dominated by MMO activity when MDH activity was inhibited and excess NADH was supplemented by the addition of sodium formate. The conversion of CH4 into CH3OH increased from 37% to 60% when the CH4 concentration in the inlet gas increases up to 20%. Therefore, the optimal CH4 concentration was 20% under the operating conditions of the IMBR.
We also examined the effect of the mass of immobilized M. capsulatus (Bath) cells on the conversion of CH4 into CH3OH in the IMBR. For this purpose, M. capsulatus (Bath) cells from 6.25 mg-DCW to 100 mg-DCW were immobilized on the filter in the IMBR, which was run in the same manner as above except that the CH4 concentration in the inlet gas was fixed at 20% (v/v). The CH4 consumption rate and consumption ratio increased from 6.6 μmol h-1 to 22.7 μmol h-1 and from 1.4% to 4.9%, respectively, as the mass of immobilized cells increased from 6.25 mg-DCW to 50 mg-DCW (Fig. 5b). On the other hand, the CH3OH production rate increased from 3.8 μmol h-1 at 6.25 mg-DCW to 10.1 μmol h-1 at 12.5 mg-DCW but decreased to 7.0 μmol h-1 at 25 mg-DCW and to 6.1 μmol h-1 at 50 mg-DCW. Consequently, the conversion from CH4 to CH3OH was approximately 60% when the mass of immobilized cells increased from 6.25 mg-DCW to 12.5 mg-DCW but decreased to 26.6% when the mass increased to 50 mg-DCW. Therefore, the mass of the cells is the optimum at 12.5 mg-DCW under the operating conditions for the IMBR.
Moreover, we investigated the effect of the concentration of cyclopropanol in the aqueous solution on the ratio of CH4 consumption and conversion of CH4 into CH3OH in the IMBR. M. capsulatus (Bath) cells at 12.5 mg-DCW were immobilized on the filter in the IMBR. The inlet gas containing 20% (v/v) CH4 in air was pumped at 1 mL min-1 into the reactor. The aqueous solution was infused into the bottom chamber and circulated at 10 mL min-1. The solution was exchanged successively every 3 h by the solution containing the same concentration of sodium formate at 10 mM but different concentrations of cyclopropanol at 0 mM, 1 mM and 10 mM. Figure 6a shows the consumption ratio of CH4 and the conversion of CH4 into CH3OH in three periods with three different concentrations of cyclopropanol. In the first 3 h of operation, CH3OH was not produced because of the absence of cyclopropanol, and the consumption ratio of CH4 remained at approximately 11.2 ± 0.5%. During the period with 1 mM cyclopropanol, a small amount of CH3OH was produced. However, the conversion of CH4 into CH3OH was not sustained and decreased from 4.6% to 1.6%. The consumption ratio of CH4 slightly decreased to 10.2 ± 0.6% in this period. At 6 h, the solution was exchanged again, and the concentration of cyclopropanol was increased to 10 mM. During this period, although the consumption ratio of CH4 greatly decreased to 3.7 ± 0.2%, CH3OH was significantly accumulated, and the conversion was initially approximately 60% at 7 h and then decreased to 21% at 9 h. With the same conditions as above except for the aqueous phase, which contained 20 mM cyclopropanol, its effects on the CH4 consumption ratio and conversion of CH4 into CH3OH are shown in Fig. 6b. A higher concentration of cyclopropanol was expected to greatly inhibit MDH activity to produce more CH3OH. However, the consumption ratio of CH4 further decreased to 2.9 ± 0.2%, while the maximum conversion decreased to approximately 20% by the increase in the cyclopropanol concentration. Thus, the optimal cyclopropanol concentration was 10 mM under the operating conditions of the IMBR. However, at all the cyclopropanol concentrations tested, the conversion of CH4 into CH3OH decreased with time, suggesting that the continuous supply of cyclopropanol is necessary to maintain the conversion.
Continuous bioconversion of methane into methanol in an IMBR
Finally, we carried out the continuous production of CH3OH from CH4 in a gas phase using the IMBR in the operating conditions that were optimized above (Fig. 7a). A mixed gas containing 20% (v/v) CH4 and air was continuously supplied into a 2.5-mL gas chamber of the IMBR, at which the glass fiber filter with immobilized M. capsulatus (Bath) cells of 12.5 mg-DCW was set up, and the same volume of supplied gas was exhausted from the gas chamber. A total volume of 10 mL of the medium containing 10 μM cyclopropanol and 10 mM sodium formate, as well as 9.9 mM nitrate as a nitrogen source, was circulated through a 2.5-mL liquid chamber of the IMBR at 10 mL min-1 using a peristaltic pump. To replenish formate and other medium components that were consumed and to supply active cyclopropanol, in this experiment, the fresh medium was continuously injected into the liquid chamber at 4 mL h-1 of the flow rate using a syringe pump, and the same volumetric flow of the liquid as that injected was discharged into a solution container. The space velocity for the fresh medium was 1.6 h-1.
First, the gas flow rate was varied from 0.2 to 2.5 mL min-1 to investigate its effect on the reaction. Figure 7b shows the effect of the gas flow rate on the CH4 consumption rate and the consumption ratio of CH4 in the initial 1 h of the reaction. The CH4 consumption rate in the IMBR remained constant at approximately 23 μmol h-1 when the gas flow rate was in the range from 0.2 to 1 mL min-1, while the consumption ratio of CH4 decreased from 24.2% to 5.1% in this range. On the other hand, the CH4 consumption rate decreased from 23 μmol h-1 to 8.6 μmol h-1 when the gas flow rate increased from 1 to 2.5 mL min-1, and the consumption ratio of CH4 further decreased to 0.7%. This suggests that the space time of CH4 in the IMBR is too short to maintain the same CH4 consumption rate as that at 1 mL min-1 when the gas flow rate is above 1.5 mL min-1. Then, we tried to run the IMBR by flowing the gas at 0.2 mL min-1 in its gas chamber for more than 3 d for the continuous bioconversion of CH4 into CH3OH by a microbial gas-phase reaction.
The time courses of CH4 concentration in a gas exhausted from the gas chamber and CH3OH concentration in a liquid discharged from the liquid chamber are shown in Fig. 7c. The CH4 consumption ratio and conversion of CH4 into CH3OH over time are shown in Fig. 7d. The CH4 concentration in the exhausted gas decreased from 20.0% (v/v) to 15.3% (v/v) in the first 4 h of the reaction, increased slightly to 16.9% (v/v) in the next 36 h, and thereafter remained at approximately 16.7% (v/v) in the steady state. In the 3-d operation, the consumption ratio of CH4 decreased from 23.4% to 17.6%. The variation in the CH3OH concentration of the discharged solution showed a trend in response to that of the exhausted CH4 concentration, although the response was slightly delayed. The produced CH3OH initially increased and reached 1.4 mM in 15 h. Then, it slowly decreased along with the drop in the consumption rate of CH4 and then remained constant at 1.0 mM after 40 h to the end of the experiment. During the continuous operation of the reactor, the production rate of CH3OH reached a maximum of 7.1 μmol h-1 at 15 h and a constant of 4.4 ± 0.2 μmol h-1 in the steady state. The average conversion in the steady state was approximately 27%. CH3OH productivity in the steady state was 0.88 mmol L-1 h-1 in the proposed IMBR.