Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Biological Methanol Production by a Type II Methanotroph Methylocystis bryophila

  • Patel, Sanjay K.S. (Institute of SK-KU Biomaterials) ;
  • Mardina, Primata (Department of Chemical Engineering, Konkuk University) ;
  • Kim, Sang-Yong (BioNgene Co., Ltd) ;
  • Lee, Jung-Kul (Department of Chemical Engineering, Konkuk University) ;
  • Kim, In-Won (Institute of SK-KU Biomaterials)
  • Received : 2016.01.08
  • Accepted : 2016.01.31
  • Published : 2016.04.28
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Abstract

Methane (CH4) is the most abundant component in natural gas. To reduce its harmful environmental effect as a greenhouse gas, CH4 can be utilized as a low-cost feed for the synthesis of methanol by methanotrophs. In this study, several methanotrophs were examined for their ability to produce methanol from CH4; including Methylocella silvestris, Methylocystis bryophila, Methyloferula stellata, and Methylomonas methanica. Among these methanotrophs, M. bryophila exhibited the highest methanol production. The optimum process parameters aided in significant enhancement of methanol production up to 4.63 mM. Maximum methanol production was observed at pH 6.8, 30℃, 175 rpm, 100 mM phosphate buffer, 50 mM MgCl2 as a methanol dehydrogenase inhibitor, 50% CH4 concentration, 24 h of incubation, and 9 mg of dry cell mass ml-1 inoculum load, respectively. Optimization of the process parameters, screening of methanol dehydrogenase inhibitors, and supplementation with formate resulted in significant improvements in methanol production using M. bryophila. This report suggests, for the first time, the potential of using M. bryophila for industrial methanol production from CH4.

Keywords

Introduction

Methane (CH4) is currently receiving great attention owing to its severe impact on the environment as a greenhouse gas (GHG). CH4 is a primary component of biogas (produced as a result of anaerobic digestion), including natural gas, shale gas, and landfill gases [1,4,6,8]. Therefore, there is an urgent need to reduce the harmful environmental effects due to the release of CH4. It is possible to capture CH4 and transform it into useful products for sustainable development. Recent studies have suggested that biogas could be a potential feed for the synthesis of biofuels and industrially relevant organic chemicals [4,35,42]. Thus, the adoption of various alternative processes is encouraged to permit efficient utilization of CH4 for the synthesis of valuable products, such as methanol. Compared with CH4, methanol can be stored and transported more safely [6]. Methanol can be synthesized industrially from CH4 via traditional chemical engineering approaches, or through the judicious use of microorganisms [34,39]. Biological conversion of methanol from CH4 has many industrial advantages over the traditional methods, including lower energy consumption, higher conversion efficiency, higher selectivity, and lower equipment costs [6,24,28,37].

Biotransformation of CH4 into methanol is carried out naturally by a group of microorganisms known as methanotrophs. Methanotrophs belong to the Proteobacteria group of prokaryotes. They can be found in diverse habitats, including soils, peat bogs, wetlands, sediments, lakes, fresh waters, and marine waters [1,6,23,37]. Methanotrophs play a key role in the natural carbon cycle and in the metabolism of CH4 for the synthesis of biomass, releasing carbon dioxide (CO2) as a final oxidation product [4,23]. Methane monooxygenase (MMO) is the enzyme involved in the single-step conversion of CH4 into methanol. Subsequently, methanol is metabolized to formaldehyde by methanol dehydrogenase (MDH), which is converted in turn to formate by formaldehyde dehydrogenase. Finally, formate is metabolized by formate dehydrogenase, releasing CO2.

Methanotrophs can possess two types of MMOs: particulate (pMMO: associated with membranes) and soluble (sMMO). Methanotrophs are broadly classified into three types (I, II, and X) on the basis of the type of MMOs they contain. Type I methanotrophs produce only pMMO. They assimilate CH4 through the ribulose monophosphate cycle, and include Methylobacter, Methylomicrobium, Methylocaldum, Methylococcus, Methyloglobulus, Methylohalobius, Methylomarinum, Methylomonas, Methylosphaera, Methylosoma, Methylosarcina, Methylothermus, and Methylovolum. Type II methanotrophs produce both pMMO and sMMO. They can assimilate CH4 through the serine cycle, and include Methylosinus, Methylocapsa, Methylocella, Methylocystis, and Methyloferula. Type X methanotrophs possess certain properties of both Types I and II. Methylococcus capsulatus is an example of a Type X methantroph [1,4,8,36].

Previous studies have examined the bioconversion of CH4 to methanol in certain methanotrophic strains, including Methylocaldum sp. [35], Methylococcus capsulatus [7], Methylosinus trichosporium [9,21,22,41], and Methylosinus sporium [33,42]. In addition, various MDH inhibitors such as phosphate buffer, ethylenediaminetetraacetic acid (EDTA), sodium chloride (NaCl), and ammonium chloride (NH4Cl) have been used to enhance methanol production. Methanol accumulates upon inhibition of its metabolism by MDH [3,7]. In light of these studies, we sought to explore the feasibility and efficiency of methanol production using a range of different methanotrophs. In this study, we screened one Type I and three Type II methanotrophic species. The Type I species we tested was Methylomonas methanica. The Type II species tested were Methylocella silvestris, Methylocystis bryophila, and Methyloferula stellata. Optimization of the process parameters, screening of MDH inhibitors, and supplementation with formate resulted in significant improvements in methanol production using M. bryophila. Our results suggest that M. bryophila, a Type II methanotroph, may have potential to be developed for industrial methanol production.

 

Materials and Methods

Bacterial Strains and Growth Conditions

Methylocella silvestris DSM 15510, Methylocystis bryophila DSM 21852, Methyloferula stellata DSM 22108, and Methylomonas methanica DSM 25384 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Cells were grown on nitrate mineral salt (NMS) medium containing 0.26 g/l KH2PO4, 0.716 g/l Na2HPO4·12H2O, 1.0 g/l KNO3, 0.20 g/l CaCl2, 1.0 g/l MgSO4·7H2O, 0.38 g/l Fe-EDTA, and 0.026 g/l Na2MO4·2H2O. Trace element solution (1 ml) was added to the medium. The trace element solution contained 0.4 g/l ZnSO4·7H2O, 0.015 g/l H3BO3, 0.05 g/l CoCl2·6H2O, 0.25 g/l Na2-EDTA, 0.02 g/l MnCl2·4H2O, and 0.01 g/l NiCl2·6H2O. The pH of the medium was adjusted to 7.0 using 1 M H2SO4 and 1 M NaOH. Millipore water (18 MΩ) was used in all the reagent preparations and for each measurement. All chemical reagents were of analytical grade and were purchased from Sigma-Aldrich (USA). Pure CH4 (99.995%) was purchased (NK Co., Busan, Korea). Cells were cultivated in a 1 L flask (Duran-Schott, Germany) with an air-tight screw cap (Suba seal) containing 200 ml of NMS under an atmosphere containing 20% of CH4. Cultures were incubated at 30℃ on a rotary shaker (Lab Champion IS-971R, USA) at 200 rpm for 5 days. During cultivation, CH4 was added every alternate day, to maintain its concentration at 20%. Cell growth was measured by determining the optical density (OD) at 595 nm with a UV/Vis spectrophotometer (Jenway Scientific, UK) [2,13]. At the completion of incubation, cells were harvested by centrifugation (Gyrozen 1580 MGR, South Korea) at 11,200 ×g for 15 min at 4℃ [5,16,25], and then washed twice with sodium phosphate buffer (20 mM, pH 7.0). Harvested cells were stored at 4℃ until use [15]. The dry cell mass (DCM) was calculated after incubation for 48 h at 70℃. The specific growth rate (μ) of M. bryophila was determined using the method described previously [35]. Strains were maintained by subculturing them every 3-4 weeks, and they were stored at 4℃ on NMS agar plates.

Methanol Production

The experiments assessing methanol production were carried out using 120 ml serum bottles (Sigma-Aldrich, USA) containing a total of 20 ml of the reaction volume, under batch culture conditions. The reaction mixture contained 10 μM of iron (II) sulfate (Fe2+) solution, 5 μM of copper (II) sulfate (Cu2+) solution, and free cells from 3 mg of DCM ml-1 as an inoculums in sodium phosphate buffer. Pure CH4 was added to the head space volume until it completely displaced the air. The reaction mixture was incubated for 120 h at 30℃, with shaking at 150 rpm. All the values presented here are based on three different experiments. All our data have been subjected to standard deviation analysis.

Assessing the Effect of the Copper Ions on Growth and Methanol Production

To assess the effect of Cu2+ metal ion concentrations on growth and methanol production, cells batches were initially cultured in growth media (NMS) containing copper ions in the range of 1-10 μM. Subsequently, the respective batches of cells were placed in 20 mM phosphate buffer (pH 7.0) and used in the methanol production experiments. In this set of copper-dependent experiments, only a portion of the headspace air volume (30%) was replaced with p u re CH4. These cultures were incubated for 24 h at 30℃ with an agitation rate of 150 rpm.

Optimization of Process Parameters

Methanol production was evaluated using buffered solutions covering a range of pH values (5.0-8.5). The reaction mixture was incubated for 24 h at 150 rpm, with 30% CH4 in the headspace as a feed. To evaluate the effect of incubation temperature and agitation rate on methanol production, the reaction mixture was incubated at optimal pH 6.8 for 24 h at different temperatures (25℃–40℃) and at different agitation rates (50–250 rpm).

Assessing the Effect of MDH Inhibitors and Na-Formate on Methanol Production

Phosphate buffer has been reported to inhibit MDH, leading to an accumulation of methanol. Initially, the effects of phosphate buffers (concentrations ranging 25–125 mM, pH 6.8) were tested under optimum conditions. For further enhancement of methanol production, incubations of 24 h using 30% CH4 were performed that screened other MDH inhibitors, in combination with phosphate buffer (100 mM, pH 6.8). These other chemicals that we tested were MgCl2 (5–75 mM), NaCl (10–100 mM), NH4Cl (5–75 mM), and EDTA (0.05–1.5 mM). A possible supportive role of Na-formate (concentration range 25–150 mM) for cofactor (NADH) regeneration during methanol production was also evaluated.

MDH Activity Measurements

MDH activity was measured spectrophotometrically by monitoring the change in absorbance at 600 nm induced by phenazine methosulfate-mediated reduction of 2,6-dichlorophenol-indophenol (DCPIP) [14]. The assay was performed in a 1 ml reaction volume containing CaCl2 (10 mM), NH4Cl (45 mM), phosphate buffer (0.3 M, pH 7.5), the whole cell supernatant (5 mg of DCM), DCPIP (0.13 μM), and phenazine methosulfate (3.3 μM).

Effect of Methane Concentration and the Inoculum Load on Methanol Production

The methanol production profile was evaluated at different concentrations of CH4 (10–50% in the headspace) for up to 120 h of incubation under optimum conditions (pH 6.8, 30℃, and 175 rpm). The effect of altering the inoculum load was investigated by adding DCM in the ranges of 1.5–9.0 mg/ml to the reaction mixture under optimum conditions, containing 30% of CH4 as a feed.

Analytical Methods

Methanol concentration was analyzed using an alcohol oxidase assay (Sigma-Aldrich), instead of the KMnO4 method described previously [40]. Methanol concentration was also analyzed using a gas chromatography (GC) system (Agilent 7890A) equipped with an HP-5 column (Agilent 19091J-413) connected to an FID detector. Helium was used as a carrier gas along with H2 at a makeup flow of 25 ml/min and air (300 ml/min). The oven temperature was initially maintained at 35℃ for 5 min. Following this, the temperature was raised at the rate of 5℃/min to 150℃, and subsequently at a rate of 20℃/min to 250℃. Injector and detector temperatures were set at 220℃ and 250℃, respectively.

 

Results

Screening of Methanotrophs for Methanol Production

One Type I methanotroph (Methylomonas methanica) and three Type II methanotrophs (Methylocella silvestris, Methylocystis bryophila, and Methyloferula stellata) were screened for their methanol production potential in NMS medium. Methanol production was observed in the range of 0.01-1.00 mM (Table S1). Among these strains, M. bryophila gave the highest methanol production, so it was selected for further production optimization.

Influence of Copper Ions on Growth and Methanol Production

The growth of M. bryophila was significantly influenced by the presence of Cu2+ metal ions, in the concentration range of 1-10 μM in the NMS growth media (Table 1). Growth rates (μ) between 0.021 and 0.031 h-1 were observed. When Cu2+ was excluded from the medium as a control, a rather low μ value of 0.017 h-1 was obtained, and the methanol production reached 1.00 mM. On the other hand, methanol production was always higher in the presence of Cu2+. Both maximum μ (0.31 h-1) and methanol production (1.18 mM) were observed at 2.5 μM of Cu2+.

Table 1.Effect of copper ions on methanol production and growth of M. bryophila.

Effect of Process Parameters on Methanol Production

The process parameters (pH, incubation temperature, and agitation rate) were optimized for methanol production (Fig. 1). At different pH values, the methanol production was observed in the range of 0.29-1.22 mM (Fig. 1A). In the pH range between 6.5 and 8.5, methanol production was steadily in the range of 1.01-1.22 mM, whereas acidic pH values of 5.0, 5.5, and 6.0 resulted in lower methanol production. The optimum temperature for methanol production was 30℃, producing a maximum concentration of 1.22 mM (Fig. 1B). As agitation rate increased from 50 rpm to 175 rpm, an increase in methanol production was observed (0.68 mM to 1.34 mM, Fig. 1C). At higher agitation rates up to 250 rpm, methanol production was quite stable, with a value of 1.27 mM. Overall, the maximum methanol production (1.34 mM) was observed at pH 6.8 after 24 h incubation at 30℃ and 175 rpm.

Fig. 1.Effect of process parameters (pH (A), temperature (B), and agitation rate (C)) on methanol production by M. bryophila. Under batch culture experiments, methanol production was performed in a serum bottle (120 ml) with a total working volume of 20 ml, containing 30% CH4 in the headspace as feed. The reaction mixture consisted of Cu2+ (5 μM), Fe2+ (10 μM), and 3 mg of DCM ml-1 (inoculum) in phosphate buffer (20 mM), incubated for 24 h. Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

Effect of MDH Inhibitors

Inhibition of MDH is a primary route to reduce the further metabolism of methanol. Initially, the effect of phosphate buffer concentration was evaluated at pH 6.8 (Table 2). As the phosphate buffer concentration increased, up to 100 mM, a significant improvement in methanol production was observed, from 1.34 to 1.88 mM. MDH activity assays demonstrated that this enhanced production in the presence of 100 mM phosphate buffer was directly related to the relative inhibition of MDH activity by 19.2% (80.8% activity remained). Furthermore, an increase in the buffer concentration to 125 mM resulted in higher relative MDH inhibition of 21.9%, but a lower methanol production of 1.76 mM. Thereafter, other MDH inhibitors were screened to enhance methanol production, in combination with phosphate buffer 100 mM (Table S2). The maximum methanol production of 2.21, 2.05, 1.96, and 2.04 mM was observed at optimum inhibitor concentration of 50 mM MgCl2, 50 mM NaCl, 10 mM NH4Cl, and 0.1 mM EDTA, respectively. The optimum concentration of these inhibitors, in combination with phosphate buffer (100 mM), resulted in higher MDH inhibition of 34.2%, 31.8%, 26.2%, and 48.4%, respectively (Fig. 2). Overall, these other MDH inhibitors, in combination with phosphate buffer, greatly enhanced methanol production.

Table 2.aMDH activity in 20 mM phosphate buffer was considered as 100%, with methanol production of 1.22 ± 0.09 mM.

Fig. 2.Relative MDH inhibition (%) by different inhibitors in phosphate buffer. The 100% activity was defined as the initial MDH activity in 20 mM phosphate buffer (pH 6.8). The relative decrease in MDH activity was measured at optimum inhibitor concentrations of MgCl2 (50 mM), NaCl (50 mM), NH4Cl (10 mM), and EDTA (0.1 mM), in combination with 100 mM of phosphate buffer. Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

Effect of Na-Formate

Supplementation of the production media with Na-formate resulted in an enhancement of methanol production (Fig. 3). An increase in methanol production from 2.21 to 4.15 mM was obtained by increasing the Na-formate concentration to 100 mM, which was the optimal concentration for methanol production. Further increase of Na-formate concentration to 150 mM resulted in lower methanol production (3.86 mM).

Fig. 3.Effect of Na-formate on methanol production. Methanol production was evaluated at different Na-formate concentrations in the range of 20-120 mM, in phosphate buffer (100 mM, pH 6.8) containing MgCl2 (50 mM) as an MDH inhibitor and 30% of CH4 as a feed, incubated for 24 h. Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

Effect of Methane Concentration

By altering the feed concentration of CH4 in the headspace of the culture vessel, significant enhancements in methanol production could be produced. These enhancements were only seen during the first 6 to 24 h of the incubation period. Methanol production decreased thereafter, up to the 120 h limit of our incubation periods (Fig. 4). Methanol production increased with increasing feed concentrations of CH4, from 10% to 50%. The greatest proportionate increase in methanol production occurred when CH4 was increased from 10% to 30% under optimum conditions; the production increased from 2.22mM to 4.15mM. Further increasing the CH4 concentration to 50% only resulted in a slightly higher methanol production of 4.24 mM.

Fig. 4.Effect of CH4 concentration on methanol production. Methanol production was performed in phosphate buffer (100 mM, pH 6.8) containing MgCl2 (50 mM) as an MDH inhibitor, and incubated up to 120 h in the presence of different feed CH4 concentrations: ● (10%), ○ (20%), ▼ (30%), and △ (50%). Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

Effect of Inoculum Load

The effect on methanol production of different M. bryophila inoculum loads was evaluated (Fig. 5). An increase in the inoculum loading from 1.5 to 9.0 mg of DCM ml-1 increased the methanol production from 3.04 mM to 4.53 mM. Here, higher inoculum loads (6 and 9 mg of DCM ml-1) resulted in a more rapid methanol production, compared with lower inoculum loads (1.5 and 3.0 mg of DCM ml-1).

Fig. 5.Effect of inoculum on methanol production. Methanol production was performed in phosphate buffer (100 mM, pH 6.8) containing MgCl2 (50 mM) as an MDH inhibitor, incubated up to 48 h with different inoculum loads (mg of DCM ml-1): ● (1.5), ○ (3.0), ▼ (6.0), and △ (9.0). Each value represents the mean of triplicate measurements that varied from the mean by no more than 10%.

 

Discussion

The suitability of various alternative energy sources, such as alcohols, hydrogen gas, and CH4, has been widely evaluated [3,10-12,17,18,20,26,27,29,30,32,34,43]. Unlike hydrogen gas, CH4 has received significant attention owing to its harmful environmental effects as a GHG. To minimize the adverse impact of CH4, it has been suggested that it can be diverted for the production of methanol, which is a starting material for the synthesis of valuable organic compounds. Methanotrophs can use CH4 to produce methanol efficiently [4,34,37]. In spite of this, only a handful of methanotrophs, such as Methylosinus trichosporium and Methylosinus sporium strains, have been characterized and shown experimentally to be able to produce methanol from CH4 [9,33,42]. In this study, we evaluated methanol production by both Type I and Type II methanotrophs. We tested the Type I methantroph Methylomonas methanica, and the Type II methanotrophs Methylocella silvestris, Methylocystis bryophila, and Methyloferula stellata. Among these strains, M. bryophila produced the most methanol. To the best of our knowledge, ours is the first study to demonstrate the production of methanol from CH4 using M. bryophila.

The growth of methanotrophs is highly influenced by metal ions such as Cu2+ in the NMS growth medium [22,38]. The maximum μ of M. bryophila was enhanced about 1.8-fold in the presence of 2.5 μM Cu2+, compared with a μ of 0.017 h-1 in the absence of any Cu2+. During methanol production, process parameters such as pH, temperature, and agitation rate were critical for the well-studied strains M. tricosporium [3,9] and Methylosinus sporium [33,42]. The optimum process parameters, resulting in maximum methanol production, for M. bryophila were observed at pH 6.8, 30℃, and 175 rpm. Inhibition of MDH can enhance methanol production by reducing methanol consumption by the CH4 metabolic pathway. A 1.4-fold increase in methanol production was observed at a concentration of 100 mM of phosphate buffer, compared with the control (phosphate buffer, 20 mM). Here, phosphate buffer (100 mM) resulted in a higher MDH inhibition of 19.2%, compared with the 9.3% inhibition reported for Methylosinus trichosporium OB3b [9]. Supplementation of another MDH inhibitor, MgCl2 (50 mM), in combination with phosphate buffer (100 mM) gave a maximum methanol production of 2.21 mM. Interestingly, greater inhibition of MDH activity did not result in higher methanol production. Similar results were reported for pure Methylosinus trichosporium OB3b [9], and mixed cultures of Methylosinus sporium NCIMB 11126, Methylosinus trichosporium OB3b, and Methylococcus capsulatus Bath [7]. Here, higher MDH inhibition might be not suitable, due to the requirement for cofactor regeneration during higher MMO activity [21]. On the other hand, a significant enhancement (1.9-fold) in methanol production was observed by M. bryophila when 100 mM Na-formate was supplied in the production mixture, using 30% of CH4 as feed. According to previous studies, the enhanced production seen upon addition of Na-formate in the Type II methanotroph Methylosinus trichosporium NCIB1113 is primarily due to the high regeneration of the NADH cofactor [19,21]. We speculate that a similar mechanism is at work for M. bryophila in our study.

In previous studies, differing feed CH4 concentrations resulted in variable methanol yields, depending on the type of methanotrophic strains, and on optimum production conditions [3,28,31,33]. Here, M. bryophila produced methanol efficiently during an incubation period of 24 h, in contrast to the longer incubations times of 27, 40, and 48 h reported for Methylosinus sporium KCTC 22312 [42], Methylosinus trichosporium OB3b [3], and Methylocaldum sp. 14B [35], respectively. The decreasing methanol production in M. bryophila thereafter, up to 120 h of incubation, might be due to the incomplete inhibition of MDH activity, which allowed further metabolism of methanol. The effect of inoculum loads on methanol production was quite variable in Methylosinus sporium and Methylosinus trichosporium strains [3,33]. A significant increase in methanol production (1.5-fold) was observed in M. bryophila upon increasing the cell biomass in the inoculum loads from 1.5 to 9.0 mg of DCM ml-1. Inoculum loads needed for enhanced methanol production were reported as being much higher (105 mg of DCM ml-1) for Methylosinus sporium B2121, with 0.35 mM of maximum methanol production [31]. Here, M. bryophila had almost a 13-fold higher methanol production than Methylosinus sporium B2121, and did so at much lower inoculum of 9.0 mg of DCM mL-1 using pure CH4. Moreover, our work has resulted in much higher methanol production than the maximum production of 0.71 mM and 0.02 mM reported for Methylosinus sporium KCTC 22312 [42] and Methylosinus trichosporium IMV 3011 [41] grown on synthetic biogas feed.

In conclusion, biological methanol production by methanotrophs seems a suitable and environmentally friendly approach to reduce GHGs such as CH4. M. bryophila, a Type II methanotroph, produced methanol in much higher concentration than other previous reports, for example using Methylosinus sporium strains and Methylosinus trichosporium IMV 3011. This is the first report of methanol production from CH4 using M. bryophila. Our results demonstrate that M. bryophila can be used as a potential methanol producer. Furthermore, methanol production using biogas, originating from anaerobic digestion, may be a promising low-cost approach.

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