Hierarchical Macroporous Particles for Efficient Whole-Cell

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Biological and Medical Applications of Materials and Interfaces

Hierarchical Macro-porous Particles for Efficient Whole-Cell Immobilization: Application in Bioconversion of Greenhouse Gases to Methanol Sanjay Kumar Singh Patel, Min Soo Jeon, Rahul K. Gupta, Yale Jeon, Vipin Chandra Kalia, Sun Chang Kim, Byung-Kwan Cho, Dong Rip Kim, and Jung-Kul Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03420 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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ACS Applied Materials & Interfaces

Hierarchical Macro-porous Particles for Efficient Whole-Cell Immobilization: Application in Bioconversion of Greenhouse Gases to Methanol

Sanjay K. S. Patela,‡, Min Soo Jeonb,‡, Rahul K. Guptaa, Yale Jeonb, Vipin Chandra Kaliaa, Sun Chang Kimc,d,e, Byung Kwan Choc,d,e, Dong Rip Kimb,f,*, Jung-Kul Leea,*

Addresses: aDepartment of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 05029, Republic of Korea; bSchool of Mechanical Engineering, Hanyang University, Seoul 04763, Republic of Korea; cDepartment of Biological Sciences and KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea; dKAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea; eIntelligent Synthetic Biology Center, Daejeon 34141, Republic of Korea; fInstitute of Nano Science and Technology, Hanyang University, Seoul 04763, Republic of Korea ‡These authors contributed equally to this work.

*Correspondence

authors.

E-mail: [email protected] (Jung-Kul Lee, Fax: +82-2-458-3504), E-mail: [email protected] (Dong Rip Kim, Fax: +82-2-2220-2299)

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ABSTRACT A viable approach for methanol production under ambient physiological conditions is to use greenhouse gases, methane (CH4) and carbon dioxide (CO2) as feed for immobilized methanotrophs. In the present study, unique macro-porous carbon particles with pore sizes in the range of ~ 1–6 µm were synthesized and used as support for the immobilization of Methylocella tundrae. Immobilization was accomplished covalently on hierarchical macro-porous carbon particles. Maximal cell loading of covalently immobilized M. tundrae was 205 mg of dry cell mass (DCM) per g of particles. Among these particles, the cells immobilized on 3.6 µm pore size particles showed the highest reusability with the least leaching and were chosen for further study. After immobilization, M. tundrae showed up to 2.4-fold higher methanol production stability at various pH and temperature values because of higher stability and metabolic activity than free cells. After eight cycles of reuse, the immobilized cells retained 18.1-fold higher relative production stability compared to free cells. Free and immobilized cells exhibited cumulative methanol production of 5.2 and 9.5 µmol mg-DCM-1 under repeated batch conditions using simulated biogas [CH4 and CO2, 4:1 (v v-1)] as feed, respectively. The appropriate pore size of macro-porous particles favors the efficient M. tundrae immobilization in order to retain better biocatalytic properties. This is the first report concerning the covalent immobilization of methanotrophs on the newly synthesized macro-porous carbon particles and its subsequent application in repeated methanol production using simulated biogas as a feed.

Keywords: Greenhouse gas; Immobilization; Macro-porous particles; Methanol; Methylocella tundrae; Simulated biogas


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INTRODUCTION The increasing emission (331 Tg year-1) of methane (CH4) as a major greenhouse gas (GHG) is a primary concern worldwide. It is caused by a significant rise in anthropogenic activities, including those from agricultural and industrial areas.1-5 Similarly, renewable source of energy is a key interest for sustainable development.6-10 Therefore, a comprehensive strategy has been discussed for effective utilization of CH4 as feedstock to produce valuable bioproducts such as methanol, in order to minimize their harmful environmental effects.11-16 Methanol is a key primary substrate for complex chemical synthesis of many materials, including transportation fuels. It has a significantly higher density (400 times) than CH4. Compared to CH4, methanol incurs significantly lower storage and transportation costs. The conversion of CH4 to methanol is largely performed via thermochemical processes, which require costly chemical catalysts and harsh reaction conditions, including very high temperature and pressure.5,17 The biological production of methanol from CH4 was found more effective than thermochemical processes due to high efficiency and selectivity, as well as ambient reaction conditions. Methanotrophs are potential candidates to use CH4 efficiently for production of methanol by utilizing methane monooxygenase (MMO) enzymes.3,18 Methanotrophs express two types of MMOs – membrane associated particulate MMO (pMMO) and cytosolic soluble MMO (sMMO). The expression of these enzymes is largely dependent on copper (Cu) metal ions.19 At low and high Cu concentrations, the relative expressions of sMMO and pMMO are dominant, respectively. Compared to sMMO, pMMO showed a higher CH4 oxidation potential.20 Briefly, methanotrophs utilize CH4 as a carbon source, producing CO2 for growth of biomass through complex

pathways

using

MMOs,

methanol

dehydrogenase

(MDH),

dehydrogenase (FalDH) and formate dehydrogenase (FDH) as shown in Eq. 1.1,17 3 ACS Paragon Plus Environment

formaldehyde


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(1)

Methanol production is low because the MDH enzyme in the subsequent step of the metabolic pathway metabolizes it. Thus, partial inhibition of MDH with inhibitors such as ammonium chloride, cyclopropanol, magnesium chloride, phosphate buffer, and sodium chloride has been suggested to enhance methanol production, as regeneration of the co-factor (nicotinamide adenine dinucleotide) is necessary for the activity of sMMO.21,22 In contrast, no co-factor is required for the catalytic activity of pMMO to convert CH4 to methanol.1 Further, immobilized whole cells have been demonstrated to improve the properties of methanotrophs for oxidation of CH4.2,23 Various kinds of supports, including activated carbon, amberlite, building materials (bricks, aerated concrete, Maastricht and Euville limestone), ceramic balls, chitosan, diethylaminoethyl-cellulose, Duolite, and polymeric matrices have been used for immobilization of methanotrophs.9,17,21,24-27 Methanotrophs are classified into types I, II, and X based on the MMOs (pMMO and sMMO) they produce.17 Both the type of methanotroph and immobilization method have produced significantly variable results for methanol production. The low cell loading on supports as well as lower efficiency of methanol production by immobilized cells are major targets for improvement.17,23,24,28 Still, efficient whole cell immobilization is a primary limiting factor for high methanol production under repeated batch conditions. Also, the macroporous nature of the supports provides a suitable microenvironment for better CH4 oxidation, improving methanotroph stability.25 Thus, immobilization of methanotrophs on macro-porous particles will be more effective at improving both cell loading and methanol production efficiency under repeated batch conditions.


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In this study, we propose an effective methanol production platform involving whole cell reactions by employing porous carbon particles in which mass transport channels from a few nanometers through to a few microns to several tens of microns are hierarchically networked. The proposed platform benefits the effective mass transport due to the hierarchically interconnected porous structures, thereby assisting the considerable enhancement of biochemical conversion for methanol production.

RESULTS AND DISCUSSION Fabrication of Hierarchically Interconnected Porous Carbon Particles Figure 1 illustrates the hierarchically interconnected porous carbon particles that enhance whole cell immobilization and methanol production. The proposed methanol production platform has three key features. First, stacking spherical porous carbon particles provides an effective mass transport platform. While the spherical shape of individual particles has a high surface to volume ratio, the piled porous carbon particles, which have hierarchical networks from the interconnected pores inside the particles to the spaces among the particles, can facilitate the mass transport of reactive materials due to their minimized transport resistance, following Murray’s law.29-31 Second, a porous carbon particle enhances whole cell loading properties. A few hundreds of micron-sized particles, with micron-scale pores that are interconnected with each other, not only provide short travel distances for bacterial cells (here, Methylocella tundrae a type II methanotroph) to reach the central part of the particle, but also effectively maintain the enhanced densities of the loaded cells. Finally, the proposed platform is fabricated by a facile, reproducible, and scalable sphere template method.32,33 Subsequent gelation, carbonization, and pulverization can be performed over large-scale sphere templates, leading to the effective 5 ACS Paragon Plus Environment


fabrication of porous carbon particles in a high-throughput manner. The proposed platform enables the effective loading and immobilization of whole cells to produce methanol in a highly efficient and stable manner via repeated batch experiments.

Figure 1. Schematic of whole cell immobilization and the methanol production system using porous carbon particles possessing hierarchically interconnected mass transport channels. 6 ACS Paragon Plus Environment

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Characterization of Porous Carbon Particles Figure 2a shows photographs of stacked porous carbon particles. The size of individual porous carbon particle ranges from 300 µm to 600 µm (Figure 2b). When the particles are piled up, the mass transport channels have sizes of several tens of microns. In addition, the porous carbon particles have a few micron-sized pores with highly interconnected channels, as seen from the scanning electron microscopy (SEM) image in Figure 2c.

Figure 2. Representative images of porous carbon particles. (a) photograph, (b) optical microscopy, (c) scanning electron microscopy (SEM), (d) transmission electron microscopy (TEM) images.



Figure 3. Pore characteristics of synthesized macro-porous carbon (3.6 µm pore size) particles. Log differential intrusion (dV/dlogD, where V is intrusion volume and D is the diameter of pores) in terms of pore size diameters (a), and CO2 uptake performance of porous carbon particles and porous carbon scaffold (b). Considering the physical size of M. tundrae, porous carbon particles with different pore sizes were fabricated by changing the size of the polystyrene (PS) spheres in the template (Figure S1). Specifically, porous particles with pore sizes of 0.9 ± 0.1 µm, 2.0 ± 0.1 µm, 3.6 ± 0.1 µm, and 6.3 ± 0.6 µm were synthesized using different sizes of PS spheres with diameters of 1.0 ± 0.1 µm, 2.0 ± 0.1 µm, 4.2 ± 0.1 µm, and 6.6 ± 0.3 µm, respectively (Figures S2 and S3). The pore sizes of the particles are smaller than the PS sphere sizes because the spheres are slightly deformed during the heating processes of the pre-pressed template fabrication. The interconnected channels among the pores of the porous carbon particles allow whole cells to populate the inner part of the particles and facilitate effective loading and immobilization of M. 8 ACS Paragon Plus Environment

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tundrae. The porous particles are made of a carbon aerogel [resorcinol and formaldehyde (RF)gel] and consist of interconnected nano-sized pores with an average diameter of 6.34 ± 0.64 nm, as shown by transmission electron microscopy (TEM) analysis (Figure 2d). These nano-sized particle pores can benefit the whole cell immobilization and methanol production system. Nanoscale structures not only enable enhanced bacterial cell adhesion,34,35 but also pore networks with macropores and mesopores can increase outstanding mass transport properties.31,36 The presence of nano-sized pores was confirmed by measurement of the porous carbon particles using the Barrett-Joyner-Halenda (BJH) method (Figure 3a) and using nitrogen adsorption analysis, showing a sharp rise in the low-pressure range (relative pressure of 0 ~ 0.2) (Figure S4). These hierarchically sized channels form passages among the particles through the interconnected micron-sized pores to the nano-sized pores inside the particles, which can enhance methanol production efficiency by minimizing the transport resistance of reactive materials (CH4, CO2) as indicated by Murray’s law.29-31 To consolidate the structural advantages of the macro-porous particles, compared to the previously reported scaffolds, we compared the reaction gas (CO2) uptake capability of the developed macro-porous particles and previously reported scaffolds.33 The carbon scaffolds were fabricated using the same materials and pore sizes by the template method.33 A higher gas adsorption capacity indicates a better mass transport performance.37,38 The macro-porous carbon particles showed a 1.2-fold higher uptake capacity, as seen in Figure 3b; this highlights the usefulness of the developed hierarchical macro-porous particles for effective mass transport, in accordance with Murray’s law. CH4 adsorption should be similar to CO2 adsorption, as shown in previous studies.39-41



Immobilization of Whole Cells To improve the stability of whole cells during the biotransformation process, immobilization of free cells is crucial.21 The immobilization of methanotrophs on solid supports and polymeric matrices varied significantly in cell loading, as well as in methanol production.17,21,23,24,28 Thus, M. tundrae were covalently immobilized on the synthesized hierarchical macro-porous carbon particles with pore sizes from 0.9 to 6.3 µm to improve methanol production efficiency. To immobilize cells covalently on synthesized supports, the particles were functionally activated

Figure 4. Immobilization of M. tundrae whole cells on synthesized hierarchical macro-porous particles with different pore sizes: (a) time profile of cells immobilization, (b) methanol production efficiency and (c-d) loading of cells on the particles of various sizes. Significant (p ≤ 0.05) differences were observed by one-way ANOVA. 10 ACS Paragon Plus Environment

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with glutaraldehyde. The optimum glutaraldehyde concentration was determined to be 1 M for the pre-functionalization of the macro-porous particles to achieve a high immobilization yield (IY) (Figure S5). Initially, the IY of whole cells on the synthesized macro-porous carbon particles was increased by extending the incubation period up to 24 h, and thereafter stabilized with longer incubation up to 32 h (Figure 4a). A similar trend was observed for the relative efficiency (RE) of methanol production by immobilized cells under the same conditions (Figure 4b). An efficient IY and RE were observed in the ranges of 26.2–52.3% and 36.9–84.9%, respectively, after 24 h incubation (Figure 4a-b). Methanol production from immobilized M. tundrae on synthesized 0.9, 2.0, 3.6, and 6.3 µm pore size particles was 0.44, 0.58, 0.98, and 1.01 µmol mgdry cell mass (DCM)-1, respectively, while free cell production was 1.19 µmol mg-DCM-1. Interestingly, both IY (%) and RE (%) increased with increasing pore size from 0.9 to 6.3 µm. The highest RE of methanol production by M. tundrae cells immobilized on 6.3 µm pore particles was 84.9%, which was greater than that previously reported after cell encapsulation in sodium-alginate

(Na-alginate).42 After

the

covalent

immobilization

of

Methylosinus

trichosporium NCIB 11131 on a diethylaminoethyl cellulose support, the RE of methanol production by free cells was 44.4%.21 In addition, RE of methanol production by the immobilized M. tundrae cells was significantly higher than that by Methylomonas sp. Z201 immobilized on various activated carbon supports synthesized using apricot shell, coconut shell, and coal (Table S1).23 Compared to previous reports on batch methanol production using immobilized methanotrophs via various supports, M. tundrae cells immobilized on the newly synthesized macro-porous carbon particles exhibited a higher RE and methanol production ability (Table S1). Cell immobilization on macro-porous supports through the covalent method 11 ACS Paragon Plus Environment


is also influenced by non-covalent interactions such as adsorption. To confirm the contribution of cell adsorption during the covalent immobilization process, the immobilized cells on the macroporous particles were pre-treated with sodium chloride (NaCl, 1 M) at room temperature for 2 h (Figure S6). Pre-treatment of the particles with a larger pore size (6.3 μm) with NaCl resulted in higher cell leaching, which can be associated with the high adsorption of cells, compared to that in case of the particles with a smaller pore size (3.6 μm) during immobilization. Hence, leaching of the cells immobilized on the 3.6 μm pore size particles was lower than that of the cells immobilized on the 6.3 μm pore size particles, proving that these particles serve as the most suitable support for further research. In order to increase whole cell immobilization on the synthesized supports, the IY was evaluated at higher cell loads up to 2 g of DCM g-1 of support (Figure 4c). An increase for immobilized cells was observed with higher loading. Maximum IY values of 117, 163, 198, and 205 mg of DCM were observed for 0.9, 2.0, 3.6, and 6.3 µm pore size particles, respectively, by loading 2 g of DCM g-1 of support. The 3.6 and 6.3 µm pore size particles allowed higher loading of immobilized cells than that of the 0.9 and 2.0 µm pore size particles, and this may be associated with the larger pore size compared to the size of M. tundrae cells (1-3 µm) that favors more cell attachment within the particles to maintain the enhanced cell density. Further, a marginal increase in the IY and cell loading with an increase in the pore size from 3.6 to 6.3 µm can be attributed to significantly larger pore size compared to that of the cells, which can be associated with the higher leaching of the attached cells over comparable pore size to the cells for 3.6 µm pore size particles (Figure S6). Additionally, 3.6 µm pore size particles provide a larger surface area for the immobilization of cells than that provided by the 6.3 µm pore size particles. IY was significantly reduced to 5.9, 8.2, 9.9, and 10.3%, respectively, at maximum 12 ACS Paragon Plus Environment

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loading, and the RE for methanol production decreased to 8.6, 13.8, 71.2, and 62.6%, respectively with an increase in the cell load (Figure 4d). Here, the significant reduction in RE might be associated with diffusion limitations. These results suggested that pore size has a significant influence on both IY and RE of whole cells. Consequently, the macro-porous particles in this study showed a 1.9-fold higher methanol production ability than the previously reported scaffolds (Figure S7). However, the macro-porous carbon scaffold exhibited a significantly lower IY and RE of 27.1 and 43.9%, respectively (Figure S7), than the macroporous carbon particles (3.6 µm pore size). Yu et al. (1998) reported the immobilization of Methylomonas sp. Z201 on different activated carbon supports, including apricot shell, coconut shell, and coal, with significantly lower cell loads.23 In addition, the amount of whole cells loaded onto the macro-porous particles in this study was higher than that in case of the immobilization of Methylosinus sporium DSMZ 17706 on amberlite (XAD-2, XAD-4, and XAD-HP7), duolite A7, and chitosan supports through covalent binding (Table S1).17 Overall, the synthesized macro-porous particles exhibited up to a 24.1-fold higher loading amount of immobilized methanotroph cells than that obtained in case of other supports.17,23 Also, the synthesized macro- porous support was found to be more effective for high cell loading than mixed methane oxidizing bacteria on solid waste-based activated carbon, which had a maximum loading of 179 mg g-1 of support. This system was used for the utilization of CH4 exhaust gas from a biogas upgrading process.9 Therefore, these results suggest that loading and RE of immobilized cells for methanol production can be significantly improved by macro-porous carbon particles. The immobilization of M. tundrae on the hierarchically synthesized macro-porous particles was confirmed by SEM images (Figure 5). Here, the higher IY of the cells immobilized on the 13 ACS Paragon Plus Environment


macro-porous particles having pores > 3 μm is possibly because of the size of the M. tundrae cells (1–3 µm). The macropores are interconnected, providing a larger surface area for the effective internalization of the cells towards the central part of the particle in order to maintain an enhanced cell density. This is also correlated with a higher cell density in particles with larger pores. The high loading of cells 117, 163, 198, and 205 mg of DCM with the 0.9, 2.0, 3.6, and 6.3 μm pore size particles was correlated with a significant reduction of relative weight to 75.52, 71.73, 68.64, and 68.83% in comparison with weight lost to 86.42, 86.65, 87.09, and 87.33% for pure particles in thermogravimetric analysis (TGA) at 410 C (Figure S8).

Figure 5. SEM images of immobilized M. tundrae cells on the hierarchically synthesized macroporous particles: (a) 0.9, (b) 2.0, (c) 3.6, and (d) 6.3 μm pore size particles.

Methanol Production Using Methane The methanol production profile of free and immobilized cells using 30% CH4 as the feed is presented in Figure 6a. Initially, methanol production increased with an increase in the 14 ACS Paragon Plus Environment

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incubation time up to 24 h for the free cells. Thereafter, a significant reduction (41.2%) in methanol production was observed up to 144 h of incubation. The decrease in production might be directly associated with further metabolism of methanol due to methanol dehydrogenase activity in the reaction mixture, as described previously.11,42-44 In contrast, immobilized cells exhibited stable methanol production up to 144 h of incubation. The maximum methanol production by free and immobilized cells on 0.9, 2.0, 3.6, and 6.3 μm pore size particles was 1.19, 0.59, 0.81, 1.15, and 1.18 µmol mg-DCM-1, respectively. At the maximum methanol production, the leaching of immobilized cells was observed as 1.5, 1.8, 2.3, and 5.7%, respectively (Figure 6b). The greater stability of methanol production at longer incubation periods by immobilized cells over free cells might be associated with higher relative MMO activity (Figure 6c). Here, the higher methanol production by cells immobilized on 3.6 μm pore particles than that of cells immobilized on 6.3 μm pore size particles exhibited a correlation with lower leaching and higher residual MMO activity. As MDH was involved in the subsequent utilization of methanol, MDH activity was measured (Figure S9). The significant increase in MDH activity from 32.8% at 24 h to 58.9% at 144 h supports the reduction in the produced methanol. In contrast, maintenance of the methanol produced by the immobilized cells on the macro-porous particles can be correlated with the relatively lower MDH activity in the range of 27.2–31.2%. Additionally, to prove the effectiveness of 3.6 μm pore size particles as whole cell immobilization supports over 6.3 μm pore size particles, the cumulative leaching of immobilized cells was measured for five cycles (Figure S10). Under optimum batch conditions, only 4.2% cumulative leaching of the cells immobilized on 3.6 μm pore size particles was observed, suggesting this scale as a more suitable support for methanol production over that of 6.3 μm pore size particles, which showed 4.0-fold higher leaching of 16.9%. 15 ACS Paragon Plus Environment


The cofactor nicotinamide adenine dinucleotide (NAD+) is required for MMO catalysis in the conversion of CH4 to methanol. Therefore, cyclic voltammetry (CV) analysis was performed using free and immobilized cell suspensions to evaluate its ratio to the reduced form, NADH (NAD+/NADH, E0 = -0.32 V) which indicates the intracellular redox state of cells at 24 h of incubation using a three-electrode experimental setup with a potentiostat at a scanning rate of 20 mV s-1 (Figure S11).43 The overall redox state of free and immobilized cells on 0.9, 2.0, 3.6, and 6.3 μm pore size particles deviated towards higher reduction, starting at a potential of -0.20 V with a maximum current density of 5.30, 4.20, 4.53, 6.32, and 6.12 µA cm-2 at a potential of 0.5 V, respectively. Among these particles, the higher current density of cells immobilized on 3.6 μm pore size particles suggests better biocatalytic activity potential of cells, which can be correlated with MMO activity (Figures 6c and S11).


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Figure 6. Methanol production by M. tundrae immobilized on synthesized macro-porous particles: (a) profile, (b) leaching, and (c) MMO activity. CH4 (30%, v v-1) was used as a feed. Significant (p ≤ 0.05) differences were observed by one-way ANOVA. 17 ACS Paragon Plus Environment


The maximum conversion yields of CH4 to methanol by free and immobilized cells on 0.9, 2.0, 3.6, and 6.3 μm pore size particles were 56.7, 43.2, 48.5, 60.5, and 55.2%, respectively. Overall, immobilized M. tundrae cells showed a higher methanol production activity compared to the M. sporium (B2119–B2123) and Methylosinus trichosporium (B2117 and B2118) strains immobilized on a polymeric matrix (Table S1).24 In addition, the encapsulated MMO enzymes caused considerably lower methanol production and required the addition of an expensive cofactor.45 Among the different particles, the 3.6 μm pore size particle showed the best methanol production and exhibited 2.5-fold lower cell leaching compared to immobilized cells on 6.3 μm pore particles. Therefore, 3.6 μm pore size particles were used for subsequent studies.

Characterization of Immobilized Cells As immobilized methanotrophs exhibited variable methanol production efficiencies under different physiological conditions,21,46 the methanol production potential of free and immobilized cells was compared at different pH and temperature values for 24 and 48 h incubations, respectively (Figure S12). The optimum pH for methanol production was 7.0 for both free and immobilized cells. Immobilized cells showed significantly higher methanol production of 0.90 and 0.72 µmol mg-DCM-1 at pH values of 6.0 and 7.8, respectively (Figure S12a), whereas free cells exhibited methanol production of 0.51 and 0.36 µmol mg-DCM-1, respectively under similar conditions. A 2.0-fold improvement in methanol production was observed at these pH values by immobilized cells compared to free cells. The optimum temperature for methanol production was 30 C for both free and immobilized cells (Figure S12b). Immobilized cells exhibited improved methanol production to 0.41 µmol mg-DCM-1 at higher temperatures up to 40 C as compared to free cells, which produced 0.17 µmol mg-DCM-1. In contrast, a similar 18 ACS Paragon Plus Environment

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methanol production of 0.81 and 0.82 µmol mg-DCM-1 was observed at 25 C by free and immobilized cells, respectively. A 2.4-fold enhancement of methanol production at a higher temperature was obtained from immobilized cells over free cells. Further, the storage stability of free and immobilized cells at 4 C was evaluated (Figure S12c). After 15 days of incubation, the free and immobilized cells retained a methanol production efficiency of 38.7 and 88.2%, respectively. Here, the higher methanol production stability of cells immobilized on 3.6 μm pore size particles is probably associated with retention of higher residual MMO activity (92.1%) over that in free cells (31.2%) (Figure S12d). Further, the immobilized cells retained a methanol production efficiency of 58.4% after 30 days at room temperature, whereas the free cells completely lost their methanol production efficiency after 14 days under the same conditions (Figure S13). Macro-porous particles were found to be a more effective support for the immobilization of M. tundrae to retain high pH and temperature stability for methanol production than other supports such as Na-alginate and silica gel used for M. sporium encapsulation. This observation might be associated with better compatibility of synthesized carbon composite or that the large porous nature of the supports may lead to better CH4 diffusion.9,28,42,46

Repeated Batch Methanol Production Repeated batch culture is an effective method to improve methanol production by whole cells, either free or immobilized.24 Methanol production by free and immobilized M. tundrae was evaluated using 30% CH4 as the feed for 24 incubations, cycled up to eight times (Figure 7a-b). Methanol production by free and immobilized cells decreased over consecutive cycles. After eight cycles of reuse, the free and immobilized cells showed relative methanol production of 19 ACS Paragon Plus Environment


3.9% and 70.7%, respectively. Remarkably, the immobilized cells showed 18.1-fold higher stability for residual methanol production than free cells. Overall, cumulative production of 4.01 and 8.13 µmol mg-DCM-1 (12.0 and 24.4 mM) was observed from free and immobilized cells, respectively. Previously, only 2.59-3.56 mM of methanol production was observed for the polymeric matrix-encapsulated M. sporium and M. trichosporium strains after three cycles of batch methanol production (Table S2).24 Furthermore, the higher methanol production stability or biocatalytic activity of immobilized cells on 3.6 μm pore size particles under repeated batch conditions was correlated with the retention of higher residual MMO activity of 76.2% compared to 6.2% activity for free cells (Figure 7c). Similarly, the higher biocatalytic potential for methanol production by immobilized cells was validated with preservation of high reduction current density of 5.55 µA cm-2 over that of free cells of 1.74 µA cm-2 after eight cycles of reuse (Figure 7d). Our results suggest that macro-porous particles are very effective for M. tundrae immobilization and subsequent methanol production. There are currently no other reports available on immobilization of methanotrophs on such unique macro-porous synthesized carbon particles.


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Figure 7. Repeated batch methanol production by M. tundrae free or immobilized on 3.6 μm pore size particles: (a) reusability, (b) cumulative production, (c) relative MMO activity and (d) reduction current density at - 0.5 V. CH4 (30%, v v-1) was used as a feed. Significant (p ≤ 0.05) differences were observed by one-way ANOVA.

Methanol Production Using Simulated Biogas Raw biogas produced through the anaerobic digestion of complex biowaste material can be used as a feed for methanol production.14,26 In raw biogas, CH4 (62.3-69.8%) is associated with CO2 (28.8-36.7%) and traces of ammonia, hydrogen, and hydrogen sulfide.14,26,44 Also, the effective ratio of CH4 and CO2 [2:1 to 4:1, (v v-1)] in the feed has been reported for methanol production by methanotrophs.14,26 To demonstrate methanol production by immobilized M. tundrae from 21 ACS Paragon Plus Environment


biogas, synthetic biogas was simulated by a mixture of CH4 and CO2 [4:1, (v v-1)] and was used as the feed. The methanol production profile of free and immobilized cells using simulated biogas is presented in Figure 8a. The maximum methanol production by free and immobilized cells using simulated biogas was 1.63 and 1.95 µmol mg-DCM-1, respectively. Here, the use of simulated biogas as the feed for methanol production enhanced methanol production by approximately 1.4- and 1.6-fold for free and immobilized cells, respectively, compared to pure CH4 (30%) as a feed. Simulated biogas produced higher methanol production stability of up to 120 h of incubation by both free and immobilized cells when compared to pure CH4. The maximum conversion yield of simulated biogas to methanol by free and immobilized cells was 49.3% and 67.1%, respectively.


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Figure 8. Methanol production by M. tundrae immobilized on 3.6 μm pore size particles from simulated biogas [CH4 and CO2 (4:1, v v-1)]: (a) production profile, (b) relative MDH activity, and (c) cumulative production. Significant (p ≤ 0.05) differences were observed by one-way ANOVA. 23 ACS Paragon Plus Environment


Here, high conversion of simulated biogas to methanol by immobilized cells may be associated with higher inhibition of MDH activity (85.6%) compared to that of free cells (91.3%) and the retention of higher MMO residual activity of 61.5% compared to that of free cells (17.9%) (Figures 8b and S14). Here, the lower MDH activity of the immobilized cells is possibly associated with mass-transfer limitations or changes in the physiological properties of the cells after immobilization.9,28,42 Further, electrochemical impedence spectroscopy (EIS) analysis showed charge transfer resistance values of 632 and 580 Ω for free and immobilized cells, respectively (Figure S15). Here, lower charge transfer resistance for immobilized cells might be positively associated with higher methanol production.43 Overall, these results signify that the cells immobilized on 3.6 μm pore size particles produce methanol more efficiently than free cells. The macro-porous particles were found to be more effective supports for immobilization of Methanotrophs to retain high pH and temperature stability for methanol production than Na-alginate and silica gel. Methanol production from simulated biogas by M. tundrae was significantly higher than that previously reported for M. sporium and M. trichosporium from similar feeds.44,47 Further, in order to improve methanol production from a simulated biogas mixture of CH4 and CO2 [4:1, (v v-1)], methanol production by free and immobilized M. tundrae was evaluated up to eight cycles (Figure 8c). After eight cycles of reuse, the free and immobilized cells showed cumulative methanol production of 5.2 and 9.5 µmol mg-DCM-1, respectively. Here, simulated biogas as feed showed better methanol production than pure CH4 (Figure 7b). These results suggested that simulated biogas could be a potential feed to improve methanol production under repeated batch conditions by immobilized cells on synthesized macro-porous carbon particles.


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CONCLUSIONS This study reports the synthesis of hierarchical macro-porous carbon particles with pore sizes in the range of 0.9-6.3 µm for efficient immobilization of M. tundrae. Covalently immobilized whole cells on particles with a pore size of 3.6 µm were found to be the most effective for methanol production. Immobilized cells showed significantly higher pH and temperature stability for methanol production over free cells. Under repeated batch methanol production, immobilized cells exhibited higher stability and superior biocatalytic activity for residual methanol production than free cells. Overall, hierarchical macro-porous particles were found to be very effective for whole cell immobilization, and methanotrophs immobilized on 3.6 µm particles produced methanol efficiently from both CH4 and simulated biogas. This work is the first report of methanotroph immobilization on synthesized macro-porous carbon particles and demonstrates that unique macro-porous particles as support for whole cell immobilization can be effectively used in methanol production from GHGs to facilitate sustainable development.

EXPERIMENTAL SECTION Materials. M. tundrae (DSMZ 15673) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). CH4 and CO2 were obtained from NK Co., Busan, Republic of Korea. 2,6-dichlorophenol-indophenol (DCPIP), copper sulfate (CuSO4), formate, glutaraldehyde, iron sulfate (FeSO4), phenazine methosulfate, and tetrazotized o-dianisidine were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ammonium chloride (NH4Cl), calcium chloride (CaCl2), magnesium chloride (MgCl2), ethanol, formaldehyde, methanol, and resorcinol were purchased from Daejung Chemicals (South Korea). Sodium carbonate was purchased from Alfa Aesar (Ward Hill, MA, USA). All chemicals and reagents were of analytical grade and obtained from commercial sources, unless otherwise stated. Fabrication of Porous Carbon Particles. PS spheres were synthesized by conventional 25 ACS Paragon Plus Environment


dispersion polymerization.33,48 The PS sphere powders were packed in a glass vial with a diameter of 1 cm by applying a pressure of ~100 Pa. In order to increase the contact areas of each sphere, the pressed PS sphere templates were then heated for 15 min at 145 °C, 150 °C, 155 °C, and 160 °C for sphere diameters of 1.0 μm, 2.0 μm, 4.2 μm, and 6.6 μm, respectively. RF solution was synthesized by mixing resorcinol, formaldehyde, sodium carbonate, and deionized water in a 1.00:2.00:0.02:5.68 molar ratio. The RF solution was infiltrated into a template in a vacuum environment, followed by heating at 60 °C for 24 h to form an RF gel. The RF gel-PS sphere composite was pulverized using a mortar and pestle and sifted through 600 µm × 600 µm mesh and then 300 µm × 300 µm mesh. Finally, the PS sphere-RF gel composite was

carbonized at 950 °C for 2 h in a nitrogen environment. The control porous carbon scaffolds were identically fabricated except the pulverization and sieve process. CO2 Uptake Measurements. CO2 uptake was investigated in the pressure range of 0–1 bar at 25 °C using a Multiport Chemisorption/Physisorption/Micropore Analyzer (3Flex 3500). Prior to the uptake measurements, the samples were degassed under vacuum at 200 °C for 3 hours. Culture Conditions. M. tundrae was grown in nitrate mineral salts medium using 20% CH4 as a feed in 1 L Erlenmeyer flasks, incubating for 5 days at 30 C as reported previously.42 Fullygrown cells were collected by centrifugation at 4000 × g for 20 min at 4 C. The DCM was determined as described previously.26 Immobilization of M. tundrae. Whole cell immobilization was done on synthesized macroporous particles through adsorption and covalent attachment. Different supports were prefunctionalized with glutaraldehyde, as reported previously.49,50 Briefly, 1 g of each type of particle was suspended in 50 mM phosphate buffer (pH 7.0) containing 1 M glutaraldehyde and incubated for 2 h at 25 °C. After functional activation, particles were recovered by centrifugation and washed two times with phosphate buffer to remove excess glutaraldehyde completely. M. tundrae was immobilized at a cell concentration of 0.25 g of DCM per g of support over an incubation period of 32 h at 4 °C. Once optimal binding conditions were determined, cell loading was further assessed with cell concentrations of 0.05 to 2.00 g of DCM per g of support in the reaction mixture. 26 ACS Paragon Plus Environment

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The IYs and REs of methanol production were calculated as follows (Eqs. 2-3): IY (%) = (amount of cells bound on the supports/total amount of cells loaded) × 100

(2)

RE (%) = ratio of methanol produced by immobilized and free cells × 100

(3)

Methanol Production. Under batch conditions, methanol production by free or immobilized cells (3 mg of DCM mL-1) was evaluated in serum bottles (120 mL) with a reaction volume of 20 mL containing 30% CH4 as the feed.42 Briefly, the reaction volume was prepared in phosphate buffer (100 mM, pH 7.0) including MgCl2 (50 mM), FeSO4 (10 µM), CuSO4 (5 µM), and formate (100 mM). Reactions were incubated at 30 C with shaking at 150 rpm. The conversion yield (%) of CH4 or simulated biogas to methanol was calculated as the ratio of moles of methanol produced to moles of CH4 consumed. Methanol production profiles of free or immobilized cells on the macro-porous particles were evaluated for incubation up to 144 h using cell inocula of 3 mg of DCM mL-1 and 30% of CH4 as a feed. Free and efficiently immobilized cells on macro-porous particle with a 3.6 µm pore size were characterized for methanol production at different pH (6.0-7.8) and temperatures (25-40 C). Further, methanol production by free and immobilized cells using 30% CH4 was performed for eight cycles under repeated batch conditions, as described previously.42 After each cycle of incubation for 24 h, cells were recovered by centrifugation and used for the next cycle.17 In order to evaluate the potential of immobilized cells for methanol production from biogas, simulated biogas was prepared using pure gases of CH4 and CO2 at a ratio of 4:1 (v v-1) and used as the feed.26 Leaching. The leaching of immobilized cells was evaluated by the measurement cell optical density (OD) at 600 nm, as follows (Eq. 4). Leaching (%) = (OD of dissociated cells in supernatants/initial immobilized cells OD) × 100 (4) Naphthalene Oxidation and MDH Activity Measurements. The sMMO activity of free or immobilized cells (3 mg of DCM) was measured in a naphthalene oxidation assay using crystals of naphthalene and freshly prepared tetrazotized o-dianisidine (10 mg mL-1) at 30 °C as described previously.42 MDH activity was determined spectrophotometrically by phenazine 27 ACS Paragon Plus Environment


methosulfate-mediated reduction of DCPIP in phosphate buffer (pH 7.5) using CaCl2 (10 mM), NH4Cl (45 mM), and phenazine methosulfate (3.3 µM) at 600 nm, as described previously.17 Electrochemical Measurements. CV analysis was evaluated using a SP-150 potentiostat (BioLogic, Knoxville, USA), with a 3 electrode system of glassy carbon, platinum and Ag/AgCl as working, counter and reference electrodes in a 20 mL reaction cell using free and immobilized cell suspensions in phosphate buffer in the potential range from -0.5 to +0.5 V at a scanning rate of 20 mV s-1.43,51 EIS was analyzed in potentiostatic mode from 100 KHZ to 10 mHZ, with an amplitude voltage of 10 mV from Nyquist plots, the charge transfer resistance was noted from the low-frequency region. Charge transfer resistance was calculated, on the basis of circular fit analysis from the instrument.43 Instrumental Analysis. The pore sizes of the synthesized macro-porous particles were determined via the BJH method (Micromeritics AutoPore IV 9500 mercury porosimeter) and the nitrogen adsorption measurements (Micromeritics TriStar II 3020). The morphological features of the synthesized macro-porous particles were investigated by high-resolution TEM (JEM-2100F) at an accelerating voltage of 200 kV. Methanol concentration was measured by a gas chromatographic (GC) system equipped with an HP-5 column (Agilent 19091J-413) connected to a flame ionization detector as described previously (Agilent 7890A; Agilent Technologies, Wilmington, DE, USA).17,26 Helium was used as the carrier gas along with H2 at a makeup flow of 25 mL min-1 and air (300 mL min-1). CH4 and CO2 were analyzed by GC equipped with a Carboxen 1010 Plot fused silica capillary column (Supelco, Bellefonte, PA, USA) and a thermal conductivity detector using N2 as the carrier gas.26 Absorption spectra were recorded on a spectrophotometer (Varian Cary 100 Bio UV-Vis spectrophotometer, Palo Alto, CA, USA).52 The thermal decomposition characteristics of the synthesized macro-porous particles and immobilized cells were measured by TGA (Seiko Exstar 6000 TG/DTA 6100, Japan). SEM images of the macro-porous particles and cell-immobilized particles were analyzed by field-emission SEM (FE-SEM, JEOL, Japan). For SEM analysis, the samples of cells immobilized on the macro-porous particles were prepared by primary fixation using Karnovsky’s fixative solution followed by samples dehydration using graded ethanol (30%, 50%, 70%, 80%, 90%, and 100%) as reported previously.53 Each value represents the mean of three sets of 28 ACS Paragon Plus Environment

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experiments and varies from the mean by less than 15%. The statistical significance was measured by analysis of variance (ANOVA, α = 0.05) using GraphPad Prism 5 software (GraphPad Software, CA, USA).54 ■ ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected] (Jung-Kul Lee) Fax: (+82) 2-458-3504 E-mail: [email protected] (Dong Rip Kim) Fax: (+82) 2-2220-2299

NOTES The authors declare no competing interest.

ACKNOWLEDGEMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2017R1A2B3011676, 2017R1A4A1014806, 2013M3A6A8073184). This work was supported by KU Research Professor program of Konkuk University. This study was also supported by the



Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Science and ICT of Korea (NRF-2012M3A6A8054889).

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(51) Patel, S. K. S.; Anwar, M. Z.; Kumar, A.; Otari, S. V.; Pagolu, R. T.; Kim, S.-Y.; Kim, I.W.; Lee, J.-K. Fe2O3 Yolk-shell Particle-Based Laccase Biosensor for Efficient Detection of 2,6-Dimethoxyphenol. Biochem. Eng. J. 2018, 132, 1-8. (52) Patel, S. K. S.; Choi, S. H.; Kang, Y. C.; Lee, J.-K. Eco-friendly Composite of Fe3O4reduced Graphene Oxide Particles for Efficient Enzyme Immobilization. ACS Appl. Mater. Interfaces 2017, 9, 2213-2222. (53) Patel, S. K. S.; Otari, S. V.; Li, J.; Kim, D. R.; Kim, S. C.; Cho, B.-K.; Kalia, V. C.; Kang, Y. C.; Lee, J.-K. Synthesis of Cross-Linked Protein-Metal Hybrid Nanoflowers and Its Application in Repeated Batch Decolorization of Synthetic Dyes. J. Harad. Mater. 2018, 347, 442-450. (54) Patel, S. K. S.; Kumar, V.; Mardina, P.; Li, J.; Lestari, R.; Kalia, V. C.; Lee, J.-K. Methanol Production from Simulated Biogas Mixtures by Co-Immobilized Methylomonas methanica and Methylocella tundrae. Bioresour. Technol. 2018, 263, 25-32.



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Hierarchical Macroporous Particles for Efficient Whole-Cell

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