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Continuous biocatalytic synthesis of epoxypropane using a biofilm reactor
Process Biochemistry 38 (2003) 1739 /1746 www.elsevier.com/locate/procbio
Continuous biocatalytic synthesis of epoxypropane using a biofilm reactor Jia-Ying Xin a, Jun-Ru Cui a, Jian-Bo Chen a, Shu-Ben Li a,*, Chun-Gu Xia a, Li-Min Zhu b a
State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China b School of Biological and Applied Sciences, University of North London, London N7 8DB, UK Received 25 June 2002; accepted 30 August 2002
Abstract Mixed culture methanotrophic attached biofilms immobilized on diatomite particles in a three-phase fluidized bed reaction system were developed. Methane monooxygenase (MMO) activity on diatomite particles increased as soon as the lag phase ended. More than 90% of the MMO activity in the fluidized bed was attached. A biofilm concentration of 3.3c3.7mg dry weight cell (dwc) per g dry solid (DS) was observed. Batch experiments were performed to explore the possibility of producing epoxypropane by a propene /methane co-oxidation process. The effect of methane on the epoxidation of propene and the effect of propene on the growth of methanotroph was also studied. In continuous experiments, optimum mixed gas containing 35 methane, 20 propene and 45% oxygen were continuously circulated through the fluidized bed reactor to deliver substrates and extract product. Initial epoxypropane productivity was 110 /150 mmol/day. The bioreactor operated continuously for 53 days without obvious loss of epoxypropane productivity. # 2002 Elsevier Ltd. All rights reserved. Keywords: Methanotroph; Attached biofilm; Fluidized bed; Epoxypropane cooxidation
1. Introduction Direct oxidation of propene to epoxypropane is an attractive chemical process[1]. Methane monooxygenase (MMO) is the first enzyme in the methane utilization pathway in methanotroph [2] and due to its broad substrate specificity, methanotrophs can oxygenate a wide range of n -alkanes and n -alkenes [3]. Methanotrophs, therefore, show promise for the transformation of propene to epoxypropane, which is difficult to prepare chemically [1]. The capability of methanotrophs to transform propene to epoxypropane may be limited by reducing power supply as well as by product toxicity. This
* Corresponding author. Fax: /86-931-827-7088. E-mail address: [email protected] (S.-B. Li). 0032-9592/02/$ - see front matter # 2002 Elsevier Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 2 6 2 - 5
transformation, however, is of little benefit to the cells as they typically derive neither reducing power nor biomass from this transformation. Epoxidation usually is terminated due to accumulation of toxic product and the exhaustion of reducing power. For maintenance of epoxidation for a long time, constant sources of reducing power for monooxygenation and continuous removal of product are critical. Prichanont and co-workers [4] performed epoxidation reaction in an aqueous-organic solvent two-liquid phase system to avoid accumulation of toxic product, but lack of reducing power still plays a limiting role in such a system, and phase toxicity may also hamper the success of the reaction. To prolong the monooxidation reaction, Furuto and co-workers [5] supplied an external source of reducing power by provision of formate in the feed stream to the reactor. However, addition of formate to maintain epoxidation for a long time was uneconomical.
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The biosynthesis of epoxypropane by methanotrophs has also emphasized the need to be able to maintain high bacterial activity for a long time. Process improvements must be provided to overcome the operational problem related to low bacterial activity and stability. Fluidized bed technology uses small, fluidized media particles to induce extensive growth cell immobilization to give high biomass hold-up and a long mean residence time [6]. It has also been shown that microorganisms, when present in biofilms, are hundreds or even thousands of times more resistant to antimicrobial agents than the same species grown in suspended culture [7]. In this work, a three-phase methanotrophic attachedfilm fluidized bed (MAFFB) reactor was developed for the biosynthesis of epoxypropane from propene. For maintenance of epoxidation for a long time, methane was selected as a co-substrate for reducing power (NADH2), regeneration and cell growth. Batch experiments were performed to explore the possibility of producing epoxypropane by a methane/propene cooxidation process. The effect of methane on the epoxidation of propene and the effect of propene on the growth of methanotroph were also studied. In continuous experiments, optimum mixed gas containing 35 methane, 20 propene and 45% oxygen were continuously circulated through the fluidized bed reactor to deliver substrates and extract product. In this slightly low growth mode, initial epoxypropane productivity was about 110 /150 mmol/day. The bioreactor operated continuously for 53 days without obvious loss of epoxypropane productivity.
2. Materials and methods 2.1. Culture of methanotrophs Methylomonas sp. GYJ3 was isolated from soil samples from the oil fields of Yumen, Gansu Province, China. Methylomonas sp. GYJ3 was classified as type methanotrophs and cultivated as described by Shen et al. [8]. Methylococcus capsulatus IMV 3021 and Methylosinus trichosporium IMV 3011 were obtained from the Institute of Microbiology and Virology (Kiev). The following nitrate mineral salt (NMS) medium was used for the methanotroph large-scale cultivation, (g/l): NH3Cl, 0.5; K2HPO4, 0.49; KH2PO4 ×/ 7H2O, 0.40; MgSO4 ×/ 7H2O, 0.3; CaCl2 ×/ 2H2O, 0.02; KNO3, 1.6; NaCl, 0.3; Fe SO4 ×/ 7H2O, 0.004; CuSO4 ×/ 5H2O, 0.004; MnSO4 ×/ H2O, 0.0004; ZnSO4 ×/ 7H2O, 0.00034; Na2MoO4 ×/ 2H2O, 0.00024; pH 7.0. Large-scale cultivation of cells was carried out in a 15l, continuous-gas feed fermentor (LH1075, UK) under an atmosphere of methane and oxygen (1:1, v/v) at 32 8C about 72/96 h. Cells were harvested at 9000 /g for 10 min and washed twice with 20 mM phosphate
Fig. 1. Schematic representation of the MAFFB reactor system.
buffer (pH 7.0). Cells were resuspended in the same buffer containing 5mM MgCl2 (at a cell concentration of 2 /3 mg dry weight cell (dwc) per ml) and used in batch experiments. 2.2. MAFFB reactor set-up A schematic of the three-phase MAFFB reactor is shown in Fig. 1. The MAFFB reactor system consists of a fluidized-bed (A), an absorber (C) and a closed gas loop containing a 2-l gasbag (D). When leaving the absorber and gasbag, the gas is depleted in the product and enriched in growth and/or bioconversion substrate. A mixed culture methanotrophic biofilm was cultivated on diatomite particles (30 g, particle size range: 200/800 mm) located inside the fluidized-bed (30 cm high, 4.0 cm i.d.). The temperature was maintained by circulating water at 32 8C around the fluidized-bed. The NMS liquid medium containing attached and detached methanotrophic cell was recycled from the fluidized bed (A) to clarifier (B) by a pump G-3 (10 ml/h). The clarifier (20 cm high, 1.4 cm i.d.) served as a segregator tube. Detached cells concomitant with effluent (F) exited from the effluent outlet above the clarifier, and were sedimented and recycled into the fluidized bed. In the fluidized bed, diatomite particles with attached methanotrophs were fluidized by an uprising flow of the gas (pump G-1, flow rate /300 ml/h), gaseous substrates were transferred to the liquid phase and volatile product (epoxypropane, bp 35 8C) was transferred to the gas phase and removed from the fluidized-bed. The absorber (C) was held at 0/4 8C and consisted of three 50-ml flasks filled with distilled water. When back in the absorber, the gas phase was stripped of the product epoxypropane and re-established by the gasbag. The gas loop was closed. The fresh NMS medium (E) was fed by
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Fig. 2. MMO activity on diatomite during the continuous culture process.
pump G-2 at a daily addition of 50 ml/day, and surplus NMS medium (F) containing detached cells was exited from the effluent outlet above the clarifier. In the absorber, the majority of the epoxypropene was accumulated and the process yielded a clear solution. The absorption of epoxypropane was accomplished by periodically removing the first flask and replacing it with a new flask at the rear. 2.3. Development of biofilm Pure culture of Methylomonas sp. GYJ3, M. capsulatus IMV 3021 and M. trichosporium IMV 3011, pregrown separately in NMS with 50 methane and 50% oxygen headspace, were used for inoculation. The reactor was not sterilized before inoculation. The reactor was operated in batch mode for 50 days and then continuous NMS medium feed was started by turning on G-2 and G-3. The gas phase was initially maintained at about 50 methane and 50% oxygen with a total pressure of 1.0 atm. As methane and O2 may be utilized at different rates in the MAFFB reactor, the methane and O2 partial pressures in the gasbag may be changed. The initial partial pressures were re-established every 24 h by replacing the gasbag. 2.4. Biosynthesis of epoxypropane in the MAFFB reactor After 65 days of continuous culture, five batch tests with 2-day intervals were conducted in the three-phase MAFFB reactor to optimize the biosynthesis of epoxypropane. In each of five batch tests, mixed gas containing 20 propene, 40% oxygen and various concentrations of methane were fed continuously over a 3day period. At each 2-day interval, growth gas of
methane and oxygen (1:1, v/v) was fed continuously for resumption of bacterial activity and reductant (NADH2) regeneration. A continuous biosynthesis of epoxypropane was conducted by long-term continuousgas feed. 2.5. Batch experiments Batch experiments were conducted in 100 ml sealed conical flasks (under atmospheric pressure) containing 20 /25 ml of washed cell suspension. The gaseous phase of the flask was replaced by the gas mixture containing various concentrations of propene, oxygen, and methane (as described above). The flasks were incubated and shaken (150 rpm) at 35 8C on a controlled temperature rotary shaker. Chromatographic determinations of the liquid phase (epoxypropane) were carried out at different periods of reaction. All batch values are averaged for the three runs with a variation of 3/5% from the mean. 2.6. Analytical methods Epoxypropane formation from propene was determined chromatographically using a gas chromatograph equipped with a capillary GC column(c{0.23 mm /30 m; stationary phase, SE-54) and a flame ionization detector (FID). Pure nitrogen served as the carrier gas at a flow rate of 75 ml/min. The temperatures of the column, detector, and injector were 60, 180, and 180 8C, respectively. Quantification was performed by the method of external standardization. The assays of MMO activity were performed by measuring epoxypropane formation from propene. An assay was performed in a total volume of 1 ml of cell
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Fig. 3. Pathway of propene (or methane) metabolism and NADH regeneration.
suspension sealed in a 10 ml reaction vial. The gaseous phase of the vial was removed by vacuum and replaced with a gas mixture of the propene and air (1:1, v/v). The reaction was started by addition of the gas mixture. Assays were performed at 35 8C for 30 min. Epoxypropane formation from propene was also measured using same gas chromatography. Specific activities were expressed as nanomoles of epoxypropane formed per minute per milligram of dwc. The optical density (OD) of the culture medium was determined by UV spectrophotometry at 550 nm (UV120-02, Shimadzu Co.). Sample preparation for scanning electron microscopy (SEM): The samples were dried by lyophilization at / 50 8C and 10 mmHg, the dried samples were gold sputtered and observed at 30 kV in a JEOL (JSM5600LU) scanning microscope.
3. Results and discussion 3.1. Development of biofilm in three-phase MAFFB reactor Many methanotrophs were reported to form capsular or slime material [9,10], which is known to play an important role in the ability of microorganisms to stick to surfaces. The immobilized cells grow, reproduce, and produce extracellular polymeric substances (EPS) that frequently extend from the cell forming a tangled mass of fibres lending structure to the entire assemblage, which shall be termed a biofilm [11]. In this work, three pure culture strains of Methylomonas sp.GYJ3, M. capsulatus IMV 3021 and M. trichosporium IMV 3011 were co-used as inocula. Biofilm development was monitored through MMO activity measurements. The living cell concentration on diatomite particles was associated with MMO activity on diatomite particles. Fig. 2 shows the dynamics of MMO activity and
biomass on diatomite particles through the biofilm formation process. The biofilm formation dynamics presented sigmoid behavior with two consecutive stages: a lag phase of 23 days and a fast increasing activity phase. The first phase was associated with the initial formation of the biofilm where the methanotrophs move from the liquid to the diatomite particle surface. The second phase was associated with the growth of biofilm. Scour action effects induced by uprising flow of the gas and particles chases detach the more exposed biofilm sections. This factor contributes to biofilm destruction. Furthermore, it is hypothesized that as cells in the biofilm are inactivated, they are detached and replaced by new cells; the cells in the biofilm are transported by advection. Methanotrophs grow on the diatomite particles and equilibrate the detached methanotrophs, a steady state was obtained. The detached methanotrophs at the steady state value corresponds to 10% of the total methanotrophs in the MAFFB reactor. This result can be seen as evidence that the immobilized methanotrophs are robust against detachment induced by scour action effects. The methanotrophic concentration on diatomite particles was 3.3 /3.7 mg dwc per g dry solid (DS).
3.2. Propene concentration Various concentrations of propene were used to examine the production of epoxypropane by cell suspensions of Methylomonas sp. GYJ3, M. capsulatus IMV 3021 and M. trichosporium IMV 3011. The initial oxygen concentration in the gaseous phase was kept constant (40%). Nitrogen gas was used to balance the remainder of the gaseous phase. The amount of epoxypropane produced was assayed after 30 min of incubation. A propene concentration of 20% supported maximum epoxypropane production. Higher propene concentration did not stimulate the production of epoxypropane (data not shown).
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Table 1 Effect of methane on the epoxidation of propene Reaction time (h)
Composition of gaseous phase
1 1 1 1 1 1 2 2 2 2 2 2
Epoxypropene (yM)
Oxygen (%)
Methane (%)
Propene (%)
Nitrogen (%)
40 40 40 40 40 40 40 40 40 40 40 40
0 5 10 20 30 40 0 5 10 20 30 40
20 20 20 20 20 20 20 20 20 20 20 20
40 35 30 20 10 0 40 35 30 20 10 0
738 945 952 1077 1139 406 1021 1327 1389 1582 1076 766
Table 2 Effect of propene on growth of methanotrophs Initial partial pressures (%)
Cell concentration after 72 h fermentation (mg dry weight cell per ml)
Propene
Oxygen
Methane
Nitrogen
0 20 20 20 20 20
50 40 40 40 40 40
50 40 35 30 20 10
0 0 5 10 20 30
0.30 0.27 0.27 0.10 0.03 B/0.01
3.3. Possibility of producing epoxypropane by a propene/ methane cooxidation process According to the Fig. 3, MMO can oxidize propane and methane to epoxypropane and methanol, respectively. As a natural substrate of methanotrophs, methane can be oxidized completely to carbon dioxide via methanol, formaldehyde and formate. Reducing power (NADH2) for monooxygenation was obtained during dehydrogenation of intermediates of methane oxidation [12]. However, epoxypropane cannot be further metabolized and accumulate extracellularly. Epoxidation usually is terminated due to accumulation of toxic product and the exhaustion of reducing power. Epoxypropane synthesis in batch reaction was carried out as described above. To retain epoxidation activity of methanotrophs, methane was selected as a co-substrate for NADH2 regeneration. A single MMO is responsible for the oxidation of both propene and methane, and the presence of methane may inhibit the oxidation of propene. However, after the initial oxidation step, methane may be further degraded to regenerate NADH2, which can promote more propene oxidation. The effect of methane addition on the epoxidation of propene was studied. The production of epoxypropane
from propene by cell suspension of Methylomonas sp. GYJ3 was assayed in the presence of varying amounts of methane. The initial partial pressures of propene and oxygen in the gaseous phase were kept constant in all of the experiments; N2 was used to balance the gaseous phase. As shown in Table 1, the epoxidation ability of Methylomonas sp. GYJ3 cell was enhanced by addition of lower concentrations of methane, suggesting substantial NADH2 limitations for the cells. The maximum production of epoxypropane occurred in an atmosphere of 30% methane. Although methane enhanced the epoxidation ability of Methylomonas sp. GYJ3 cells under an atmosphere of 30% methane, this effect diminished when methane concentration was higher than 30%, suggesting that the positive effect of methane (NADH2 regeneration) can be overwhelmed by its competitive inhibition effect for the higher methane concentration. As for M. capsulatus IMV 3021 and M. trichosporium IMV 3011, almost the same result was observed. 3.4. The effect of propene on the growth of methanotroph Propene did not support the growth of methanotroph. As a single MMO molecule is responsible for the
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Fig. 4. Biosynthesis of epoxypropene in the MAFFB reactor; Feed gas composition: 1, propene, oxygen, nitrogen, 20, 40, 40(kPa); fed continuously over a 3-day period; 2, propene, methane, oxygen, nitrogen, 20, 10, 40, 30(kPa); fed continuously over a 3-day period; 3, propene, methane, oxygen, nitrogen, 20, 20, 40, 20 (kPa); fed continuously over a 3-day period; 4, propene, methane, oxygen, nitrogen, 20, 30, 40, 10 (kPa), fed continuously over a 3-day period; 5, propene, methane, oxygen, 20, 40, 40 (kPa), fed continuously over a 3-day period; 6, propene, methane, oxygen, 20, 35, 45 (kPa); fed continuously for 53 days.
oxidation of both types of substrates, the presence of nonbeneficial propene can inhibit the oxidation rate of growth substrate methane (competitive inhibition). The competition of methane and propene for MMO determined the efficiency of conversion of methane by the cell, which determined the growth rate of methanotrophs. At the same time, during the growth of methanotrophs on methane plus propene, part of the reducing power (NADH2) was used for the epoxidation of propene. To determine if the addition of 20% propene could influence growth of methanotrophs, the bacteria were grown in the presence of 20% propene and various partial pressure of methane in a gas-continuous feed fermentor, respectively. The epoxypropane produced was extracted continuously from the fermentor by gasfeed. Methanotrophic bacteria growth was monitored by measuring OD550 and expressed as dwc. As shown in Table 2, when the partial pressure of methane was more than 35%, addition of 20% propene slightly inhibited the growth of methanotrophs. 3.5. Epoxypropane inhibition test To test whether epoxypropane alone had an inhibitory (or toxic) effect on epoxypropane synthesis with methanotrophs, cells of Methylomonas sp. GYJ3, M. capsulatus IMV 3021 and M. trichosporium IMV 3011 were incubated with both propene and 0.015% of epoxypropane, respectively. The results showed that
the rate of epoxidation was slower than in the standard assay system in which no external epoxypropane was added (data not shown). This result indicates that the epoxypropane produced has an inhibitory effect on epoxidation of propene with methanotrophs. Hence, the process for epoxypropane synthesis should minimize exposure of the cell. In order to avoid product inhibition, continuous removal of toxic product epoxypropane is critical. 3.6. Biosynthesis of epoxypropane in three-phase MAFFB reactor After 65 days of continuous culture to develop the biofilm, five batch tests with 2 days interval were conducted in the three-phase MAFFB reactor to optimize the biosynthesis of epoxypropane. In each of five batch tests, mixed gas containing 20 propene, 40% oxygen and various partial pressures of methane were fed continuously over a 3-day period. At each 2-day interval, growth gas of methane and oxygen (1:1, v/v) was fed continuously for bacterial activity and reducing power (NADH2) regeneration. After this growth mode ended, almost all of the initial MMO activity resumed (Data not shown). This result suggests that rapid processes such as NADH2 regeneration were responsible, rather than slower processes such as the generation of additional cells or MMO. As shown in Fig. 4, when mixed gas-containing 20 of propene, 40 of oxygen and
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40 kPa of nitrogen were fed continuously over a 3-day period, the methanotrophs gradually lost the ability to oxidize propene. Based on earlier batch studies, this was attributed to depletion of reducing power (NADH2). The presence of low amounts of methane enhanced epoxypropane current yield and reduced the decay rate of epoxypropane current yield, presumably due to the regeneration of reducing power (NADH2). However, the effect of enhanced epoxypropane current yield was diminished when the methane concentration was increased, presumably due to competitive inhibition. Comparing the epoxypropane yield and the decay rate of epoxypropane yield of the five tests, 30 kPa of methane versus 20 kPa of propene exhibited the highest formation capacities of epoxypropene. However, as shown in Table 2, under this atmosphere, methanotrophic growth inhibition occurred. To determine whether the slightly lower growth mode could maintain sufficient methanotrophic cells on diatomite particles for biosynthesis of epoxypropane, continuous biosynthesis of epoxypropane was conducted under long term continuous 20 kPa of propene versus 35 kPa of methane feed. As shown in Fig. 4, the reaction yielded epoxypropane with a yield of 110/150 mmol/day. The MAFFB reactor was operated continuously for 53 days without obvious loss of productivity, presumably due to constant sources of reducing power for monooxygenation, continuous removal of toxic product and stable maintenance of methanotrophic cells concentration on diatomite particles. 3.7. SEM studies of the biofilm SEM studies (Fig. 5) showed that there were interstices, pleats and crevices that furrowed a rough surface on the bare diatomite particles. The surface was clearly suitable for colonization and apparently offered a large surface. At the end of the biofilm formation process, the particles were colonized in crevices, pleats and surfaces and showed a covered biofilm. After several days of epoxypropane biosynthesis, the biofilm appeared unchanged.
4. Conclusions
Fig. 5. SEM microphotographs of the methanotroph attached-biofilm immobilized on diatomite. (a) Bare diatomite particles; (b) biofilmcovered diatomite particles; (c) biofilm-covered diatomite particles after 53 days of epoxypropane biosynthesis.
The results reported in this study show that it is possible to accumulate and maintain control of large amounts of methanotrophic cells in a stable attachedfilm fluidized bed reactor system during both growth mode and methane /propene co-oxidation mode. The feasibility of using the three-phase attached-film methanotrophs for continuous biosynthesis of epoxypropane by methane/propene co-oxidation process was described. The effect of methane on the epoxidation of
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propene and the effect of propene on the growth of the methanotroph were also studied. The significant increase in the initial epoxypropane current yield by the addition of an optimal concentration of methane, suggests that NADH2 regeneration was responsible. Further, the diminished initial epoxypropane current yield observed for higher than 30% methane suggests that the positive effect of methane (NADH2 regeneration) can be overwhelmed by its competitive inhibition effect at higher concentrations. Propene did not support growth of methanotroph. Since a single MMO is responsible for the oxidation of both types of substrates, the presence of nonbeneficial propene inhibits the oxidation rate of growth substrate methane. The competition of methane and propene for MMO determined the efficiency of conversion of methane by the cells, which determined the growth rate of methanotrophs. When 20% propene was added into the gas phase in the presence of 30% methane, growth inhibition of methanotrophs was observed. However, when 20% propene was added into the gas phase in the presence of 35% methane, the growth inhibition of methanotrophs was not significant. In this slightly lower growth mode, a sufficient number of methanotrophic cells for cooxidation were still maintained and the MMO activity of the methanotrophic population in the MAFFB reactor system was stable. Propene was epoxidated by the methanotrophs and epoxypropane was accumulated during the reaction. The epoxidation of propene was inhibited if the epoxypropane concentration was enhanced. The biocommunities in the MAFFB, however, continued to epoxidate the propene without obvious inhibition, presumably due to continuous removal of epoxypropane and resistance of sessile methanotrophic populations to epoxypropane. In conclusion, the cooxidation process described in this work shows the potential for commercial applications and can be readily scaled up since it is efficient and tolerates long-term operation.
Acknowledgements The authors would like to thank the National Nature Science Foundation of Chinese (29933040), the Special Funds for Major State Basic Research of China and the Royal Society of UK for the supporting.
References [1] Leak DJ, Aikens PJ, Seyed-Mahmoudian M. The microbial production of epoxides. TIBTECH 1992;10:256 /61. [2] Westerheide L, Pascaly M, Krebs B. Methane monooxygenase and its related biomimetic models. Curr Opin Chem Biol 2000;4:235 /41. [3] Burrows KJ, Cornish A, Scott D, Higgins IJ. Substrate specificities of the soluble and particulate methane monooxygenases of Methylosinus trichosporium OB3b. J Gen Microbiol 1984;5:335 / 42. [4] Prichanont S, Leak DJ, Stucky DC. Alkene monooxygenasecatalyzed whole cell epoxidation in a two-liquid phase system. Enzyme Microb Tech 1998;22:471 /9. [5] Furuto T, Takeguchi M, Okura I. Semicontinuous methanol biosynthesis by Methylosinus trichosporium OB3b. J Mol Catal A: Chem 1999;144:257 /61. [6] Meraz M, Alvarez-Ramirez J. A simple rate model for the dynamics of immobilization of anaerobic bacteria on a plastic support. J Chem Technol Biotechnol 2000;75:152 /8. [7] LeChevallier MW, Cawthon CD, Lee RG. Inactivation of biofilm bacteria. Appl Environ Microbiol 1988;54:2492 /9. [8] Shen RN, Yuchi L, Ma QQ, et al. Direct Evidence for a soluble methane monooxygenase from type methanotrophic bacteria:purification and properties of a soluble methane monooxygenase from Methylomonas sp. GYJ3. Arch Biochem Biophys 1997;345(2):223 /9. [9] Whittenbury R, Phillips KC, Wilkinson JF. Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol 1970;6x:205 /18. [10] Dworkin M, Foster JW. Studies on Pseudomonas mmethanica (sohngen) nov comb. J Bacteriol 1956;72:646 /59. [11] Turakhia MH, Characklis WG. Activity of Pseudomonas aeruginosa in biofilms: effect of calcium. Biotech Bioeng 1989;33:406 / 14. [12] Dalton H. Biological methane activation-lessons for the chemists. Catal Today 1992;13:455 /61.
Continuous biocatalytic synthesis of epoxypropane using a biofilm reactor Jia-Ying Xin a, Jun-Ru Cui a, Jian-Bo Chen a, Shu-Ben Li a,*, Chun-Gu Xia a, Li-Min Zhu b a
State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China b School of Biological and Applied Sciences, University of North London, London N7 8DB, UK Received 25 June 2002; accepted 30 August 2002
Abstract Mixed culture methanotrophic attached biofilms immobilized on diatomite particles in a three-phase fluidized bed reaction system were developed. Methane monooxygenase (MMO) activity on diatomite particles increased as soon as the lag phase ended. More than 90% of the MMO activity in the fluidized bed was attached. A biofilm concentration of 3.3c3.7mg dry weight cell (dwc) per g dry solid (DS) was observed. Batch experiments were performed to explore the possibility of producing epoxypropane by a propene /methane co-oxidation process. The effect of methane on the epoxidation of propene and the effect of propene on the growth of methanotroph was also studied. In continuous experiments, optimum mixed gas containing 35 methane, 20 propene and 45% oxygen were continuously circulated through the fluidized bed reactor to deliver substrates and extract product. Initial epoxypropane productivity was 110 /150 mmol/day. The bioreactor operated continuously for 53 days without obvious loss of epoxypropane productivity. # 2002 Elsevier Ltd. All rights reserved. Keywords: Methanotroph; Attached biofilm; Fluidized bed; Epoxypropane cooxidation
1. Introduction Direct oxidation of propene to epoxypropane is an attractive chemical process[1]. Methane monooxygenase (MMO) is the first enzyme in the methane utilization pathway in methanotroph [2] and due to its broad substrate specificity, methanotrophs can oxygenate a wide range of n -alkanes and n -alkenes [3]. Methanotrophs, therefore, show promise for the transformation of propene to epoxypropane, which is difficult to prepare chemically [1]. The capability of methanotrophs to transform propene to epoxypropane may be limited by reducing power supply as well as by product toxicity. This
* Corresponding author. Fax: /86-931-827-7088. E-mail address: [email protected] (S.-B. Li). 0032-9592/02/$ - see front matter # 2002 Elsevier Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 2 6 2 - 5
transformation, however, is of little benefit to the cells as they typically derive neither reducing power nor biomass from this transformation. Epoxidation usually is terminated due to accumulation of toxic product and the exhaustion of reducing power. For maintenance of epoxidation for a long time, constant sources of reducing power for monooxygenation and continuous removal of product are critical. Prichanont and co-workers [4] performed epoxidation reaction in an aqueous-organic solvent two-liquid phase system to avoid accumulation of toxic product, but lack of reducing power still plays a limiting role in such a system, and phase toxicity may also hamper the success of the reaction. To prolong the monooxidation reaction, Furuto and co-workers [5] supplied an external source of reducing power by provision of formate in the feed stream to the reactor. However, addition of formate to maintain epoxidation for a long time was uneconomical.
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The biosynthesis of epoxypropane by methanotrophs has also emphasized the need to be able to maintain high bacterial activity for a long time. Process improvements must be provided to overcome the operational problem related to low bacterial activity and stability. Fluidized bed technology uses small, fluidized media particles to induce extensive growth cell immobilization to give high biomass hold-up and a long mean residence time [6]. It has also been shown that microorganisms, when present in biofilms, are hundreds or even thousands of times more resistant to antimicrobial agents than the same species grown in suspended culture [7]. In this work, a three-phase methanotrophic attachedfilm fluidized bed (MAFFB) reactor was developed for the biosynthesis of epoxypropane from propene. For maintenance of epoxidation for a long time, methane was selected as a co-substrate for reducing power (NADH2), regeneration and cell growth. Batch experiments were performed to explore the possibility of producing epoxypropane by a methane/propene cooxidation process. The effect of methane on the epoxidation of propene and the effect of propene on the growth of methanotroph were also studied. In continuous experiments, optimum mixed gas containing 35 methane, 20 propene and 45% oxygen were continuously circulated through the fluidized bed reactor to deliver substrates and extract product. In this slightly low growth mode, initial epoxypropane productivity was about 110 /150 mmol/day. The bioreactor operated continuously for 53 days without obvious loss of epoxypropane productivity.
2. Materials and methods 2.1. Culture of methanotrophs Methylomonas sp. GYJ3 was isolated from soil samples from the oil fields of Yumen, Gansu Province, China. Methylomonas sp. GYJ3 was classified as type methanotrophs and cultivated as described by Shen et al. [8]. Methylococcus capsulatus IMV 3021 and Methylosinus trichosporium IMV 3011 were obtained from the Institute of Microbiology and Virology (Kiev). The following nitrate mineral salt (NMS) medium was used for the methanotroph large-scale cultivation, (g/l): NH3Cl, 0.5; K2HPO4, 0.49; KH2PO4 ×/ 7H2O, 0.40; MgSO4 ×/ 7H2O, 0.3; CaCl2 ×/ 2H2O, 0.02; KNO3, 1.6; NaCl, 0.3; Fe SO4 ×/ 7H2O, 0.004; CuSO4 ×/ 5H2O, 0.004; MnSO4 ×/ H2O, 0.0004; ZnSO4 ×/ 7H2O, 0.00034; Na2MoO4 ×/ 2H2O, 0.00024; pH 7.0. Large-scale cultivation of cells was carried out in a 15l, continuous-gas feed fermentor (LH1075, UK) under an atmosphere of methane and oxygen (1:1, v/v) at 32 8C about 72/96 h. Cells were harvested at 9000 /g for 10 min and washed twice with 20 mM phosphate
Fig. 1. Schematic representation of the MAFFB reactor system.
buffer (pH 7.0). Cells were resuspended in the same buffer containing 5mM MgCl2 (at a cell concentration of 2 /3 mg dry weight cell (dwc) per ml) and used in batch experiments. 2.2. MAFFB reactor set-up A schematic of the three-phase MAFFB reactor is shown in Fig. 1. The MAFFB reactor system consists of a fluidized-bed (A), an absorber (C) and a closed gas loop containing a 2-l gasbag (D). When leaving the absorber and gasbag, the gas is depleted in the product and enriched in growth and/or bioconversion substrate. A mixed culture methanotrophic biofilm was cultivated on diatomite particles (30 g, particle size range: 200/800 mm) located inside the fluidized-bed (30 cm high, 4.0 cm i.d.). The temperature was maintained by circulating water at 32 8C around the fluidized-bed. The NMS liquid medium containing attached and detached methanotrophic cell was recycled from the fluidized bed (A) to clarifier (B) by a pump G-3 (10 ml/h). The clarifier (20 cm high, 1.4 cm i.d.) served as a segregator tube. Detached cells concomitant with effluent (F) exited from the effluent outlet above the clarifier, and were sedimented and recycled into the fluidized bed. In the fluidized bed, diatomite particles with attached methanotrophs were fluidized by an uprising flow of the gas (pump G-1, flow rate /300 ml/h), gaseous substrates were transferred to the liquid phase and volatile product (epoxypropane, bp 35 8C) was transferred to the gas phase and removed from the fluidized-bed. The absorber (C) was held at 0/4 8C and consisted of three 50-ml flasks filled with distilled water. When back in the absorber, the gas phase was stripped of the product epoxypropane and re-established by the gasbag. The gas loop was closed. The fresh NMS medium (E) was fed by
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Fig. 2. MMO activity on diatomite during the continuous culture process.
pump G-2 at a daily addition of 50 ml/day, and surplus NMS medium (F) containing detached cells was exited from the effluent outlet above the clarifier. In the absorber, the majority of the epoxypropene was accumulated and the process yielded a clear solution. The absorption of epoxypropane was accomplished by periodically removing the first flask and replacing it with a new flask at the rear. 2.3. Development of biofilm Pure culture of Methylomonas sp. GYJ3, M. capsulatus IMV 3021 and M. trichosporium IMV 3011, pregrown separately in NMS with 50 methane and 50% oxygen headspace, were used for inoculation. The reactor was not sterilized before inoculation. The reactor was operated in batch mode for 50 days and then continuous NMS medium feed was started by turning on G-2 and G-3. The gas phase was initially maintained at about 50 methane and 50% oxygen with a total pressure of 1.0 atm. As methane and O2 may be utilized at different rates in the MAFFB reactor, the methane and O2 partial pressures in the gasbag may be changed. The initial partial pressures were re-established every 24 h by replacing the gasbag. 2.4. Biosynthesis of epoxypropane in the MAFFB reactor After 65 days of continuous culture, five batch tests with 2-day intervals were conducted in the three-phase MAFFB reactor to optimize the biosynthesis of epoxypropane. In each of five batch tests, mixed gas containing 20 propene, 40% oxygen and various concentrations of methane were fed continuously over a 3day period. At each 2-day interval, growth gas of
methane and oxygen (1:1, v/v) was fed continuously for resumption of bacterial activity and reductant (NADH2) regeneration. A continuous biosynthesis of epoxypropane was conducted by long-term continuousgas feed. 2.5. Batch experiments Batch experiments were conducted in 100 ml sealed conical flasks (under atmospheric pressure) containing 20 /25 ml of washed cell suspension. The gaseous phase of the flask was replaced by the gas mixture containing various concentrations of propene, oxygen, and methane (as described above). The flasks were incubated and shaken (150 rpm) at 35 8C on a controlled temperature rotary shaker. Chromatographic determinations of the liquid phase (epoxypropane) were carried out at different periods of reaction. All batch values are averaged for the three runs with a variation of 3/5% from the mean. 2.6. Analytical methods Epoxypropane formation from propene was determined chromatographically using a gas chromatograph equipped with a capillary GC column(c{0.23 mm /30 m; stationary phase, SE-54) and a flame ionization detector (FID). Pure nitrogen served as the carrier gas at a flow rate of 75 ml/min. The temperatures of the column, detector, and injector were 60, 180, and 180 8C, respectively. Quantification was performed by the method of external standardization. The assays of MMO activity were performed by measuring epoxypropane formation from propene. An assay was performed in a total volume of 1 ml of cell
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Fig. 3. Pathway of propene (or methane) metabolism and NADH regeneration.
suspension sealed in a 10 ml reaction vial. The gaseous phase of the vial was removed by vacuum and replaced with a gas mixture of the propene and air (1:1, v/v). The reaction was started by addition of the gas mixture. Assays were performed at 35 8C for 30 min. Epoxypropane formation from propene was also measured using same gas chromatography. Specific activities were expressed as nanomoles of epoxypropane formed per minute per milligram of dwc. The optical density (OD) of the culture medium was determined by UV spectrophotometry at 550 nm (UV120-02, Shimadzu Co.). Sample preparation for scanning electron microscopy (SEM): The samples were dried by lyophilization at / 50 8C and 10 mmHg, the dried samples were gold sputtered and observed at 30 kV in a JEOL (JSM5600LU) scanning microscope.
3. Results and discussion 3.1. Development of biofilm in three-phase MAFFB reactor Many methanotrophs were reported to form capsular or slime material [9,10], which is known to play an important role in the ability of microorganisms to stick to surfaces. The immobilized cells grow, reproduce, and produce extracellular polymeric substances (EPS) that frequently extend from the cell forming a tangled mass of fibres lending structure to the entire assemblage, which shall be termed a biofilm [11]. In this work, three pure culture strains of Methylomonas sp.GYJ3, M. capsulatus IMV 3021 and M. trichosporium IMV 3011 were co-used as inocula. Biofilm development was monitored through MMO activity measurements. The living cell concentration on diatomite particles was associated with MMO activity on diatomite particles. Fig. 2 shows the dynamics of MMO activity and
biomass on diatomite particles through the biofilm formation process. The biofilm formation dynamics presented sigmoid behavior with two consecutive stages: a lag phase of 23 days and a fast increasing activity phase. The first phase was associated with the initial formation of the biofilm where the methanotrophs move from the liquid to the diatomite particle surface. The second phase was associated with the growth of biofilm. Scour action effects induced by uprising flow of the gas and particles chases detach the more exposed biofilm sections. This factor contributes to biofilm destruction. Furthermore, it is hypothesized that as cells in the biofilm are inactivated, they are detached and replaced by new cells; the cells in the biofilm are transported by advection. Methanotrophs grow on the diatomite particles and equilibrate the detached methanotrophs, a steady state was obtained. The detached methanotrophs at the steady state value corresponds to 10% of the total methanotrophs in the MAFFB reactor. This result can be seen as evidence that the immobilized methanotrophs are robust against detachment induced by scour action effects. The methanotrophic concentration on diatomite particles was 3.3 /3.7 mg dwc per g dry solid (DS).
3.2. Propene concentration Various concentrations of propene were used to examine the production of epoxypropane by cell suspensions of Methylomonas sp. GYJ3, M. capsulatus IMV 3021 and M. trichosporium IMV 3011. The initial oxygen concentration in the gaseous phase was kept constant (40%). Nitrogen gas was used to balance the remainder of the gaseous phase. The amount of epoxypropane produced was assayed after 30 min of incubation. A propene concentration of 20% supported maximum epoxypropane production. Higher propene concentration did not stimulate the production of epoxypropane (data not shown).
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Table 1 Effect of methane on the epoxidation of propene Reaction time (h)
Composition of gaseous phase
1 1 1 1 1 1 2 2 2 2 2 2
Epoxypropene (yM)
Oxygen (%)
Methane (%)
Propene (%)
Nitrogen (%)
40 40 40 40 40 40 40 40 40 40 40 40
0 5 10 20 30 40 0 5 10 20 30 40
20 20 20 20 20 20 20 20 20 20 20 20
40 35 30 20 10 0 40 35 30 20 10 0
738 945 952 1077 1139 406 1021 1327 1389 1582 1076 766
Table 2 Effect of propene on growth of methanotrophs Initial partial pressures (%)
Cell concentration after 72 h fermentation (mg dry weight cell per ml)
Propene
Oxygen
Methane
Nitrogen
0 20 20 20 20 20
50 40 40 40 40 40
50 40 35 30 20 10
0 0 5 10 20 30
0.30 0.27 0.27 0.10 0.03 B/0.01
3.3. Possibility of producing epoxypropane by a propene/ methane cooxidation process According to the Fig. 3, MMO can oxidize propane and methane to epoxypropane and methanol, respectively. As a natural substrate of methanotrophs, methane can be oxidized completely to carbon dioxide via methanol, formaldehyde and formate. Reducing power (NADH2) for monooxygenation was obtained during dehydrogenation of intermediates of methane oxidation [12]. However, epoxypropane cannot be further metabolized and accumulate extracellularly. Epoxidation usually is terminated due to accumulation of toxic product and the exhaustion of reducing power. Epoxypropane synthesis in batch reaction was carried out as described above. To retain epoxidation activity of methanotrophs, methane was selected as a co-substrate for NADH2 regeneration. A single MMO is responsible for the oxidation of both propene and methane, and the presence of methane may inhibit the oxidation of propene. However, after the initial oxidation step, methane may be further degraded to regenerate NADH2, which can promote more propene oxidation. The effect of methane addition on the epoxidation of propene was studied. The production of epoxypropane
from propene by cell suspension of Methylomonas sp. GYJ3 was assayed in the presence of varying amounts of methane. The initial partial pressures of propene and oxygen in the gaseous phase were kept constant in all of the experiments; N2 was used to balance the gaseous phase. As shown in Table 1, the epoxidation ability of Methylomonas sp. GYJ3 cell was enhanced by addition of lower concentrations of methane, suggesting substantial NADH2 limitations for the cells. The maximum production of epoxypropane occurred in an atmosphere of 30% methane. Although methane enhanced the epoxidation ability of Methylomonas sp. GYJ3 cells under an atmosphere of 30% methane, this effect diminished when methane concentration was higher than 30%, suggesting that the positive effect of methane (NADH2 regeneration) can be overwhelmed by its competitive inhibition effect for the higher methane concentration. As for M. capsulatus IMV 3021 and M. trichosporium IMV 3011, almost the same result was observed. 3.4. The effect of propene on the growth of methanotroph Propene did not support the growth of methanotroph. As a single MMO molecule is responsible for the
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Fig. 4. Biosynthesis of epoxypropene in the MAFFB reactor; Feed gas composition: 1, propene, oxygen, nitrogen, 20, 40, 40(kPa); fed continuously over a 3-day period; 2, propene, methane, oxygen, nitrogen, 20, 10, 40, 30(kPa); fed continuously over a 3-day period; 3, propene, methane, oxygen, nitrogen, 20, 20, 40, 20 (kPa); fed continuously over a 3-day period; 4, propene, methane, oxygen, nitrogen, 20, 30, 40, 10 (kPa), fed continuously over a 3-day period; 5, propene, methane, oxygen, 20, 40, 40 (kPa), fed continuously over a 3-day period; 6, propene, methane, oxygen, 20, 35, 45 (kPa); fed continuously for 53 days.
oxidation of both types of substrates, the presence of nonbeneficial propene can inhibit the oxidation rate of growth substrate methane (competitive inhibition). The competition of methane and propene for MMO determined the efficiency of conversion of methane by the cell, which determined the growth rate of methanotrophs. At the same time, during the growth of methanotrophs on methane plus propene, part of the reducing power (NADH2) was used for the epoxidation of propene. To determine if the addition of 20% propene could influence growth of methanotrophs, the bacteria were grown in the presence of 20% propene and various partial pressure of methane in a gas-continuous feed fermentor, respectively. The epoxypropane produced was extracted continuously from the fermentor by gasfeed. Methanotrophic bacteria growth was monitored by measuring OD550 and expressed as dwc. As shown in Table 2, when the partial pressure of methane was more than 35%, addition of 20% propene slightly inhibited the growth of methanotrophs. 3.5. Epoxypropane inhibition test To test whether epoxypropane alone had an inhibitory (or toxic) effect on epoxypropane synthesis with methanotrophs, cells of Methylomonas sp. GYJ3, M. capsulatus IMV 3021 and M. trichosporium IMV 3011 were incubated with both propene and 0.015% of epoxypropane, respectively. The results showed that
the rate of epoxidation was slower than in the standard assay system in which no external epoxypropane was added (data not shown). This result indicates that the epoxypropane produced has an inhibitory effect on epoxidation of propene with methanotrophs. Hence, the process for epoxypropane synthesis should minimize exposure of the cell. In order to avoid product inhibition, continuous removal of toxic product epoxypropane is critical. 3.6. Biosynthesis of epoxypropane in three-phase MAFFB reactor After 65 days of continuous culture to develop the biofilm, five batch tests with 2 days interval were conducted in the three-phase MAFFB reactor to optimize the biosynthesis of epoxypropane. In each of five batch tests, mixed gas containing 20 propene, 40% oxygen and various partial pressures of methane were fed continuously over a 3-day period. At each 2-day interval, growth gas of methane and oxygen (1:1, v/v) was fed continuously for bacterial activity and reducing power (NADH2) regeneration. After this growth mode ended, almost all of the initial MMO activity resumed (Data not shown). This result suggests that rapid processes such as NADH2 regeneration were responsible, rather than slower processes such as the generation of additional cells or MMO. As shown in Fig. 4, when mixed gas-containing 20 of propene, 40 of oxygen and
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40 kPa of nitrogen were fed continuously over a 3-day period, the methanotrophs gradually lost the ability to oxidize propene. Based on earlier batch studies, this was attributed to depletion of reducing power (NADH2). The presence of low amounts of methane enhanced epoxypropane current yield and reduced the decay rate of epoxypropane current yield, presumably due to the regeneration of reducing power (NADH2). However, the effect of enhanced epoxypropane current yield was diminished when the methane concentration was increased, presumably due to competitive inhibition. Comparing the epoxypropane yield and the decay rate of epoxypropane yield of the five tests, 30 kPa of methane versus 20 kPa of propene exhibited the highest formation capacities of epoxypropene. However, as shown in Table 2, under this atmosphere, methanotrophic growth inhibition occurred. To determine whether the slightly lower growth mode could maintain sufficient methanotrophic cells on diatomite particles for biosynthesis of epoxypropane, continuous biosynthesis of epoxypropane was conducted under long term continuous 20 kPa of propene versus 35 kPa of methane feed. As shown in Fig. 4, the reaction yielded epoxypropane with a yield of 110/150 mmol/day. The MAFFB reactor was operated continuously for 53 days without obvious loss of productivity, presumably due to constant sources of reducing power for monooxygenation, continuous removal of toxic product and stable maintenance of methanotrophic cells concentration on diatomite particles. 3.7. SEM studies of the biofilm SEM studies (Fig. 5) showed that there were interstices, pleats and crevices that furrowed a rough surface on the bare diatomite particles. The surface was clearly suitable for colonization and apparently offered a large surface. At the end of the biofilm formation process, the particles were colonized in crevices, pleats and surfaces and showed a covered biofilm. After several days of epoxypropane biosynthesis, the biofilm appeared unchanged.
4. Conclusions
Fig. 5. SEM microphotographs of the methanotroph attached-biofilm immobilized on diatomite. (a) Bare diatomite particles; (b) biofilmcovered diatomite particles; (c) biofilm-covered diatomite particles after 53 days of epoxypropane biosynthesis.
The results reported in this study show that it is possible to accumulate and maintain control of large amounts of methanotrophic cells in a stable attachedfilm fluidized bed reactor system during both growth mode and methane /propene co-oxidation mode. The feasibility of using the three-phase attached-film methanotrophs for continuous biosynthesis of epoxypropane by methane/propene co-oxidation process was described. The effect of methane on the epoxidation of
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propene and the effect of propene on the growth of the methanotroph were also studied. The significant increase in the initial epoxypropane current yield by the addition of an optimal concentration of methane, suggests that NADH2 regeneration was responsible. Further, the diminished initial epoxypropane current yield observed for higher than 30% methane suggests that the positive effect of methane (NADH2 regeneration) can be overwhelmed by its competitive inhibition effect at higher concentrations. Propene did not support growth of methanotroph. Since a single MMO is responsible for the oxidation of both types of substrates, the presence of nonbeneficial propene inhibits the oxidation rate of growth substrate methane. The competition of methane and propene for MMO determined the efficiency of conversion of methane by the cells, which determined the growth rate of methanotrophs. When 20% propene was added into the gas phase in the presence of 30% methane, growth inhibition of methanotrophs was observed. However, when 20% propene was added into the gas phase in the presence of 35% methane, the growth inhibition of methanotrophs was not significant. In this slightly lower growth mode, a sufficient number of methanotrophic cells for cooxidation were still maintained and the MMO activity of the methanotrophic population in the MAFFB reactor system was stable. Propene was epoxidated by the methanotrophs and epoxypropane was accumulated during the reaction. The epoxidation of propene was inhibited if the epoxypropane concentration was enhanced. The biocommunities in the MAFFB, however, continued to epoxidate the propene without obvious inhibition, presumably due to continuous removal of epoxypropane and resistance of sessile methanotrophic populations to epoxypropane. In conclusion, the cooxidation process described in this work shows the potential for commercial applications and can be readily scaled up since it is efficient and tolerates long-term operation.
Acknowledgements The authors would like to thank the National Nature Science Foundation of Chinese (29933040), the Special Funds for Major State Basic Research of China and the Royal Society of UK for the supporting.
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