Environmental Sciences, 13, 4 (2006) 185 192 R. Mitsui et al 185 MYU Tokyo ES620 Formaldehyde Uptake by Methylobacterium sp. MF1 and Acidomonas Methanolica MB 58 with the Different Formaldehyde Assimilation Pathways Ryoji Mitsui, Hideaki Kitazawa, Takayuki Sato and Mitsuo Tanaka * Department of Biochemistry, Faculty of Science, Okayama University of Science 1-1 Ridai-cho, Okayama city, Okayama 700-0005, Japan (Received October 17, 2005; accepted March 7, 2006) Key words: pathways methylotrophic bacteria, formaldehyde uptake, continuous cultivation, C1 metabolic Methylobacterium sp. MF1 (an obligate methylotrophic bacterium isolated newly by the authors) and Acidomonas methanolica MB58 (a facultative methylotrophic bacterium) uptake formaldehyde similarly. It was found that the former assimilated formaldehyde via the serine pathway whereas the latter did so via the ribulose-monophosphate pathway from the measurement of the key enzyme activities in each assimilation pathway. That is, hydroxy pyruvate reductase was detected in only the above-mentioned MF1 strain, but hexulose phosphate synthase (HPS) was not. The efficiencies of formaldehyde consumption by both strains under a continuous chemostat cultivation in the steady state were almost the same in spite of their different assimilation pathways. That is, the consumption efficiencies of the MF strain and the MB58 strain were ca. 1.2 g/l/d and ca. 1.8 g/l/d, respectively, under the experimental conditions. In the future, optimal continuous operating conditions will be investigated. 1. Introduction Formaldehyde is widely used and is important in the chemical industry as a solvent and raw material for the production of phenolic polymers. The domestic output of Japan is about 1.1 million tons per year. (1) At the end of the production process, formaldehyde-containing * E-mail: mtanaka@dbc.ous.ac.jp 185
186 Environmental Sciences, 13, 4 (2006) 185 192 R. Mitsui et al. wastewater is generated and wasted 16,000 tons per year in Japan. (2) Such wastewater cannot be discarded into the environment and is accepted in wastewater treatment plants only when it is below certain chemical oxygen demand (COD) and toxicity limits. Formaldehyde is highly toxic to most living organisms because it reacts nonspecifically with protein and nucleic acids and is known to cause cancer and sick-house syndrome. Therefore, various methods of formaldehyde removal from wastewater have been investigated. (3 5) There are several reports on formaldehyde removal using microorganisms, for example, Rhodococcus erythropolis (6) and Hansenula polymorpha. (7) Formaldehyde is a metabolic intermediate of C1 compounds, such as methane and methanol, in methylotrophs. Methanol is oxidized by methanol dehydrogenase (MDH) to give intermediate formaldehyde in the C1 metabolic pathway. Since formaldehyde in microorganic cells is highly toxic, it must be oxidized or assimilated immediately. It has been reported that formaldehyde-fixation enzymes, such as hexulose phosphate synthase (HPS) and hexulose phosphate isomerase (PHI) are involved in the detoxification system. (8) In this study, we show that Methylobacterium sp. MF1 (an obligate methylotrophic bacterium isolated by the authors) and Acidomonas methanolica MB58 (a facultative methylotrophic bacterium) can grow on formaldehyde as the sole carbon source and that formaldehyde is efficiently consumed in continuous culture by these microorganisms. Furthermore, we show that these microorganisms assimilate formaldehyde by different C1 metabolic pathways from the analysis of enzymes involved in the formaldehyde metabolic system. 2. Materials and Methods 2.1 Microorganisms used The microorganisms used were Methylobacterium sp. MF1 and Acidomonas methanolica MB58. The former was newly isolated from a soil sample using a medium containing formaldehyde as the sole carbon source. The composition of the medium was 0.3 1.5 g/l formaldehyde, 0.2% NaNO 3, 0.2% (NH 4 ) 2 SO 4, 0.2% K 2 HPO 4, 0.1% KH 2 PO 4, 0.02% MgSO 4 7H 2 O, 0.02 % yeast extract and trace vitamin and metal solutions. The ph of the medium was adjusted to 7.0. Cultivation was carried out for 5 7 days at 28 C with shaking, and repeated several times for enrichment. The enrichment culture was spread on the above medium solidified with agar. The taxonomical characteristics of the isolated bacterium were investigated. The sequence of the 16S rdna showed that the bacterium belonged to the genus Methylobacterium, although it did not match any known species. On the basis of these results, this strain was named Methylobacterium sp. MF1. (8) Although the MB58 strain was grown on several multicarbon sources, the MF1 strain could grow on only methanol and formaldehyde. The following compounds were not utilized by the MF1 strain: glucose, L- arabinose, D-mannitol, maltose, D-mannose, gluconate, n-capric acid, adipic acid, citrate, ethanol, acetaldehyde, phenyl acetate and N-acetyl D-glucosamine. Methylobacterium sp. MF1 was found to be an obligate methylotroph from the above results and Acidomonas methanolica MB58, (9) purchased from the Japan Collection of Microorganisms, was a facultative methylotroph.
Environmental Sciences, 13, 4 (2006) 185 192 R. Mitsui et al 187 2.2 Cultivation Using fed-batch cultivation as a preliminary test, the basic conditions of continuous cultivation were estimated. The components of the culture medium were as follows: formaldehyde (HCHO), 0.45 9 g/l; NaNO 3, 2.0 g/l; (NH 4 ) 2 SO 4, 2.0 g/l; K 2 HPO 4, 2.0 g/l; KH 2 PO 4, 1.0 g/l; MgSO 4 7H 2 O, 0.2 g/l; yeast extract, 0.2 g/l; vitamin solution, 2.0 ml/ L and metal solution, 10 ml/l. The vitamin solution contained Ca-pantothenate, 0.4 g/l; inositol, 0.2 g/l; niacin, 0.4 g/l; p-aminobenzoate, 0.2 g/l; pyridoxine, 0.2 g/l; thiamin, 0.2 g/l; biotin, 2.0 mg/l; and vitamin B 12, 0.5 mg/l. The metal solution contained CaCl 2 2H 2 O, 2.0 g/l; H 3 BO 3, 2.5 g/l; CuSO 4 5H 2 O, 0.2 g/l; KI, 0.5 g/l; FeSO 4 7H 2 O, 1.0 g/l; MnSO 4 4 7H 2 O, 2.0 g/l; and NaMoO 4 7H 2 O, 0.5 g/l. First, continuous cultivation with Methylobacterium sp. MF1 was performed using the apparatus shown in Fig. 1. In the continuous chemostat cultivation, formaldehyde was the limiting substrate. The initial cell turbidity was adjusted to 0.2 at a wavelength of 610 nm. The aeration rate was 0.018 Nm 3 /h and the working volume of the culture tank was 1000 ml. The culture liquid, containing 6 g/l formaldehyde in the culture liquid reservoir, was supplied to the tank via a microtube pump @, which controlled the dilution rate from 0.003 h 1 to 0.024 h 1. The ph of the cultivation was 7.0. Culture broth containing the cells was recovered using a separate microtube pump A. All cultivations were performed at 28 C. Optimal steady-state continuous chemostat cultivation was performed under the conditions of an initial cell turbidity of 3.0, an initial formaldehyde concentration of 0.45 g/l in the tank, a supply rate of 8.0 ml/h of the culture liquid containing 6 g/l formaldehyde to the tank (dilution rate was 0.008 h 1 ) and an aeration rate of 0.018 Nm 3 /h. On the basis of the results of fed-batch cultivation and continuous chemostat cultivations, an optimal steady-state continuous chemostat cultivation of Acidomonas methanolica MB58 was performed under the conditions of an initial cell Fig. 1. Outline of apparatus for continuous chemostat cultivation.
188 Environmental Sciences, 13, 4 (2006) 185 192 R. Mitsui et al. turbidity of 1.0, an initial formaldehyde concentration in the tank, a supply rate of 9 ml/h of the culture liquid containing 9 g/l formaldehyde to the tank (dilution rate was 0.009 h 1 ) and an aeration rate of 0.018 Nm 3 /h. The ph of the cultivation was 4.0. It was confirmed by a preliminary test that formaldehyde was not lost in the experiment. 2.3 Measurement of formaldehyde concentration The concentration of formaldehyde was estimated using a standard enzyme assay. The oxidation of formaldehyde by glutathione (GSH)-independent formaldehyde dehydrogenase was followed by measuring the formation of NADH spectrophotometrically at a wavelength of 340 nm. 2.4 Cell-free extract A continuous cultivation similar to that of 2.2 was performed with methanol or formaldehyde as a carbon source. Cells in the steady state were harvested by centrifugation at 7,000 x g and the cell pellet was washed twice with 50 mm potassium phosphate buffer (ph 7.0). The washed cell pellet was resuspended in the same buffer. The wet cells (10 w/v% in 50 mm potassium phosphate buffer, ph 7.0) were disrupted by sonication at 9 khz and 170 W for 10 min with icing to protect enzyme activity. The sonicate was clarified by centrifugation at 15,000 x g for 20 min at 4 C. The resultant supernatant was used as the cellfree extract. 2.5 Enzyme assay Protein concentrations were determined by the Lowry method using bovine serum albumin as a standard. Methanol dehydrogenase activity was measured by a dye-linked assay with phenazine ethosulfate. Hydroxypyruvate reductase, as the marker enzyme of the serine pathway for formaldehyde assimilation, was assayed by measuring the formation of NADH spectrophotometrically at 340 nm. Hexulose phosphate synthase (HPS) was measured by the rate of the Ru5P (ribulose 5-phosphate)-dependent disappearance of formaldehyde. (10) 3. Results and Discussion 3.1 Formaldehyde-limited chemostat cultivation First, we investigated formaldehyde uptake by Methylobacterium sp. MF1. In the continuous chemostat culture, several dilution rates were used. The results are shown in Fig. 2. When the dilution rate was lower than 0.012 h 1, the formaldehyde in the culture tank was completely consumed and cell turbidity increased, reaching ca. 2.0 as dilution rate increased. However, when the dilution rate was higher than 0.018 h 1, formaldehyde gradually accumulated in the culture tank resulting in a monotonic decrease in cell turbidity. These results suggest that the optimal dilution rate was around 0.012 h 1. On the basis of this result, as outlined in 2.2, optimal continuous chemostat cultivation was performed. As shown in Fig. 3, in the initial culture period, a decrease in cell turbidity and a slight accumulation of formaldehyde (ca. 6 mg/l) was detected. However, from 50 h to at least
Environmental Sciences, 13, 4 (2006) 185 192 R. Mitsui et al 189 Fig. 2. Chemostat culture of the MF1 strain for obtaining the optimal conditions. Symbols:, Cell turbidity (-);, Formaldehyde (g/l). Fig. 3. Continuous culture of the MF1 strain under the optimal conditions. Symbols:, Cell turbidity (-);, Formaldehyde (g/l).
190 Environmental Sciences, 13, 4 (2006) 185 192 R. Mitsui et al. 600 h of cultivation time, cell turbidity and the rate of formaldehyde decomposition remained in the steady state. The efficiency of formaldehyde consumption was ca. 1.2 g/l/d. Secondly, we investigated formaldehyde uptake by Acidomonas methanolica MB58. In a continuous chemostat culture, several dilution rates were used. The results are shown in Fig. 4. When the dilution rate was lower than 0.009 h 1, the formaldehyde in the culture tank was remarkably consumed and cell turbidity increased, reaching ca.1.0 as dilution rate increased. However, when the dilution rate was higher than 0.021 h 1, formaldehyde did not accumulate in the culture tank, even though cell turbidity in the tank decreased slightly. These results suggest that the optimal dilution rate was at least around 0.012 h 1 0.024 h 1. On the basis of this result, as outlined in 2.2, optimal continuous chemostat cultivation was performed. In this case, the culture liquid containing 9 g/l formaldehyde in the culture liquid reservoir was supplied, even though the dilution rate was 0.009 h 1. As shown in Fig. 5, in the initial culture period, a decrease in cell turbidity occurred. From around 100 h to at least 300 h of cultivation, cell turbidity and the decomposition rate of formaldehyde remained in the steady state. Although a small amount of formaldehyde remained in the tank (ca. 18 mg/l), the efficiency of formaldehyde consumption was ca. 1.8 g/l/d. That there was residual formaldehyde shows that the operating conditions can be optimized further. By lowering the dilution rate, the formaldehyde in the tank was completely consumed, for example, when the supply rate of the culture liquid containing 12 g/l formaldehyde to the tank was 3 ml/h Fig. 4. Chemostat culture of the MB58 strain for obtaining the optimal conditions. Symbols:, Cell turbidity (-);, Formaldehyde (g/l).
Environmental Sciences, 13, 4 (2006) 185 192 R. Mitsui et al 191 Fig. 5. Continuous culture of the MB58 strain under the optimal conditions. Symbols are the same as in Fig. 3. (dilution rate: 0.003 h 1 ). The formaldehyde uptake behaviors of the above-mentioned two methylotrophs (one obligate and the other facultative) were almost the same, nevertheless the bacteria have different assimilation pathways. 3.2 Comparison of formaldehyde metabolic pathways It was demonstrated that the formaldehyde metabolic pathways were different between Methylobacterium sp. MF1 and Acidomonas methanolica MB58 from the analysis of enzymes involved in formaldehyde assimilation. Methylotrophic bacteria utilize methanol as a sole carbon source and assimilate formaldehyde as a metabolic intermediate using methanol dehydrogenase. Tables 1 and 2 show each enzyme activity when the carbon substrate was methanol or formaldehyde. The key enzyme of the ribulose-monophosphate pathway is HPS, which was not detected in the MF1 strain. However, the marker enzyme of the serine pathway, hydroxy pyruvate reductase, was detected in this strain, (8) nevertheless HPS was not. This result shows that formaldehyde was assimilated via the serine pathway in the MF1 strain. On the other hand, it was reported that Acidmonas methanolica MB58 assimilates formaldehyde via the ribulose-monophosphate pathway. (9) Even though the assimilation pathways were different between these strains, formaldehyde in the medium was consumed effectively on the chemostat cultivation. It is interesting that the above two strains having different properties (one is an obligate methylotroph and the other is a facultative methylotroph) incorporate and assimilate formaldehyde similarly as a substrate
192 Environmental Sciences, 13, 4 (2006) 185 192 R. Mitsui et al. Table 1 Enzyme activities of cell-free extract from methanol or formaldehyde-grown cells of MF1 strain. Activity (U/mg) Carbon source MDH HPS Hydroxy pyruvate reductase Methanol 0.84 N.D. 0.31 Formaldehyde 0.78 N.D. 0.34 N.D.: Not detected Table 2 Enzyme activities of cell-free extract from methanol or formaldehyde-grown cells of MB58 strain. Activity (U/mg) Carbon source MDH HPS Hydroxy pyruvate reductase Methanol 2.25 4.86 N.D. Formaldehyde 0.99 4.73 N.D. N.D.: Not detected from outside cells via either the serine pathway or the ribulose-monophosphate pathway. Methylotropic bacteria may have an excess expression activity of the enzymes involved in the assimilation of formaldehyde, because formaldehyde accumulation causes the death of bacterial cells. References 1 The Chemical Daily (Japanese), Jan., (2005). 2 PRTR data in Japan (2003). 3 Eiroa, M., Vilar, A., Kennes, C. and Veiga, M.C. (2006): Biological treatment of industrial wastewater containing formaldehyde and formic acid. Water SA. 32: 115 118. 4 Eiroa, M., Kennes, C. and Veiga, M.C. (2004): Formaldehyde biodegradation and its inhibitory effect on nitrification. J. Chem. Technol. & Biotechnol. 79: 499 504. 5 Mingbo, Qu. and Bhattacharya, S.K. (2000): Toxicity and biodegradation of formaldehyde in anaerobic methanogenic culture. Biotechnol.and Bioeng. 55: 727 736. 6 Hidalgo, A., Lopated, A., Prieto, M., Serra J. L. and Llama, M. J. (2002): Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1. Appl. Microbiol. Biotechnol. 58: 260 263. 7 Kaszycki, P., Tyszka, M., Malec, P. and Koloczek, H. (2001): Formaldehyde and methanol biodegradation with the methylotrophic yeast Hansenula polymorpha. An application to real wastewater treatment. Biodegradation 12: 167 177. 8 Mitsui, R., Omori, M., Kitazawa, H. and Tanaka, M. (2005): Formaldehyde-limited cultivation of a newly isolated methylotrophic bacterium, Methylobacterium sp. MF; Enzymatic analysis related to C1 metabolism. J. Biosci. Bioeng. 99: 18 22. 9 Grundig, M.W. and Babel, W. (1989): Detoxification of formaldehyde by acetic acid bacteria. Zentralbl. Hyg. Umweltmed. 188: 466-474. 10 Kato, N., Ohashi, H., Tani, Y. and Ogata, K. (1978): 3-Hexulosephosphate synthase from Methylomonas aminofaciens 77a. Biochim. Biophys. Acta. 523: 236 244.