Selective Immobilization of Aceticlastic Methanogens to Support Material†
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Selective Immobilization of Aceticlastic Methanogens to Support Material† Toshiyuki Nomura*, Takanori Nagao, Akinori Yoshihara, Hayato Tokumoto, Yasuhiro Konishi Department of Chemical Engineering Osaka Prefecture University1 Abstract The effect of electrostatic and hydrophobic properties of microbes in anaerobic sludge on immobilization to support materials was examined. The most popular aceticlastic methanogen, Methanosaeta concilii, was uncharged and hydrophobic. Methanosarcina barkeri of a methyltrophic methanogen, and acidogens cultivated selectively from anaerobic sludge, were negatively charged and hydrophobic. Immobilized microbes on support materials were incubated with sodium acetate. Methanogens were dramatically immobilized to bamboo charcoal, in contact with hydrophilic alumina. Methanosaeta-like microbes were immobilized to bamboo charcoal. These results indicate that the hydrophobic and negatively-charged support material that can suppress the immobilization of microbes except for Methanosaeta species is suitable for selective immobilization of Methanosaeta species, which is the most important microbe in methane fermentation. Keywords: biocolloid, immobilization, methanogen, electrophoretic mobility, hydrophobicity slag, resin, foam stones, and zeolites have been used 1. Introduction for methanogen immobilization. It was reported that The establishment of a “recycling society” in Japan suitable support material has a hydrophobic sur- is rapidly advanced due to improvement in various re- face, or a shape to which microbial cells can easily cycling laws (e.g., “Fundamental Law for Establishing adhere. Contrary reports have also been published; a Sound Material-Cycle Society”). Methane fermenta- the mechanism of cell immobilization is incompletely tion has received renewed attention as a technology understood [1]. The reason is that bacterial strains for producing energy from organic waste with high immobilized on support materials were different water content. Conventional methane fermentation because the type of treated waste water was differ- has various problems: (i) fermentation efficiency is ent; or the composition of waste water was different low, and the treatment of undigested residues is nec- because of the season, even if the same type of waste essary because the growth rate of the methanogens water was used. Many researchers have examined is extremely low; and (ii) wash-out of methanogens in the surface properties of the support material used to the fermenter is carried out. High concentrations of immobilize microbial cells, but few scholars have fo- methanogens must be immobilized in a fermenter to cused on the surface properties of various microbes realize highly efficient methane fermentation. in the methane fermenter. Immobilization of methanogens as a method to If a microbe is considered to be a living particle, a maintain a high concentration in the fermenter has fine-particle technology approach aids elucidation of been investigated. Support materials such as glass, the microbial adhesion phenomenon. When microbes adhere to solid surfaces, an electrostatic, hydropho- † This paper, appeared originally in Japanese in J. Soc. bic, or specific interaction between the surfaces of Powder Technology, Japan, 43, 653-659 (2006), is pub- lished in KONA Powder and Particle Journal with the the solid and the microbial cells are related. In this permission of the editorial committee of the Soc. Powder study, the electrostatic and hydrophobic interactions Technology, Japan were noted as the first step to clarify the mechanism 1 1-1 Gakuen-cho, Sakai, Naka-ku, Osaka, 599-8531, Japan of immobilization of various microbes in the liquid ＊ Corresponding author TEL: 072-254-9300 FAX: 072-254-9911 phase to a solid surface. Selective immobilization of E-mail: [email protected] aceticlastic methanogens (a rate-limiting step in the 246 KONA Powder and Particle Journal No.26 (2008) methane fermentation process) to support materials 3. Materials and Methods was investigated using anaerobic sludge collected from an anaerobic treatment plant. 3.1 Microbial cells and support materials Five typical microbe species were selected to inves- tigate the surface characteristics of microbes living in 2. Process of Methane Fermentation an anaerobic digester. Because the complex organic The methane fermentation process is an artificial materials mainly comprised proteins, carbohydrates, ecosystem in which many types of microbes exist at and lipids, three acidogens that decompose these high density. Anaerobic digestion of complex organic materials were enriched from anaerobic sludge. Pure materials to produce methane comprises a cascade of cultures of Methanosarcina barkeri JCM 10043 and biochemical conversions catalyzed by different physi- Methanosaeta concilii DSM 3671 were the acetate-uti- ological groups of interacting microbes (Fig. 1) [2]. lizing methanogens. Methanosarcina barkeri isolated Complex organic compounds are first hydrolyzed to by Bryant et al. [6] was purchased from the Japan simpler organic compounds before being fermented Collection of Microorganisms (Wako, Japan). Metha- to volatile acids by acidogens. Volatile acids are sub- nosaeta concilii isolated by Patel [7] was purchased sequently converted to acetate and hydrogen gas by from the Deutsche Sammlung von Mikroorganismen hydrogen-producing acetogens. Finally, acetate or und Zellkulturen (Braunschweig, Germany). Anaero- hydrogen is converted to methane and carbon diox- bic sludge was collected from the anaerobic treat- ide by methanogens [3]. In this process, acetate is ment plant at the Yagi Bio-Ecology Center, Kyoto, the precursor for about 70% of the methane produced Japan. during the anaerobic digestion of complex organic Three acidogens (proteolytic bacteria, amylolytic materials [4]. Decarboxylation of acetate is the rate- bacteria, lipolytic bacteria) were enriched at 37℃ and limiting step in anaerobic digestion [1]. Methanosaeta neutral pH in a specific medium supplemented with and Methanosarcina species are the only methano- a specific substrate per 1 L of PGY medium (peptone gens capable of acetate catabolism [5]. High concen- 2 g/L, yeast extract 1 g/L, glucose 0.5 g/L). The spe- trations of acetate-utilizing methanogens must be im- cific substrate for proteolytic bacteria was skimmed mobilized in the anaerobic digester to achieve highly milk (10 g/L), it was amylogen (2 g/l) for amylolytic efficient anaerobic digestion. bacteria, and it was tributyrin (5 g/L) for lipolytic bac- teria. Methanosarcina barkeri and Methanosaeta con- cilii were grown under anaerobic condtions without shaking at 37℃ and neutral pH in pressure culture Fig. 1 Process of methane fermentation (schematic). KONA Powder and Particle Journal No.26 (2008) 247 bottles sealed with a butyl rubber stopper and alumi- cells were resuspended in phosphate buffer (pH 7.0; num crimp seal [8]. A pure culture of Escherichia coli ionic strength, 100 mol/m3). JM 109 was the control microbe. 3.4 Hydrophobicity measurements Precultured microbes were filtered through AD- Surface hydrophobicity of microbial cells was VANTEC No.2 Toyo paper filter to remove residues. determined by microbial adhesion to hydrocarbon Cells were harvested by centrifugation at 10,000 (MATH) assay [9]. Washed cells were resuspended rpm for 10 min, and washed thrice using 0.9% (w/v) in PUM buffer (pH 7.1, K2HPO4•3H2O 22.2 g/L, sterile NaCl aqueous solution. Washed cells were KH2PO4 7.26g/L, Urea 1.8 g/L, MgSO4•7H2O 0.2 resuspended in sterile solution to evaluate the physi- g/L). Subsequently, 0.4 mL of hydrocarbon (n-hexa- cochemical properties of microbial cells. decane) was added to a test tube containing 2.4 mL of Two support materials were used. Bamboo char- washed cell suspension. Mixtures were vortexed uni- coal is a hydrophobic and negatively-charged par- formly for 2 min. The solution was allowed to stand ticle; alumina is a hydrophilic and positively-charged for 15 min to ensure complete separation of the two particle. The size of these support materials was phases. Absorbance of the aqueous cell suspension about 5 mm in diameter. was measured at 400 nm using a spectrophotometer 3.2 Methane fermentation (UVmini-1240, Shimadzu). Hydrophobicity of micro- Methane fermentation was done as follows. Four bial cells and support materials was calculated using milliliters of anaerobic sludge and 1 mL of substrate the following equation: solution were placed into serum bottles of capacity 21 F = (1 – At/A0) ×100 (1) mL (20-CV, Perkin Elmer). Bottles were capped with butyl rubber stoppers and crimped with aluminum where A0 is the initial absorbance of the microbial seals. After sealing, headspaces of the bottles were suspension before mixing, and At is the absorbance purged using a deoxygenized gas pressure injector after mixing. Surface hydrophobicity of support ma- (IP-8, Sanshin) with an oxygen-free 80% N2/20% CO2 terials was evaluated using the crushed ones by the gas mixture at 120 kPa. Serum bottles were subse- same method. quently incubated at 37℃ under the N2/CO2 atmo- 3.5 Immobilization tests of microbes sphere. The initial concentration of the substrate was Immobilization of microbes in anaerobic sludge 20 mol/m3 of the sodium acetate or methanol. Biogas on support material was carried out using the ex- production was determined by thermal conductivity perimental design shown in Fig. 2. The immobiliza- (TCD) gas chromatography (GC-8APT, Shimadzu). tion test comprised three steps: immobilization