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, 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 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- 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 of Control cultures were prepared in the same way, but microbes, washing of support materials, and incuba- without dissolving substrates in the content of the tion using immobilized methanogens. Four millili- bottle. Each experiment was done in triplicate. ters of DSM 120 medium inoculated with 5.0% (v/v) 3.3 Measurements of electrophoretic mobility of anaerobic sludge and 1.0 g of support material (EPM) were placed into each of the 21-mL serum bottles. The EPM of microbial cells was measured using Bottles were subsequently capped with butyl rubber an electrophoretic light-scattering spectrophotometer stoppers and crimped with aluminum seals. After (ELS-800, Otsuka Electronics). Washed microbial sealing, headspaces of the bottles were purged with

Fig. 2 Immobilization of microbes in anaerobic sludge and methane fermentation using immobilized microbes on support material.

248 KONA Powder and Particle Journal No.26 (2008) oxygen-free N2/CO2 gas. Sodium acetate was used acetate was the substrate. Generation of methane gas as substrate at an initial concentration of 20 mol/m3. was observed two days after the culture was started Serum bottles were incubated at 37℃ under anaero- when methanol was used. Methanosaeta and Metha- bic conditions. Biogas production was measured to nosarcina species are the only methanogens capable check the activity in each of the three cultures. After of acetate catabolism. Methanosaeta species can use saturation of biogas production (20 days’ later), sup- acetate as sole growth substrate; Methanosarcina spe- port materials were removed from the bottles. Sup- cies can use acetate, H2/CO2, methanol, and methyl- port materials were washed with 0.9% (w/v) sterile amines as growth substrates [5]. When acetate and NaCl aqueous solution to remove the residues on the methanol are in the culture simultaneously, Methano- support materials. Washed support materials were sarcina species suppress the metabolizing of acetate, put into new 21-mL serum bottles filled with 4.0 mL and use methanol preferentially [10]. Methanosaeta of DSM 120 medium dissolved in 20 mol/m3 of sub- species were the dominant species in the anaerobic strate. Cultures were grown at 37℃ in an atmosphere sludge used in our study. of N2/CO2. The substrates used were the same at the Methane conversion ratio using different sub- first and final steps. Biogas production was deter- strates with incubation time is shown in Fig. 4. The mined by TCD gas chromatography final ratio of methane conversion of acetate and meth- 3.6 Direct observation of microbes anol was 55% and 75%, respectively. The stoichiomet- Microbes immobilized onto support materi- ric equations and free energy change ∆G0’ for stan- als were observed directly using a field emission dard conditions of substrates converted to methane scanning electron microscope (JSM-6700F, JEOL). are [11]: – － Samples were fixed with glutaraldehyde solution, and CH3COO ＋H2O→CH4＋HCO3 dehydrated in a graded series of acetone. Samples (∆G0’= –30 kJ/mol) (2) － + were washed in tert-butyl alcohol to remove acetone 4CH3OH→3CH4＋HCO3 ＋H ＋H2O and frozen in a refrigerator. The frozen sample was (∆G0’= –314 kJ/mol) (3) freeze-dried using a vacuum freeze drier (ES-2020, The substrates of sodium acetate and methanol in Hitachi). the bottles were almost fully converted to methane gas because the theoretical methane conversion ratio based on carbon balance was 50% and 75%, respec- 4. Results and Discussion tively. 4.1 Methane fermentation 4.2 Electrostatic interaction Fig. 3 shows methane production using different Table 1 shows the EPM of five typical microbes substrates with incubation time. Methane gas was living in an anaerobic digester. EPM measurement generated from the start of incubation when sodium was conducted in 100 mol/m3 phosphate buffer at pH

Fig. 3 Methane production using different substrates with incubation Fig. 4 Methane conversion ratio using different substrates with incuba- time. tion time.

KONA Powder and Particle Journal No.26 (2008) 249 Table 1 Electrophoretic mobility of microbes measured by Laser Doppler method (pH 7.0; ionic strength, 100 mol/m3)

7.0. Zeta potentials calculated by the Smoluchowski Table 2 Hydrophobicity of microbes and support materials measured equation are shown in Table 1. Polymer chains grew by MATH method on the surface of microbial cells like seaweed. Solu- tion flow in this polymer layer cannot be disregarded, so the zeta potential calculated from the Smoluchows- ki equation is a reference value [12–14]. Proteolytic bacteria, amylolytic bacteria, and lipolytic bacteria enriched from the anaerobic sludge were found to be negatively charged. Methanosarcina barkeri was also negatively charged. In general, most microbial cells are negatively charged at neutral pH (e.g., Esch- erichia coli). The EPM of Methanosaeta concilii was significantly smaller than that of the three acidogens, as well as Methanosarcina barkeri. It was shown EPM measurements into consideration, we can spec- that Methanosaeta concilii was uncharged. This is ulate that Methanosaeta species (the most important a unique result compared with that from general microbes in the anaerobic sludge) selectively adhere microbes. There are only few kinds of microbes re- onto bamboo charcoal. ported to be uncharged at neutral pH [15]. 4.4 Immobilizing of acetate-utilizing methano- Based on EPM measurements, we can postulate gens the following. The acidogens and Methanosarcina Fig. 5 shows methane production using immobi- barkeri easily adhere to alumina by electrostatic at- lized microbes with incubation time. Methane pro- tractive forces, but not to bamboo charcoal. Methano- duction increased gradually when bamboo charcoal saeta concilii easily adheres to bamboo charcoal and was used as a support material. Methane production alumina by van der Waals interaction because Metha- was barely observed when alumina was used. These nosaeta concilii is uncharged. results indicated that acetate-utilizing methanogen 4.3 Hydrophobic interaction adhered to the surfaces of bamboo charcoal, but not Table 2 shows the hydrophobicity of microbes to alumina. This agreed well with the prediction led and support materials measured by MATH method. by the measurements of EPM and hydrophobicity of The percentage of hydrophilic Escherichia coli ad- five typical microbes. hering to hydrocarbon was 6.4%. The adherence of Fig. 6 shows scanning electron microscope imag- Methanosaeta concilii, Methanosarcina barkeri and es of microbes and immobilized microbes on support the three acidogens was >50%. These five typical mi- material. Many rod-shaped bacteria were in the cul- crobes were hydrophobic. The adherence of bamboo ture solution (Fig. 6a), but the rod-shaped Methano- charcoal to n-hexadecane was 86.6%, and to alumina saeta-like species adhered to bamboo charcoal (Fig. was 7.3%. It was confirmed that bamboo charcoal was 6b), and microbes barely adhered to alumina (Fig. hydrophobic, and alumina was hydrophilic. 6c). Methanosaeta species were found to selectively Based on hydrophobicity measurements, we can adhere to bamboo charcoal because the proportion postulate the following. The acidogens and Methano- of Methanosaeta species on bamboo charcoal was ob- sarcina barkeri easily adhere to bamboo charcoal by viously higher than that in the culture solution. hydrophobic interaction, but not to alumina. Taking Microbial adhesion will be energetically more

250 KONA Powder and Particle Journal No.26 (2008) Fig. 5 Methane production using immobilized microbes with incubation time.

ence in their hydrophobicity. Methanosaeta species is the most important microbe in the methane fermen- tation, and is uncharged and hydrophobic. Microbes except for Methanosaeta species in the fermenter are negatively charged and hydrophobic. The hydro- phobic and negatively charged support material that can suppress immobilization of microbes except for Methanosaeta species is suitable for selective immo- bilization of Methanosaeta species.

5. Conclusions

Selective immobilization of aceticlastic methano- gens, which was a rate-limiting step in the methane fermentation process, onto support materials was investigated using anaerobic sludge. Acidogens cultivated selectively from the anaerobic sludge and Fig. 6 Scanning electron microscope images of microbes and immobi- lized microbes on support material. Methanosarcina barkeri JCM 10043 were negatively charged and hydrophobic. Methanosaeta concilii DSM 3671 was uncharged and hydrophobic. This is favorable because the surface tension of the solid a unique result compared with that seen in general increases if the surface tension of the liquid is larger microbes. Immobilized microbes on support materi- than that of the bacterium [16]. Based on the above als (bamboo charcoal or alumina) were incubated reports and the immobilization test, we can postu- with sodium acetate. Methanogens were dramatically late the following. The electrostatic repulsive force immobilized to bamboo charcoal (negatively charged between Methanosaeta species and bamboo charcoal and hydrophobic) in contract to alumina (positively is barely present. Methanosaeta species approach charged and hydrophilic). It was also proven that bamboo charcoal by van der Waals force and adhere Methanosaeta-like microbes were immobilized to to bamboo charcoal by hydrophobic attractive force. bamboo charcoal. These results indicate that the hy- Because the microbes except for Methanosaeta drophobic and negatively charged support material species are negatively charged and hydrophobic, that can suppress the immobilization of microbes ex- adhesion to bamboo charcoal can be suppressed by cept for Methanosaeta species is suitable for selective the electrostatic repulsive force, but a part of the mi- immobilization of Methanosaeta species, which is the crobes adhered by the hydrophobic attractive force. most important microbe in methane fermentation. The reason why microbes hardly adhered to alumina is thought to be because microbes can approach alu- Acknowledgment mina by the electrostatic attractive force, but cannot stably immobilize onto the surface due to the differ- This work was supported by a Grant-in-Aid for

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252 KONA Powder and Particle Journal No.26 (2008) Author’s short biography

Toshiyuki Nomura Dr. Toshiyuki Nomura is an Associate Professor of the Department of Chemical Engineering at Osaka Prefecture University. He received his B.S. in 1993 and M.S. in 1995 degrees from the Department of Chemical Engineering at Kyoto Univer- sity. He obtained the Doctor degree of Engineering at Osaka Prefecture University in 1999. Currently, he is an editor of Journal of the Society of Powder Technology, Japan and Journal of Advanced Powder Technology. His major research interests are microbial adhesion and its application, shape and size control of nanoparticles, and microcapsule preparation.

Takanori Nagao Mr. Takanori Nagao received his B.S. in 2004 and M.S. in 2006 from the Depart- ment of Chemical Engineering at Osaka Prefecture University. Since 2006, he has been working at Aica Kogyo Co., Ltd.

Akinori Yoshihara Mr. Akinori Yoshihara received his B.S. in 2006 and M.S. in 2008 degrees from the Department of Chemical Engineering at Osaka Prefecture University. Since 2008, he has been working at Sumitomo Bakelite Co., Ltd.

Hayato Tokumoto Dr. Hayato Tokumoto was appointed in 2005 to a Research Associate in the Depart- ment of Chemical Engineering at the Osaka Prefecture University. He received his Doctor degree in Division of Biology & Geosciences, Faculty of Sciences at Osaka City University in 2002. He has as a background in biology, physiology, biological organic chemistry and biochemistry.

Yasuhiro Konishi Yasuhiro Konishi is a professor of chemical engineering at Osaka Prefecture Uni- versity, where he earned his doctor’s degree in 1983. Konishi joined the faculty of the Department of Chemical Engineering at Osaka Prefecture University in 1983, and he has spent 25 years in research and lecturing. Also, he was a postdoctoral scholar in the Department of Materials Science and Mineral Engineering at the University of California at Berkeley from 1988 to 1989. Konishi’s research focuses on biochemical engineering in the processing and behavior of materials, natural minerals, wastes and effluents.

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