Accumulation of polyhydroxybutyrate by a Serratia sp.

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Abstracts / Journal of Biotechnology 136S (2008) S402–S459

2002). It has been drawing much attention as a good candidate for biodegradable and biocompatible plastic material which can be produced from renewable raw materials. Possible applications of PHB include the following: packaging films and containers, biodegradable carriers for controlled chemical and drug release, disposable items, surgical pins and sutures, wound dressings and bone replacements (Lee and ve Choi, 1999). In this study, production capability of Bacillus sp. which was isolated from various areas in Turkey was examined. The hydrolysis products were chosen as carbon sources for the production of poly-3-hydroxybutyric acid (PHB) by Bacillus sp. Accumulation of PHB granules in the organism was analyzed by Sudan black method. In shaking flask experiments, the utilization of molasses and peach pulp as a cheap substrate was compared to the utilization of mineral medium for bacterial growth under balanced conditions as well as for the production of PHB under nitrogen limitation. The amount of synthesized PHB was determined as crotonic acid by spectrophotometer (Gerhardt et al., 1994). Highest PHB (72.2%) production was found in minimal synthetic medium. Highest yield of PHB were 7.92% and 7.78% in the containing molasses and pulp media, respectively. Keywords: Polyhydroxyalkanoates; Bacillus sp.; Molasses; Peach pulp References Choi, J.I., Lee, S.Y., 1997. Process analysis and economic evaluation for poly(3hydroxybutyrate) production by fermentation. Bioprocess. Eng. 17, 335–342. Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R., 1994. Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, DC, p. 628. Jacquel, N., Lo, C.-W., Wei, Y.-H., Wu, H.-S., Wang, S.S., 2008. Isolation and purification of bacterial poly(3-hydroxyalkanoates. Biochem. Eng. J. 39 (1), 15–27. Lee, S.Y., ve Choi, J.I., 1999. Polyhydroxyalkanoates biodegradable polymer. Man. Ind. Microbiol. Biotechnol., 616–627 (Chapter 51). Ustian, T., 2002. Bacterial plastics. Wisc. Edu. Biojournals 21, 1–4.

doi:10.1016/j.jbiotec.2008.07.937

lulose and 20.4% lignin, respectively (Astima et al., 2002). After treating with alkali-microwave, cellulose composition increased to 64%, while hemicellulose and lignin composition reduced to 26% and 8%, respectively. Cellulose composition is also much higher in microwave treatment compared with conventional pre-treatment. Alkali-microwave pre-treated EFB fibre gave 30% soluble glucose higher than conventional pre-treatment when it was hydrolyzed with combination of cellulase and Novozyme 188. The optimum conditions obtained for hydrolysis process were at pH 5, 50 ◦ C and 5:1 cellulase to Novozyme 188 ratio. Keywords: Alkali; Microwave; Pre-treatment; Cellulose; Enzymatic hydrolysis References Astima, A.A., Husin, M., Anis, M., 2002. Preparation of cellulose from oil palm empty fruit bunches via ethanol digestion: effect of acid and alkali catalysts. J. Oil Palm Res. 14, 9–14. Vlasenko, E.Y., Ding, H., Labavitch, J.M., Shoemaker, S.P., 1996. Enzymatic hydrolysis of pre-treated rice straw. Bioresour. Technol. 59, 109–119. Xu, Z., Wang, Q., Jiang, Z.H., Yang, X.X., Ji, Y.Z., 2007. Enzymatic hydrolysis of pretreated soybean straw. Biomass Bioenergy 31, 162–167.

doi:10.1016/j.jbiotec.2008.07.938

V4-O-069 Accumulation of polyhydroxybutyrate by a Serratia sp. Lynne E. Macaskie 1,∗ , Ping Yong 1 , Marion Paterson-Beedle 1 , Harriet Lugg 2 , Rachel L. Sammons 2 , Peter M. Marquis 2 , Mitra Kashani 3 , Mike Jenkins 3 , Artemis Stamboulis 3 1

Unit of Functional Bionanomaterials, School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK 2 School of Dentistry, University of Birmingham, Birmingham B4 6NN, UK 3 School of Metallurgy and Materials Science, The University of Birmingham, Birmingham B15 2TT, UK

V4-O-063

E-mail address: [email protected] (L.E. Macaskie).

Enzymatic hydrolysis of treated palm oil empty fruit bunches fibre (EFB) using combination alkali-microwave techniques

Polyhydroxyalkanoates (PHAs) are microbial storage polymers typically deposited during unbalanced growth, e.g. by limitation for nitrogen or phosphorus in the presence of an excess of carbon source (Anderson and Dawes, 1990). Of these, polyhydroxybutyrate (PHB) (Anderson and Dawes, 1990) has received attention as a potential precursor for biodegradable plastics. A Serratia sp. has been used to biomanufacture nanoscale hydroxyapatite (HA) with potential use in dental and orthopaedic applications and for water purification (Thackray et al., 2004). Bio-synthesis of HA uses calcium, citrate and glycerol 2-phosphate (G2P). G2P is hydrolysed enzymatically with biomineralization of resulting HPO4 2− ions and Ca2+ in the structured exocellular space, with consumption of the citrate by the bacteria. The residual cells show large intracellular electron-transparent inclusion bodies. These were identified as polyhydroxybutyrate (PHB) by analysis of molecular fragments by GC–MS and by FTIR spectroscopy of the isolated bio-PHB in comparison with a commercial reference material. Mass balance analysis (citrate consumed) together with measurement of the extracted material indicated PHB accumulation to up to 77% of the bacterial dry weight. As far as we are aware this is the first report of PHB accumulation by an enterobacterial strain, although the use of genetically modified Escherichia coli has been used previously (Binstock and Schulz, 1981). The use of a natural Serratia sp. overcomes the constraints of using genetically modified organisms, while the economic attractiveness is enhanced by the

Fazlena Hamzah, Ani Idris ∗ Department of Bioprocess, Faculty of Chemical Engineering and Natural Resources Engineering, Universiti Teknologi Malaysia, 41020 Skudai, Johor, Malaysia E-mail address: [email protected] (A. Idris). Combination of alkali-microwave pre-treatment on empty fruit bunches (EFB) fibre changed the morphology and properties of the EFB fibre as observed through scanning electron microscope (SEM) and Fourier transformed infrared spectroscopy (FT-IR). Pre-treatment process raptures the lignin and hemicellulose component inside the fibre, thus leaving available cellulose for hydrolysis process (Vlasenko et al., 1996). Most of the silica component and any impurities on the surface of the raw EFB fibre were removed during pre-treatment process, leaving an empty cauldron on the treated EFB surface. Furthermore, an internal structure of pre-treated EFB fibre showed a clear macrofibril compared to the untreated EFB fibre. The microfibrils of pre-treated fibre were separated from the initial connected structure and this exposed the cellulose to hydrolysis, thus increased the external surface area and the porosity of the pre-treated fibre (Xu et al., 2007). Generally, raw EFB fibre consists of 44.2% alpha cellulose, 33.5% hemicel-

Abstracts / Journal of Biotechnology 136S (2008) S402–S459

co-production of high-value extracellular nanoscale HA and cellbound PHB which are easily separated.

References Anderson, A.J., Dawes, E.A., 1990. Occurrence, metabolism, metabolic role and industrial uses of bacterial poly hydroxyalkanoates. Microbiol. Rev. 54, 450–472. Binstock, J.F., Schulz, H., 1981. Fatty acid oxidation complex from Escherichia coli. Methods Enzymol. 71, 403–411. Thackray, A., Sammons, R.L., Macaskie, L.E., Yong, P., Lugg, H., Marquis, P.M., 2004. Bacterial biosynthesis of a calcium phosphate bone substitute material. J. Mater. Sci.: Mater. Med. 15, 403–406.

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Keywords: Microbial electrolysis cell (MEC); Exoelectrogenic consortia; Non-exoelectrogenic organism; Cooperation; Hydrogen production Reference Liu, H., Gort, S., Logan, B.E., 2005. Electrochemically assisted microbial production of hydrogen from acetate. Environ. Sci. Technol. 39, 4317–4320.

doi:10.1016/j.jbiotec.2008.07.940 V4-O-143

doi:10.1016/j.jbiotec.2008.07.939

Hydrogen bio-production through anaerobic microorganism fermentation using kitchen wastes as substrate

V4-O-142

Yue Shi 1,∗ , Xiu-tao Zhao 1 , Xiao-wen Liu 2 , Nan-qi Ren 2

Functional microorganisms and their cooperation in microbial electrolysis cell (MEC) for H2 production Aijie Wang ∗ , Dan Sun, Lihong Liu, Nanqi Ren, Wenzong Liu, Haoyi Cheng State Key Lab of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China It is well known that microbial electrolysis cell (MEC) was first developed by Liu et al. (2005) for hydrogen production. However, till now, little information presents the remarkable microorganisms that function in a microbial electrochemical assisted hydrogen production reactor, especially, the cooperation of different kinds of functional organisms. In this study, a medium contained poorly crystalline Fe(III)-oxide was used to enrich exoelectrogenic bacteria based on a serial dilution method. Using this method we obtained the simplest-functional-unit (exoelectrogenic consortia) from a double-chamber MEC. Cultivation tests showed that the exoelectrogenic consortia could completely reduce 100 mmol poorly crystalline Fe(III)-oxide using acetate as electron donors and form Fe3 O4 after 216 h cultivation. When the exoelectrogenic consortia was inoculated into a mediator-less microbial fuel cell fed with acetate as sole electron donor, it could utilize acetate to produce electricity, and the maximum current density is 150 mA/m2 , which is equivalent to that produced by sewer sludge under the same conditions. Meanwhile, six non-exoelectrogenic isolates, named W1, W2, W3, W4, W5 and W7 were obtained from the same double-chamber MEC, using Hungate anaerobic rolling-tube technique. Analysis of physiological-biochemical characterization and 16S rRNA gene sequence showed that strain W1 is a member of the genus Citrobacter and the others are members of the genus Bacteroides. Cultivation tests indicated that none of the six isolates could use conventional electron donors (acetate, lactate, formate, propionate, butyrate, citrate, ethanol, cellobiose, and glucose) to reduce Fe(III). Also, none of them could produce electricity in microbial fuel cells using acetate as electron donors. To further investigate the cooperation of different kinds of bacteria, the enrichment of exoelectrogenic consortia and that of Bacteroides strain W7 were simultaneously inoculated to a microbial fuel cell. The result demonstrated that the current density of co-culture of exoelectrogenic consortia and strain W7 could reach 180 mA/m2 , which is higher than that of exoelectrogenic consortia (150 mA/m2 ) about 20% under the same conditions. This indicated that though in such an oligotrophic environment (acetate as the sole electron donor) in microbial fuel cell, there still exist complicated intercellular/extracelluar communications and cooperations between different functional bacteria so as to meet the requirement of electrons and protons transfer.

1

Harbin Engineering University, College of Power & Energy Engineering, Harbin, Hei Long-Jiang Province, China 2 Harbin Institute of Technology, School of Municipal & Environmental Engineering, Harbin, Hei Long-Jiang Province, China E-mail address: [email protected] (Y. Shi). Nowadays, research on the anaerobic fermentation system to produce hydrogen became a hotspot at home and abroad (Chen et al., 2006; Showa et al., 2004). In order to bring down the costs, researchers tried to using low-cost substrates (Han and Shin, 2002; Kim et al., 2004). In this experiment, kitchen wastes were used as substrate to produce hydrogen. The experiment results showed that the fermentation type changed from mixed acid fermentation to ethanol fermentation after a continuous stirred tank reactor (CSTR) started-up for 20 days; the maximum efficiency of hydrogen bio-production of the CSTR was 3.52 L H2 /(L reactor d); the best controlling strategy of operation was as followed: organic loading rate (OLR) of 32–50 kg COD/(m3 d), oxidation reduction potential (ORP) of −450 to −400 mV, influent pH value of 5.5–6.0, effluent pH value of 4.0–4.5, influent alkalinity of 500–650 mg/L, effluent alkalinity of 300–600 mg/L, temperature of 35 ± 1 ◦ C, hydraulic retention time (HRT) of 7 h. An artificial neural network (ANN) model was established, and each parameter influencing the performance of the reactor was compared using the method of partitioning connection weights (PCW). The weight of the influence factors was: OLR > pH values > ORP > alkalinity. The results of experiment showed that producing hydrogen through anaerobic microorganism fermentation using cheap kitchen wastes as substrate was feasible. References Chen, W.H., Chen, S.Y., Khanal, S.K., et al., 2006. Kinetic study of biological hydrogen production by anaerobic fermentation. Int. J. Hydrogen Energy 31 (15), 2170–2178. Han, S.-K., Shin, H.-S., 2002. Biohydrogen production by anaerobic fermentation of food waste. Int. J. Hydrogen Energy 29, 569–577. Kim, S.-H., Han, S.-K., Shin, H.-S., 2004. Feasibility of biohydrogen production by anaerobic co-digestion of food waste and sewage sludge. Int. J. Hydrogen Energy 29 (15), 1607–1616. Showa, K.-Y., Zhang, Z.-P., Tay, J.-H., et al., 2004. Production of hydrogen in a granular sludge-based anaerobic continuous stirred tank reactor. Int. J. Hydrogen Energy 29, 1–10.

doi:10.1016/j.jbiotec.2008.07.941