Denitrification inhibiting sulfate reducing bacteria (SRB) activity in an anaerobic baffled reactor (ABR): Effect factors and mechanism analysis

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Abstracts / Journal of Biotechnology 136S (2008) S506–S518

Shang, L.A., Fan, D.D., Kim, M.I., Choi, J., Chang, H.N., 2007. Modelling of poly(hydroxybutyrate) production by high cell density culture of Ralstonia eutropha. Biotechnol. Bioprocess Eng. 12, 417–423.

doi:10.1016/j.jbiotec.2008.07.632 V7-P-009 Displacement of endogenous iron in lipoxygenase by exogenous copper ions Yan Cai 2,∗ , Yong-Mei Xia 1,2 , Yun Fang 2 , Ya-Fen Su 2 1

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China 2 School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China E-mail address: [email protected] (Y. Cai).

The interaction between exogenous and endogenous metal ions of metalloenzyme is very important in biocatalysis. Each molecule soybean lipoxygenase (LOX) contains one atom of endogenous Fe(II) (Cai et al., 2004), which is trapped in octahedral crystal cave and its activity can be accelerated by exogenous Cu2+ (André et al., 2004). However, the mechanism of the interaction remains unclear. According to Irving–Williams series theory and the fact that Cu2+ can also be trapped in octahedral crystal cave, we proposed that Cu2+ in solution trends to displace the endogenous Fe(II) in native LOX. To verify the hypothesis, one of the key technologies is to prove Cu2+ does displace Fe(II) instead of simply associating with the native LOX, qualitively and quantitively. Two indicators, p-carboxylantipyrylazo and 9-(4-carboxyphenyl)-2,3,7trihydroxyl-6-fluorone(CTF) (Li et al., 2004) were used to track the exchanging. After mixing LOX with copper sulfate solution, a missing of 0.53 ␮mol/L Cu2+ in the solution was observed while an increase of 0.56 ␮mol/L Fe2+ was detected in the solution meanwhile, which indicates that the exogenous Cu2+ displaced the endogenous Fe(II), and the replacement ratio of Fe(II) with Cu2+ was 3.48%.

Epoxide hydrolases from microbial sources have been increasingly recognized as highly versatile biocatalysts for the preparation of enantiopure epoxides and vicinal diols an important group of enantiomerically pure compounds to the chemical and pharmaceutical industries (Jaeger et al., 2001; Liu et al., 2007b). In our previous work, we have reported that a newly isolated Rhodococcus opacus ML-0004 producing the epoxide hydrolase. Also, a new gene encoding the epoxide hydrolase from R. opacus ML-0004 was cloned and expressed in Escherichia coli. The aim of this study is to further increase and optimize the epoxide hydrolase activity using the recombinant E. coli harboring epoxide hydrolase gene by response surface methodology. The maximal activity of epoxide hydrolase activity (5.5 U/mg) was predicted to occur when temperature = 30 ◦ C, inducer (IPTG) concentration = 0.5 mM, and duration of the induction period = 18 h. A repeat production of recombinant epoxide hydrolase by E. coli was carried out in a 5-L fermentor under the optimized conditions for the verification of optimization. The maximum epoxide hydrolase activity was 5.9 U/mg, which was significantly higher than that obtained under unoptimized conditions. References Jaeger, K.E., Eggert, T., Eipper, A., Reetz, M.T., 2001. Directed evolution and the creation of enantioselective biocatalysts. Appl. Microbiol. Biotechnol. 55, 519–530. Liu, Z.Q., Li, Y., Ping, L.F., Xu, Y.Y., Cui, F.J., Xue, Y.P., Zheng, Y.G., 2007a. Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production. Process Biochem. 42, 889–894. Liu, Z.Q., Li, Y., Xu, Y.Y., Ping, L.F., Zheng, Y.G., 2007b. Cloning, sequencing, and expression of a novel epoxide hydrolase gene from Rhodococcus opacus in Escherichia coli and characterization of enzyme. Appl. Microbiol. Biotechnol. 74, 99–106. Steinreiber, A., Faber, K., 2001. Microbial epoxide hydroxylases for preparative biotransformations. Curr. Opin. Biotechnol. 12, 552–558. Weijers, C.A.G.M., de Bont, J.A.M., 1999. Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis. J. Mol. Catal. B. 6, 199–214.

doi:10.1016/j.jbiotec.2008.07.634 V7-P-011

References André, K., Rayko, H., Ian, T.B., 2004. Silencing the jasmonate cascade: induced plant defenses and insect populations. Science 305, 665–668. Cai, K., Fang, Y., Xia, Y.M., Su, Y.F., 2004. Effect of exogenous iron on aerobic catalytic mechanism of soybean lipoxygenase. J. Mol. Catal. B: Enzym. 32, 21–26. Li, Z.J., Fan, Y., Liu, Z.Y., Tang, J., 2004. Spectrophotometric determination of iron(III)-dimethyldith-iocarbamate (ferbam) using 9-(4-carboxyphenyl)-2,3,7trihyoxyl-6-fluorone. Talanta 63, 647–651.

doi:10.1016/j.jbiotec.2008.07.633 V7-P-010 Optimization of the recombinant epoxide hydrolase activity from Escherichia coli using response surface methodology Zhiqiang Liu, Yuguo Zheng ∗ Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, PR China E-mail address: [email protected] (Y. Zheng). Epoxide hydrolases (EHs; EC 3.3.2.x) can hydrolyze an epoxide to its corresponding vicinal diol with the addition of a water molecule and without the need for any cofactors and prosthetic groups or metal ions for keeping their activities (Steinreiber and Faber, 2001). They were found in various mammals, insects, plants and microorganisms (Weijers and de Bont, 1999; Liu et al., 2007a).

Denitrification inhibiting sulfate reducing bacteria (SRB) activity in an anaerobic baffled reactor (ABR): Effect factors and mechanism analysis Zhaohan Zhang 1,2,∗ , Guangmin Liu 1 , Zhongxi Chen 3 , Zhiming Shu 3 , Yujie Feng 2 1 Department of Environmental Engineering, Harbin Engineering University, Harbin 150001, China 2 State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China 3 Daqing Oilfield Design and Research Institute, Daqing 163712, China

E-mail address: [email protected] (Z. Zhang). For controlling the equipment corrosion caused by the microbial production of hydrogen sulfide in oilfield land system, an experiment of denitrification inhibiting sulfate reduction was conducted in the continuous-flow ABR. Influential factors and running effect of denitrification inhibiting sulfate reduction were investigated. Batch experiments were conducted to study the inhibitory mechanisms. SO4 2− /NO3 − ratio and relative COD content were the most two important ecological factors affecting the denitrification inhibiting sulfate reduction. With the decreasing of SO4 2− /NO3 − ratio, inhibitory effect increased. The lower COD content benefited to increase the inhibitory effect. There were some effective action sections in the process of denitrification inhibiting sulfate reduction, and the effective inhibitory time was at 2.3 h–6.9 h. The

Abstracts / Journal of Biotechnology 136S (2008) S506–S518

system ORP could reflect the effect of denitrification inhibiting sulfate reduction. When ORP was at −50 mV–150 mV, denitrification predominated; but when ORP was at −50 mV–150 mV, sulfate reduction predominated. Three inhibitory mechanisms were found in the experiments, such as competitive inhibition for carbon source, nitrite-N inhibition and oxidation of autotrophic denitrification bacteria. References Anne, D.M., Mcinerney, M.J., Sublette, K.L., 1990. Microbial control of the production of hydrogen sulfide by sulfate reducing bacteria. Biotechnol. Bioeng. 35, 533–539. Eckford, R.E., Fedorak, P.M., 2002. Chemical and microbiological changes in laboratory incubations of nitrate amendment “sour” produced waters from three western Canadian oil fields. J. Ind. Microbiol. Biotechnol. 29, 243–254. Hubert, C., Nemati, M., Jenneman, G.E., et al., 2003. Containment of biogenic sulfide production in continuous up-flow packed-bed bioreactors with nitrate or nitrite. Biotechnol. Prog. 19, 338–345.

doi:10.1016/j.jbiotec.2008.07.635 V7-P-012 Control of ammonia oxidizing bacteria activity for ammonia removal during landfill leachate treatment Tao Bai 1,∗ , Hengyi Lei 1 , Guangwei Yu 2 , Zhong Li 1 , Xin Feng 1 , Hualiang Li 1 1 School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275, China 2 Department of Environmental Science and Engineering, South China Agriculture University, Guangzhou 510642, China

E-mail address: [email protected] (T. Bai). Ammonia oxidizing bacteria (AOB) was dominantly growing in an autocontrol hybrid bed reactor which was used to remove ammoniacal-N from municipal landfill leachate. The carriers of hybrid bed reactor were composed of fixed multiple flexible carriers and suspended particle carriers (Alvarez-Vazquez et al., 2004). The culture conditions and the impact factors were systematically investigated. Dissolved oxygen (DO), pH value, oxidation reduction potential (ORP) and temperature were used as on-line fuzzy control parameters of the automatic control system (Bai et al., 2008). Under the action of AOB, most of the ammonia nitrogen was removed by means of partial nitrification (Nakano et al., 2004). when the influent ammoniacal-N concentration was kept at 985 ± 20 mg/L, the maximum NH4 + -N removal rate was 0.54 kgNH4 + -N/m3 d at the optimized control condition of the hybrid reactor, DO = 2.0 ± 0.2 mg L−1 , pH 8.0 ± 0.2 and periodical influent quantity was 50 L. With the increasing concentration of ammonia nitrogen in influent, the accumulation rate of nitrite decreased gradually. The results indicated that the ammonia loading should be lower than 1.56 kgNH4 + -N/m3 d in order to ensure the effluent quality. The autocontrol system is therefore an effective tool for hybrid bed reactor to treat landfill leachate high with ammonia concentration. References Alvarez-Vazquez, H., Jefferson, B., Judd, S.J., 2004. Membrane bioreactors vs conventional biological treatment of landfill leachate: a brief review. J. Chem. Technol. Biotechnol. 79, 1043–1049. Bai, T., Lei, H.Y., Yu, G.W., Song, X.Q., Li, Z., 2008. Shortcut nitrification-denitrification for landfill leachate treatment with an automatic control two-stage hybrid reactor [J]. Acta Sci. Circumst. 28 (5), 916–924. Nakano, K., Iwasawa, H., Ito, O., Lee, T.J., Matsumura, M., 2004. Improved simultaneous nitrification and denitrification in a single reactor by using two different

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immobilization carriers with specific oxygen transfer characteristics. Bioprocess Biosyst. Eng. 26, 141–145.

doi:10.1016/j.jbiotec.2008.07.636 V7-P-013 Optimization of culture conditions for tautomycin production in shaking flasks with Streptomyces spiroverticillatus Xiaolong Chen ∗ , Xiaotao Chai, Yuguo Zheng, Yinchu Shen Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310032, PR China Tautomycin, owing a unique 2,3-dialkylmaleic anhydride or diacid moiety, has strong antifungal activity against Sclerotinia sclerotiorum and inhibitory activity to protein phosphatase of type 1 and 1A (Chen et al., 2007; Cheng et al., 1990; Favre et al., 1997; Hori et al., 1991). How to obtain bulk tautomycin is becoming necessary for structure modification of tautomycin and other uses. However, there was no report about optimization of the culture conditions for Streptomyces spiroverticillatus. The effects of cultural parameters (initial pH, temperature, and broth volume in 500 mL flasks) and medium compositions (carbon sources, nitrogen sources, metal ions, and precursors) on tautomycin formation in S. spiroverticillatus in 500 mL shaking flasks were investigated. The results showed that the optimal culture conditions in 500 mL Erlenmeyer flasks were temperature of 28 ◦ C, initial pH of 6.5 and broth volume 50 mL in a medium containing (as in % (W/V)) 2 glucose, 2 soluble starch, 2 soy meal, 0.3 dry yeast extract meal, 0.1 meat extract, 0.2 NaCl, 0.005 K2 HPO4 , 0.05 MnSO4 ·5H2 O, 0.05 MgSO4 ·7H2 O, 0.05 CaCl2 , and 0.2 glycine. The tautomycin concentration reached a maximum level of 1.85 g/L after 3.5 days of fermentation under the optimal conditions, which was 31.2% higher than that of the basal medium.

References Chen, X.L., Zheng, Y.G., Shen, Y.C., 2007. Natural products with maleic anhydride structure: nonadrides, tautomycin, chaetomellic anhydride, and other compounds. Chem. Rev. 107, 1777–1830. Cheng, X.C., Ubukata, M., Isono, K., 1990. The structure of tautomycin, a dialkylmaleic anhydride antibiotic. J. Antibiot. 43, 809–819. Favre, B., Turowski, P., Hemmings, B.A., 1997. Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycin. J. Biol. Chem. 272, 13856–13863. Hori, M., Magae, J., Han, Y.G., Hartshorne, D.J., Karaki, H., 1991. A novel protein phosphatase inhibitor, tautomycin. Effect on smooth muscle. FEBS Lett. 285, 145–148.

doi:10.1016/j.jbiotec.2008.07.637 V7-P-014 Increase hydrogen production by mutant Ethanoligenens harbinense YR-3 in comparison with the wide-type parent strain Guoxiang Zheng 1,2,∗ , Nanqi Ren 1 , Aijie Wang 1 , Wenzhe Li 2 1

School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China 2 Institute of Engineering, Northeast Agricultural University, Harbin 150030, China E-mail address: [email protected] (G. Zheng). To improve hydrogen-producing capacity of wild parent strain Ethanoligenens harbinense ZGX4, reformative traditional mutagenesis technology was applied in this study. A high efficient H2 -producing mutant, E. harbinense YR-3 was obtained by exposure to ultraviolet rays and nitrous acid after stable hydrogen-producing