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VII4-P-066

Novel assay for determination of activity of enzyme phy-tochelatin synthase

Václav Trojan 1,∗, Ladislav Havel 1, Karel Stejskal 2, Ivo Fabrik 2, JiríBaloun 1,2, Vojtech Adam 2, Josef Zehnálek 2, René Kizek 2

1 Department of Plant Biology, Faculty of Agronomy, Mendel Univer-sity of Agriculture and Forestry, Zemedelska 1, CZ-613 00 Brno, CzechRepublic2 Department of Chemistry and Biochemistry, Faculty of Agronomy,Mendel University of Agriculture and Forestry, Zemedelska 1, CZ-61300 Brno, Czech Republic

E-mail address: xtrojan@node.mendelu.cz (V. Trojan).

Phytochelatins (PCs) are post-translationally synthesized thiolpeptides with general structure (�-Glu-Cys)n-Gly, where n isgenerally in the range two to five, playing important roles in detox-ification of heavy metal in plants and other living organisms. PCsare synthesized enzymatically from glutathione (GSH) by enzymeglutamylcysteine dipeptidyl transpeptidase (phytochelatin syn-thase, PCS) in the presence of heavy metal ions (Rauser, 1990).The aim this work was to suggest new approach for determinationof PCS activity. The possible way of the study of PCS activity inthe plant cell exposed by heavy metal is via determination ofincreasing phytochelatins concentration. The first step was tooptimize high performance liquid chromatography coupled with12-channel electrochemical detector to detect five most occurringphytochelatins in plants (desGlyPC, PC2, PC3, PC4 and PC5) (Potesilet al., 2005). Further we employed the optimized method toanalyze extracts from early somatic embryos of spruce, fir, andpine (ESEs) treated with different concentrations of cadmium(II)ions. The viability of treated ESEs decreased for more than 10% after48 h long treatment. The activity of PCS in extracts of treated ESEswas determined after addition of substrate (GSH) and activator

(cadmium(II) ions). The activity of enzyme was enhanced for morethan 50% in ESEs treated with cadmium(II) ions. Based on theresults obtained it can be concluded that the proposed techniquecan be employed for determination of PCS activity in plants.

Acknowledgement

The financial support from GACR 522/07/0692 is highlyacknowledged.

References

Potesil, D., Petrlova, J., Adam, V., Vacek, J., Klejdus, B., Zehnalek, J., Trnkova, L., Havel,L., Kizek, R., 2005. Simultaneous femtomole determination of cysteine, reducedand oxidized glutathione, and phytochelatin in maize (Zea mays L.) kernelsusing high-performance liquid chromatography with electrochemical detection.J. Chromatogr. A 1084, 134–144.

Rauser, W.E., 1990. Phytochelatins. Annu. Rev. Biochem. 59, 61–86.

doi:10.1016/j.jbiotec.2008.07.1563

gy 136S (2008) S647–S677

VII4-P-067

Polyurethane immobilized Corynebacterium glutamicumbiomass for biosorption of Reactive Yellow 2

M. Sathishkumar ∗, Sung Wook Won, Bong Woo Chung, YeoungSang Yun

Division of Environmental and Chemical Engineering, Research Insti-tute of Industrial Technology, Chonbuk National University, Jeonju561-756, South Korea

E-mail address: sathishkumar77@gmail.com (M. Sathishkumar).

The potential of bacteria to adsorb organic/inorganic pollutantshas been well established (Vijayaraghavan and Yun, 2008). Butusing free-biomass has various problems like tough handling,poor mechanical strength etc. at industrial level. Corynebacteriumglutamicum, a nucleic acid fermentation industry waste, showedpromise for the removal of reactive dyes from aqueous solution(Vijayaraghavan and Yun, 2008). Due to practical difficulties insolid–liquid separation and poor mechanical stability, the freebiomass was immobilized using polyurethane. Thus produced,polyurethane-immobilized C. glutamicum biomass was used tostudy the biosorption of Reactive Yellow 2, which was used asa model dye. The pH edge experiments revealed that strongacidic conditions are mandatory to achieve maximum reactivedye biosorption. Amine groups of C. glutamicum were responsibleto accommodate negatively charged reactive dye anions throughelectrostatic interaction. Isotherm experiments were conducted toexplore the saturation potential of the biosorbent. Kinetics of theexperiment were also studied which showed a rapid increase indye-uptake till 60 min and gradually attained saturation there-after. In an effort to enhance process economy, the immobilizedbiomass was regenerated and reused for number of cycles. Decreasein adsorption efficiency was less than 10% at the end of 5th cycle.The study proved the effective use of immobilized bacterial wastefor dye sequestration in liquid phase and its reusability.

Reference

Vijayaraghavan, K., Yun, Y.S., 2008. Bacterial sorbents and biosorption. Biotech. Adv.26, 266–291.

doi:10.1016/j.jbiotec.2008.07.1564

VII4-P-068

A two-stage scheme for pesticide degradationIntegration of photocatalysis and biological oxidation

José Antonio Sánchez Pérez 1,∗, María de la Menta BallesterosMartín 1, Sixto Malato Rodríguez 2, José Luis Casas López 1, JoséLuis García Sánchez 1

1 Department of Chemical Engineering, University of Almería, AlmeríaE-04120, Spain2 Plataforma Solar de Almería-CIEMAT, Tabernas E-04200, Spain

E-mail address: jsanchez@ual.es (J.A.S. Pérez).

Water polluted with pesticides is not compatible with classicalbiological treatment, due to the toxicity shown towards micro-organisms involved in those processes. Advanced OxidationProcesses (AOPs) can be used for the complete mineralization ofthese substances (Esplugas and Ollis, 1997). Even though AOPshave been shown to be highly efficient, their operation is quiteexpensive (tens of D /m3). An attractive option is a short timepre-treatment with an AOP (Gogate and Pandit, 2004), the recal-citrant pollutants becoming biodegradable intermediates that

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Abstracts / Journal of Biotec

subsequently can be degraded in a biological process. A mixture offive pesticides (oxamyl, methomyl, imidacloprid, dimethoate andpirimethanyl) was chosen to assess photo-Fenton and biologicaltreatment integration in a two-stage process. Photo-Fenton wasconducted using a compound parabolic solar collector in a 50-Lpilot plant. The change of wastewater biodegradability at increas-ing photo-oxidation treatment times was evaluated with thebacteria Pseudomonas putida as a test microorganism (BallesterosMartín et al., 2008). At a 57% of mineralization by photo-Fentonwastewater was biologically treated in a 6-L stirred tank bioreactorwith activated sludge, reaching an overall C-removal efficiencyof 94%. Therefore combined solar photo-Fenton and biologicalsystem has been demonstrated to be an effective approach forthe treatment of wastewaters polluted with a mixture of pesticides.

Acknowledgements

Authors acknowledge the financial support of Spanish Ministe-rio de Educación y Ciencia (CTQ2006-14743-C03-03/PPQ).

References

Ballesteros Martín, M.M., Sánchez Pérez, J.A., Acién Fernández, F.G., Casas López,J.L., García-Ripoll, A.M., Arques, A., Oller, I., Malato Rodríguez, S., 2008. Com-bined photo-Fenton and biological oxidation for pesticide degradation: effect ofphoto-treated intermediates on biodegradation kinetics. Chemosphere 70 (8),1476–1483.

Esplugas, S., Ollis, D.F., 1997. Economic aspects of integrated (chemical + biological)processes for water treatment. Journal of Advances in Oxidation Technology 2,197–202.

Gogate, P.R., Pandit, A., 2004. A review of imperative technologies for wastewatertreatment I: oxidation technologies at ambient conditions. Advances in Envi-ronmental Research 8, 501–551.

doi:10.1016/j.jbiotec.2008.07.1565

VII4-P-069

Susceptibility of mixed populations in rotating biological con-tactors to isothiazolone biocide and treatment efficiency of thesystem

Prapaparn Sirikhansaeng 1,∗, Pattana Laopaiboon 1, KanitVichitphan 1, Lakkana Laopaiboon 1,2

1

Department of Biotechnology, Faculty of Technology, Khon Kaen Uni-versity, Khon Kaen 40002, Thailand2 Research Center for Environmental and Hazardous Substance Man-agement, Khon Kaen University, Khon Kaen 40002, Thailand

E-mail address: lakcha@kku.ac.th (P. Sirikhansaeng).

Isothiazolone (IT) biocide achieves its biocidal activity by reactionwith thiol-containing enzymes (Collier et al., 1990). The effectsof IT on treatment efficiency of laboratory-scale rotating biolog-ical contactors (RBCs) as well as biocide efficacy were studied.Biofilms were established on the RBCs (Laopaiboon et al., 2006)and then exposed to the synthetic wastewater containing 0 (con-trol) and 6 ppm IT. The results showed that the degree of chemicaloxygen demand (COD) removal and biocide degradation of thesystem depended on hydraulic loading rate (HLR) and hydraulicretention time (HRT). Acclimatized biofilms could degrade IT via co-metabolism using lab-lemco broth as a growth substrate. Biofilmson the discs could retain their microbial activities in the pres-ence of 6 ppm IT, while bio-oxidation of the planktonic cells inthe RBCs expressed as biochemical oxygen demand (BOD5) wasalmost totally inhibited in the presence of 0.6 ppm IT. The plank-tonic cells sloughed from the acclimatized biofilms on IT showed

gy 136S (2008) S647–S677 S675

less susceptibility to IT than that of the planktonic cells sloughedfrom the non-acclimatized biofilms. The results also showed thatbiodegradability of IT did not totally relate with the susceptibilityof cells to the biocide indicating by minimum lethal concentration(MLC) values. The resistant bacterium to IT was tentatively iden-tified as predominantly of the species Burkholderia cepacia. Dueto the severe inhibition of bio-oxidation by the biocide, BOD testwas not an appropriate analytical procedure for investigating theefficiency of wastewater treatment units contaminated with thebiocide even though adapted seed was used and/or the incubationtime was prolonged.

References

Collier, P.J., Ramsey, A., Waigh, R.D., Douglas, K.T., Austin, P., Gilbert, P., 1990. Chemicalreactivity of some isothiazolone biocides. J. Appl. Bacteriol. 69, 578–584.

Laopaiboon, L., Phukoetphim, N., Laopaiboon, P., 2006. Effect of glutaraldehyde bio-cide on laboratory-scale rotating biological contactors. Electron. J. Biotechnol. 9,1–12.

doi:10.1016/j.jbiotec.2008.07.1566

VII4-P-070

Decolorization of azo dye with Pseudomonas luteola in a mem-brane bioreactor

Jyh-Ping Chen ∗, Yung-Sheng Lin

Department of Chemical and Materials Engineering, Chang Gung Uni-versity, Taoyuan 333, Taiwan

E-mail address: jpchen@mail.cgu.edu.tw (J.-P. Chen).

In this study, Pseudomonas luteola was used in a membranebioreactor (MBR) employing a cross-flow microfiltration unit forcontinuous decolorization of Reactive Red 22 azo dye in wastew-ater (Fan et al., 2006). The influences of biomass loading, feeddye concentration, and feed rate on decolorization efficiency (DE)and overall decolorization rate (ODR) were studied (Chen and Lin,2007). The DE and ODR increased with the increase in biomassloading until reaching 1 g and a biomass concentration of 2 g/l isthe limit in the current system. The DE decreased with increas-ing feed dye concentration and feed rate. At a constant feed dyeconcentration, a higher feeding rate led to a higher dye loadingrate, but lowered the retention time and led to incomplete dyedecolorization. The amount of dye needed to be processed in a

unit time increased with the rise of feed dye concentration whenthe system was operated at a constant feed rate. Therefore, theincrease in either feed dye concentration or feed rate subsequentlyled to a higher residual dye concentration in the effluent, furtherdecreasing the DE. In contrast, the ODR increased with increasingfeed dye concentration and feed rate. This behavior arises from theMichaelis-Menten kinetic model where reaction rate will be depen-dent on the residual dye concentration in the bioreactor. A lowerDE gave higher residual dye concentrations in the bioreactor, thusresulting in a higher ODR. In conclusion, the DE and ODR were influ-enced by biomass loading, feed dye concentration, and feed ratefor decolorization of azo dye in the MBR. Excess cell loading in thebioreactor with limited working volume would not raise DE andODR. The ODR increased with increasing feed dye concentrationand feed rate at the expense of DE, which would set a lower limiton the dye concentration in the effluent stream.

References

Chen, J.P., Lin, Y.S., 2007. Decolorization of azo dye by immobilized Pseudomonas lute-ola entrapped in alginate-silicate sol-gel beads. Process Biochem. 42, 934–942.