Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47 S31

euglenozoan host and a photosynthetic eukaryotic chlorophyceanalga E. longa possess a circular 73 kb plastid genome, which isabout half the size of the E. gracilis chloroplast genome. The com-position of its nuclear genome is still a big mystery. Here, wepresent the analysis of a comprehensive E. longa transcriptomewhich should lead to a better understanding of this organism. Weobtained ca. 30.2 million paired-end Illumina reads (Illumina Hi-seq 2000 sequencer) from two mRNA samples — one cultivated onlight and the other in the dark. Transcriptome assembly was thenperformed with two different programs (Abyss/Trans-Abyss andTrinity). Results indicate that light does not significantly alter thetranscription pattern as both mRNA samples expressed roughly thesame groups of studied genes. Nevertheless, results of the E. longatranscriptome analysis may have implications for understandingthe gene expression in its close relatives.

http://dx.doi.org/10.1016/j.copbio.2013.05.050

Bioprocess Engineering

Production of shikimic acid: a potential candidate for develop-ing drug formulation for Avian/Swine flu

Garima Rawat, Rajendra Kumar Saxena

Department of Microbiology, University of Delhi South Campus, NewDelhi 110021, IndiaE-mail address: garima 205@yahoo.com (G. Rawat).

Shikimic acid has wide use in pharmaceuticals due to its appli-cation in the synthesis of drug Tamiflu used in the treatment ofAvian/Swine flu. The high cost and limited availability of shikimicacid isolated from plants has impeded the use of this valuable build-ing block of the drug. In this context, fermentation route to produceshikimic acid from renewable resources like glucose presents anexcellent alternative has become increasingly attractive. The goal ofthis research was to confer an efficient and sustainable productionof shikimic acid using simple fermentation technology. In particu-lar, the present study was embarked upon the isolation of a noveland a potent wild type bacterium which initially produces 0.54 g/Land later identified as Citrobacter sp. Different strategies of processoptimization were employed followed by fed batch studies whichfinally resulted in 40-fold increase in the yield of shikimic acid. Thepurification of the shikimic acid was also successfully carried outwherein ion exchange chromatography was employed to obtainpure shikimic acid for its other industrial applications. Further, thescalability of the process was also confirmed in large size bioreac-tor (30 L) to validate the production. Thus, the process developedis economic, efficient, fast and even more sustainable to meet thecurrent market volume of shikimic acid at competitive price. Thesefindings becomes much more important realizing well the recentoutbreaks of Swine/Avian flu in world and the blockage by Chinesefirms and sudden shoot up of the drug demand in market.

http://dx.doi.org/10.1016/j.copbio.2013.05.051

Identification and application of CaCO3 dissolving bacteria inmagnesite quarries

Furkan Orhan 1, Derya Yanmis 2, Mehmet Karadayi 2, HakanOzkan 3, Medine Gulluce 2

1 Agri Ibrahim Cecen University, Central Research and Application Lab-oratories, TR-04100 Agri, Turkey2 Ataturk University, Faculty of Science, Department of Biology, 25240Erzurum, Turkey3 Erzincan University, Faculty of Science and Arts, Department of Biol-ogy, 24100 Erzincan, TurkeyE-mail address: furkan orhan@hotmail.com (F. Orhan).

Magnesite is the main source for magnesium and its com-pounds. One of the major problem with magnesite usage is itsimpurities as it contains high amount of silicium and calcium.Some magnesite ores in Turkey are non-utilizable due to their highamount of CaCO3 (≥3%). Conventional decalcification/purificationmethods for magnesite are not impractical and not economical.In the current study, bacteria dissolving only CaCO3 were isolatedfrom two different magnesite quarries (Ercis, Mecidiye) in Erzincanprovince. The isolated bacteria were identified and characterizedby conventional and molecular techniques (fatty acid methyl esterprofiles (FAME), BOX PCR and 16S rRNA). According to sequencingresults, these isolates were identified as Bacillus sp., Exiguobac-terium sibiricum, Exiguobacterium aurantiacum and Lactococcus sp.Among the isolates, Bacillus sp. was used for biotechnological dis-solution of rich CaCO3 content of magnesite. The results showedBacillus sp. reduced the amount of CaCO3 from 3.03% to 0.68%,which means enrichment about 77.56%. In conclusion, the use ofmicroorganisms in magnesite enrichment seems to be possible andpracticable.

http://dx.doi.org/10.1016/j.copbio.2013.05.052

Advanced protein separations and multiplex molecular diag-nostics using a disposable high-throughput microcapillary film

Alexander Daniel Edwards 1, Malcolm Robert Mackley 2, NunoMiguel Reis 3

1 Reading School of Pharmacy, University of Reading, Whiteknights,Reading RG6 6AP, UK2 Department of Chemical Engineering and Biotechnology, Universityof Cambridge, Cambridge CB2 3RA, UK3 Department of Chemical Engineering, Loughborough University,Loughborough LE11 3TU, UKE-mail address: n.m.reis@lboro.ac.uk (N.M. Reis).

A novel melt-extrusion process has been recently invented atthe University of Cambridge, which allows the continuous produc-tion of a plastic ribbon from thermoplastic polymers containing anumber of parallel microcapillaries embedded in it, called Micro-Capillary Film (MCF). We have discovered that the unique opticalproperties of the MCF derived from the flat surfaces and goodtransparency of thermoplastics suits a number of preparativeand analytical applications in bioprocessing, immunochemistryand biomarker discovery. MCFs have been developed and testedfor a number of bioprocessing operations which includes pro-tein separations using conventional ion-exchange and capillaryelectrophoresis, as well for rapid multiplex detection of biomark-ers and inflammatory molecules. We succeeded in producing a19-bore 150 �m i.d. cation-exchange MCF from EVOH which deliv-ered sharp breakthrough curves and separation of proteins withclose isoelectric points. A 10-bore 200 �m i.d. MCF producedfrom FEP has also allowed fast-sharp separation of proteins using

S32 Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47

electrophoresis, which compared well to separations using smallbore fused silica capillaries. Finally, the large surface-area-to-volume ratio and exceptional optical properties of the FEP MCFwere explored for rapid multiplex detection of molecules usingstandard immunoassay chemistry, with resulted in reduced vari-ability and comparable sensitivity to microtiter plates for differenttargeted molecules. MCFs are a cost-effective high-throughputmicro-engineered material which allows non-invasive monitoringof bioseparations, therefore suitable for a wide spectrum of appli-cations across the broad biotechnology field.

http://dx.doi.org/10.1016/j.copbio.2013.05.053

Biosensors

Engraving micro-/nano-structures onto a quartz crystalmicrobalance facilitates the multiplexing sequential-acousticdetection of biomolecules

Rodica Elena Ionescu

Lnio, Université de Technologie de Troyes, Troyes, FranceE-mail address: elena rodica.ionescu@utt.fr.

Over the years the detection of biomolecules is continueaddressed through various biosensing techniques (optical, elec-trochemical, mass variation, and so on) with the final aim ofexploring the analytical performances of different parameters suchas analyte low limit of detection, time of response, sensitivity andselectivity over a large range of interfering compounds. Moreover,the quantity of analyte is usually reported as an extreme impor-tant parameter in designing biosensors. By far, the generation ofstructured surface based sensors integrated or not integrated inlab-on-chip devices would be an elegant solution for efficient andreal-time medical investigation assays. In the present work, a newconcept of engraving microstructure/nanostructures onto a com-mercial quartz crystal microbalance by using a cheap-transmissionelectron microscopy (TEM) grid mask in combination with a thinlayer of metal-evaporation create the remarkable possibility ofsequential acoustic multiplexing detection of various biomolecules(nonylphenol, atrazine, bovine serum albumin, and cholera toxin)on the same crystal. The advantages of such structured crystal whencompared with a standard QCM system it will be discussed.

http://dx.doi.org/10.1016/j.copbio.2013.05.054

Amperometric Baeyer–Villiger monooxygenase biosensor

Andrea Schenkmayerová, Marek Bucko, Peter Gemeiner, JaroslavKatrlík

Slovak Academy of Sciences, Institute of Chemistry, Department ofGlycobiotechnology, Bratislava, SlovakiaE-mail address: schenkan@gmail.com (A. Schenkmayerová).

Baeyer–Villiger (BV) oxidations are widely performed viawhole-cell genetically modified Escherichia coli overexpressing oneof the Baeyer–Villiger monooxygenases (BVMOs). BVMOs enableregioselective and enantioselective oxidation of ketone substratesleading to esters or lactones that might be used in pharmaceut-icals synthesis. The progress of the reaction is usually monitoredwith gas chromatography. We focused on biosensors develop-ment as they are small in size, permit rapid monitoring, theyare easily prepared and cheap in construction and operation.Miniaturized oxygen electrode with stirrer was chosen as thebiosensor transducer. This set up allowed to carry out highly sen-sitive measurements with low noise. Genetically modified E. coli

cells overexpressing cyclopentanone monooxygenase were usedas biorecognition element. The bacteria were immobilized in poly-electrolyte gel membrane which can be easily attached to theoxygen electrode forming the biosensor. The biosensor responsetime was about 30 s, the linear range of the calibration curve was2–130 �m of model synthetic substrate (±)-cis-bicyclo [3.2.0]hept-2-en-6-one and the sensitivity was 1.8 nA/�m. No interferenceswere detected. The biosensor sensitivity remained stable withinone week of storage at 4◦C. The biosensor was successfully used foroff-line monitoring of whole course of BV oxidation. Our biosen-sor is novel both in the device construction and biotechnologicalapplication. This work was supported by the Slovak Grant Agencyfor Science VEGA 1/0229/12. This contribution is the result of theproject implementation: Applied research in the field of industrialbiocatalysis, ITMS code: 26240220079 supported by the Research& Development Operational Program funded by the ERDF.

http://dx.doi.org/10.1016/j.copbio.2013.05.055

Whole-cell biosensor for detection of environmental pollu-tion — enhancement of detected bioluminescence

Hana Kalabova 1, Marie Pospisilova 1, Marcel Jirina 1, GabrielaKuncova 2

1 Faculty of Biomedical Engineering, Czech Technical University,Kladno, Czech Republic2 Institute of Chemical Process Fundamentals, AV CR, v.v.i., Prague,Czech RepublicE-mail address: hana.kalabova@fbmi.cvut.cz (H. Kalabova).

On-line in situ monitoring of the environmental pollution inremote localities is enabled by whole-cell optical fiber sensors.An optical fiber element (OFE) comprising tapered up quartz fiberwith an active layer of immobilized bioluminescent bioreporters onbroader end of the OFE ensures effective coupling of low intenselight produced by cells to an optical fiber. Bioluminescent biore-porters are genetically engineered microorganisms that producedbioluminescence selectively in response of contaminants in the cellenvironment. With aim to increase detected bioluminescence bymodification of OFE shape a mathematical model simulating lighttransmission applying methods of geometric optics was developed.The simulation shows a part of input rays reaching the detectoras a function of broader end and OFE length. Theoretical resultswere compared with measured transmission by OFE and PCS fiberwith core 600 �m. In the demonstration experiments bacteria Pseu-domonas fluorescens HK44 selective to presence of salicylic acidand naphthalene were immobilized in the active layers. The mea-sured intensity of bioluminescence using the OFE was six timeshigher as compared to PCS fiber, which was in agreement with theresult of mathematical modeling. The applicability of biosensingwas demonstrated by detection of BTEX with Pseudomonas putidaTVA8 in twelve samples of the wastewater in laboratory conditions.

http://dx.doi.org/10.1016/j.copbio.2013.05.056