Optimizations and Interpretations of Signal from Liquid Distribution Setup in Shake Flask

Transcription

1 Proceedings of the World Congress on Engineering 9 Vol II WCE 9, July -, 9, London, U.K. Optimiztions nd Interprettions of Signl from Liquid Distriution Setup in Shke Flsk Amizon Azizn, Rungnp Vorvichn nd Jochen Büchs Astrct Shke flsks hve een used for screening process in fermenttions for long time. The oxygen trnsfer rtes of the fermenttion medi chnge with viscosity. The liquid distriution in hydrophilic shke flsk ecomes symmetricl s the viscosity of the liquid increses. Therefore, non-invsive opticl technique hs een developed to collect ngulr informtion on the shpe of the liquid t different operting conditions like shking frequency, shking dimeter, nd filling volume. This technique uses light emitting diodes, photomultiplier tue nd fluorescent solution. sets of opticl fiers with ech set comprises of opticl fiers rrnged in certin ngle of incident light etween the fiers re ttched t different heights of the outside wll of shke flsk. Blue lights from the light emitting diodes re switched lterntely t different heights nd excite the fluorescent molecules in the flsk. The emission lights re then sent through the opticl fiers ck to the hed-on photomultiplier tue. With LVIEW progrm, utomtic nd mnul mode of signl dt collections from the photomultiplier tues re ville. The digitl signls received nd sved indicting the liquid distriution re then nlyzed in MATHLAB for the shpe. However, prior to the identifiction of the shpe of the liquid distriution in shke flsk, the optimiztions of the signl prmeters re investigted. The influences of the intensity of the light emitting diodes nd the photomultiplier tue s sensitivity level to the liquid distriution s signl t certin shking frequency nd t ech height t fixed filling volume re oserved. Vrying these prmeters chnge the output voltges from the photomultiplier tue. However, the shpes of the liquid distriutions remin the sme. The vrying numers of rottions collected in reference to the men show cceptle percentge devitions ut slight shrp increses of the percentge re shown especilly t the strt of the ulk liquid. With the informtion, signls otined nd interpreted from this setup depict invlule informtion on the hydrodynmic of the liquid distriution in shke flsk. Index Terms Liquid distriution, optimiztion, shke flsk. I. INTRODUCTION Liquid distriution comprises of rotting ulk liquid nd Mnuscript received Mrch, 9. This work ws supported in prt y Achener Verfhrenstechnik-Biochemicl Engineering, RWTH Achen University, Germny. A. Azizn is currently with Achener Verfhrenstechnik-Biochemicl Engineering, RWTH Achen University, Achen, Germny, on leve from Fculty of Chemicl Engineering, Universiti Teknologi MARA, Shh Alm, Mlysi (phone: +9 ()88; fx: +9 ()85; e-mil: mizon.zizn@ vt.rwth-chen.de). R. Vorvichn is with Thi Germn Grdute School t King Mongkut s University of Technology North Bngkok, Bngkok, Thilnd (e-mil: v_ying@hotmil.com). J. Büchs is with Achener Verfhrenstechnik-Biochemicl Engineering, RWTH Achen University, Achen, Germny (e-mil: jochen.uechs@vt.rwth-chen.de). stgnnt liquid film on the wll nd t the se of the shke flsk. According to Higie s penetrtion theory, the gs-liquid mss trnsfer sensitively depends on the thickness of the thin liquid lyer on the flsk wll nd the contct time [], []. It explins tht the mss trnsfer occurs only close to the gs-liquid interfce specificlly t the liquid film. Hence, this significntly contriutes to the specific mss trnsfer re nd to the mximum oxygen trnsfer cpcity of microorgnisms in fermenttion. The sic hydrodynmic informtion representing the gs-liquid interfce is still unville nd there re still few informtion on the power input consumption, volumetric mss trnsfer coefficient nd interfcil re []. Currently, the informtion on the liquid distriution including shpe nd thickness in shke flsk is still scrce nd might only e descried sed on fundmentl understnding of fluid flow. Liquid distriution clcultions nd model hve een introduced with the ssumption of frictionless liquid giving n insight of the liquid distriution []. However, in relity, t different prmeters during fermenttions, the liquid distriution considering frictions etween liquid nd wll chnges nd influences momentum trnsfer re nd mss trnsfer re in shke flsk. In high viscosity solution in shke flsk which is minly shown during fermenttion, the liquid distriution is lso gretly influenced. Therefore, setup is designed to determine the liquid distriution in shke flsk for further understnding of the trnsport phenomen nd hydrodynmics of the liquid in shke flsk. II. NON-INVASIVE TECHNIQUE OF LIQUID DISTRIBUTION A. Technique The technique involved the excittion of the fluorescent solution y lue light from light emitting diode (LED) t wvelength of 8 nm. The emission light from the fluorescent ws received y compct hed-on photomultiplier tue (PMT, Photosensor module H787 Hmmtsu, Jpn) t 55 nm. The technique ws non-invsive hving sets of plstic opticl fiers (POF, Conrd, Germny) positioned outside of the wll of the flsk. Ech set hs opticl fiers seprted y n ngle degree of 5 s shown in Fig.. This specific ngle of fiers positioned t the wll enled optimum light exposure close to wll detecting the coming of the strt nd end of the ulk liquid. The plstic opticl fier (POF) ws mm in dimeter with.5 mm insultor t ech side. The fier ws locted in hollow rss chnnel with mm nd mm of inside nd outside dimeter respectively. In ISBN: WCE 9

2 Proceedings of the World Congress on Engineering 9 Vol II WCE 9, July -, 9, London, U.K. Fig., A nd B (lck nd white shde) were the fiers tht chnneled the light from LED to the outside wll nd received the emitted light from fluorescent solution sending to PMT respectively. A nd B were locted in hollow tues s the holders for LEDs s shown shded in digonl rick in Fig.. C nd D (sphere shded) were the glss thickness nd fluorescent solution in shke flsk respectively. Fig. : Position rrngement of opticl fiers outside of the wll of shke flsk (top view). 5 um of fluorescent solution from fluorescein sodium slt (Acid Yellow 7, Aldrich, Steinheim, Germny) ws prepred t ph 8. The solution ws uffered with mm phosphte uffer of NH PO.H O (Crl Roth GmH, Krlsruhe, Germny). Viscous solutions were lso prepred t different concentrtions using highly polymeric inert sustnce polyvinylpyrrolidone Luviskol K9 (PVP, BASF SE, Ludwigshfen, Germny) in fluorescent solution (fluorescent-pvp solution). A hll sensor tht cted s triggering in n utomtic mesurement technique ws used for the detection of the strting of one rottion. H C A D G E C A B F 5 o D Emission light to PMT Excittion light (lue) from LED Fig. : Schemtic digrm of the liquid distriution mesurement technique in shke flsk. A: oritl shker in clockwise rottion, B: hll sensor for triggering, C: LEDs J I K B with utomtic lternte switching, D: shke flsk nd fluorescent solution, E: undle of POFs, F: top view (Fig.), G: PMT with low noise mplifier, H: mgnified imge of POF rrngement in undle, I: dt cquisition module with nlog-digitl converter, J: mgnified imge of effective re t PMT with POF undle, K: computer with LVIEW softwre. This schemtic digrm of Fig. shows the non-invsive technique for liquid distriution. Mesurements were ville for heights of. to.7 cm from se of the flsk in n increment of.5 cm ech outside the wll of typicl 5 ml nominl volume Erlenmeyer shke flsk (DURAN, DURAN Group, Minz, Germny). B. Dt collection With LVIEW (Ntionl Instrument, Austin, TX, USA) softwre, utomtic nd mnul dt collections were enled for different shking frequencies. Options of hving certin numer of rottions for men dt for one rottion ws ville. The numer of rottions used in this pper rnges from to numer of rottions for men result for one rottion. The signl from PMT ws converted to digitl dt y Dt Acquisition Crd (NI-PCI-MIO-XE-5 Ntionl Instrument, Austin, TX, USA) nd sved to mximum output voltge of V. III. INTERPRETATIONS OF DATA Results otined from the experiments show liquid distriution t respective heights s mentioned in Sect. II. Dt interprettions were conducted y tking these steps: A. Plots of liquid distriutions t respective heights The results were plotted s shown in Fig. depicting typicl liquid distriution plots for rottion of o of ngle. B. Identifictions of the liquid distriutions Liquid distriution plot ws nlyzed sed on the strt nd end of ulk liquid. Both strt nd end of ulk liquid were identified y slope nlysis s shown in (). A Grphicl User Interfce ws creted in MATHLAB (R8, Mthworks, USA) for n utomtic detection of strt nd end of ulk liquid. The detection of the strt of ulk liquid ws sed on the shrp increse of the slope. The sme principle ws used to the end of the ulk liquid y detecting the increse of slope ut from the opposite direction. The slope ws clculted etween two consecutive points in ech dt mesurement using the following eqution: V V m = () θ θ where m is slope, V is second output voltge, V is first output voltge, θ is second ngle respective to V nd θ is first ngle respective to V. Fig. is n exmple of typicl of liquid distriution showing the strt nd end of the ulk liquid t respective heights. The ngle positions determined were then plotted in three-dimensionl (D) plots in MATHLAB showing the ISBN: WCE 9

3 Proceedings of the World Congress on Engineering 9 Vol II WCE 9, July -, 9, London, U.K. momentum trnsfer re of the liquid distriution in shke flsk. These re shown in section V with vrying filling volumes nd viscous conditions rnging from 5 to ml nd to 7 g/l of PVP concentrtion respectively Fig. : Typicl liquid distriution plots t different heights for 5 um fluorescent, ph 8, mm phosphte uffer, rpm of shking frequency, 5 mm of shking dimeter nd 5 ml of filling volume. Symols in : ( ). cm; ( ).7 cm; ( ). cm; (+).7 cm. Symols in : ( ). cm; ( ).7 cm; ( ). cm; ( ).7 cm; ( ). cm. Symols in () nd (): ( ) strt of ulk liquid nd (/) end of the ulk liquid. volume sed on certin numer of rottions of,, 5, nd rottions using the following equtions: n N ABD Rn [( = = R) / N ], () n= n N AVD = = ABD / N, () n= n = /[( = N PD AVD R) / N ] Χ. () n= where R is output voltge reding, n is numer of reding nd N is the totl numer of different redings. V. RESULTS AND DISCUSSION A. Identifictions of liquid distriutions Tle I shows the degree determined y the slope nlysis for the strt nd end of ulk liquid t different heights only for rpm of shking frequency, 5 mm of shking dimeter nd 5 ml of filling volume s shown in Fig.. The degrees were then plotted in ngulr plots respective to the heights nd dimeter of the flsk. Tle I: List of the degree positions in one rottion for the strt nd end of ulk liquid. IV. OPTIMIZATIONS OF LIQUID DISTRIBUTION DATA COLLECTION The optimiztions of the non-invsive technique were investigted on three spects. A. Influence of intensity of light emitting diode (LED) Chnging the input voltge of LED chnges the level of intensity of LED. The purpose of this ws to oserve the effect of different input voltge level to the detection of liquid distriution. Experimentl conditions of rpm of shking frequency, 5 mm of shking dimeter nd 5 ml of filling volume were implemented. B. Influence of level of sensitivity of photomultiplier tue (PMT) With the increse of the sensitivity level of PMT, the more sensitive the detection of the light emitted from the fluorescent solution. The level of the sensitivity ws chosen s such tht it ws not overloded eing more thn mximl output voltge of V. The prmeters used were t rpm of shking frequency, 5 mm of shking dimeter nd 5 ml of filling volume. C. Repetility nd reproduciility test Repetility nd reproduciility of the results were evluted y the percentge of devition (PD). The clcultions followed in sequence of solute devition (ABD) using (), verge devition (AVD) using () nd finlly percentge devition (PD) using (). The evlutions were conducted t rpm of shking frequency, 5 mm of shking dimeter nd 5 ml of filling Heights from ottom [cm] Strt of ulk [degree] End of ulk [degree] Hving ll the degrees plotted t ech respective ngulr plot t ech height, the shpe of the liquid cn e clerly seen for the strt nd end of ulk liquid s shown in Fig.. This represents the shpe of liquid sickle nd the liquid distriution t the wll of the shke flsk. Fig. specificlly shows the increse of the momentum trnsfer re of the liquid s the filling volume increses. Büchs et l. 7 showed some shpes of the liquid distriution in shke flsk tken y photogrphs. With these results from this method, the shpe of the strt of ulk liquid is similr to the shpe shown nd it justifies the effectiveness ISBN: WCE 9

4 Proceedings of the World Congress on Engineering 9 Vol II WCE 9, July -, 9, London, U.K. of the method to determine the liquid distriution in shke flsk. This is very useful for further study of the mss trnsfer of gs-liquid in shke flsk during fermenttions y investigting the liquid distriution with different viscosity solutions. This is further shown in Fig. 5 showing the liquid distriution for different viscosity conditions in shke flsk. Z [cm] - - Y [cm] X [cm] ml ml 5 ml ml 5 ml ml Fig. : Three-dimensionl representtions of liquid distriution for 5 um fluorescent solution, ph 8, mm phosphte uffer, rpm of shking frequency, 5 mm of shking dimeter nd t different filling volume rnging from 5 to ml. Symols: (+) 5 ml; ( ) ml; ( ) 5 ml; ( ) ml; ( ) 5 ml; ( ) ml. Z [cm] X [cm] Y [cm] - g/l PVP g/l PVP g/l PVP g/l PVP g/l PVP 7 g/l PVP Ref. Fig. 5: Three-dimensionl representtions of liquid distriution for 5 um fluorescent solution, ph 8, mm phosphte uffer, rpm of shking frequency, 5 mm of shking dimeter, 5 ml of filling volume nd t different PVP concentrtions. Symols: (+) g/l; ( ) g/l; ( ) g/l; ( ) g/l; ( ) g/l; ( ) 7 g/l. The liquid distriutions of different viscous solutions of,,,,, nd 7 g/l of fluorescent-pvp s shown in Fig. 5 depict tht s the viscosities increse, the positions of the liquid shift to the right from the reference point t zero coordinte t y-xis (shown s Ref.). The shpes of the liquid sickles for,,, nd g/l of fluorescent-pvp remin in-tct, however, the shpes for the liquid sickles for nd 7 g/l chnge most proly due to the chnge of the rheology of the viscous solutions. It lso showed very wide shift of these high viscous solutions of nd 7 g/l of fluorescent-pvp solution to the right (counter clockwise direction) from the reference point in reference to,,, nd g/l of fluorescent-pvp solution. This my e due to less movement of the liquid in prllel to the oritl shking of the shke flsk s descried s out-of-phse condition [5]. B. Intensity of light emitting diodes (LEDs) Mesurements t. nd.7 cm for different heights from the se of the flsk t vrying input voltge of LEDs re shown in Fig.. At oth positions, liquids circulting in the flsk re detected through opticl fiers y the PMT. With vrying input voltges, the liquid positions especilly the strt of the ulk liquid re t the sme point t oth heights. The shpes of the plots re lso lmost the sme hving percentge devitions of less thn 5% for plots with voltge,.5, nd 5. ut not for voltge of.5 for oth heights of. nd.7 cm from the se of the flsk. For the plot t n input voltge of.5, the mximum output voltge is lower thn other voltges Fig. : Influence of input voltge of LED to the liquid distriution positions for 5 um fluorescent solution, ph 8, mm phosphte uffer, rpm of shking frequency, 5 mm of shking dimeter nd 5 ml of filling volume t different heights of. cm (shown in ) nd.7 cm (shown in ) from the se of the flsk. Symols: ( ) voltge.5; ( ) voltge.; ( ) voltge.5; ( ) voltge Fig. 7: Influence of PMT sensitivity level to the liquid distriution positions for 5 um fluorescent solution, ph 8, mm phosphte uffer, rpm of shking frequency, 5 mm of shking dimeter nd 5 ml of filling volume t different heights of. cm (shown in ) nd.7 cm (shown in ) from the se of the flsk. Symols: ( ) sensitivity.; ( ) sensitivity.; ( ) sensitivity.; ( ) sensitivity.; ( ) sensitivity.5; ( ) sensitivity 5.; ( ) sensitivity.. ISBN: WCE 9

5 Proceedings of the World Congress on Engineering 9 Vol II WCE 9, July -, 9, London, U.K. Theoreticlly, the higher the input voltge set up for LED, the higher the intensity of the light. This excites more fluorescent molecules from fluorescent solution resulting in high intensity of emission light eing emitted. Therefore, the output voltge ecomes higher. Significntly, despite the chnge of the voltge prmeter, the liquid distriution remins the sme especilly t the strt point of the ulk liquid. C. Level of sensitivity of photomultiplier tue (PMT) By incresing the level of the sensitivity of PMT, the more sensitive the mesurement of the emission light from the fluorescent solution y PMT. Therefore, for oth heights of. nd.7 cm from the se of the flsk, the higher the PMT sensitivity level, the higher the output voltge of the mesurements. From Fig. 7, the liquid distriution plot shpes re the sme nd the positions of the strt of the ulk liquid lso remin the sme for oth heights despite the increse of the PMT sensitivity level. These show tht the liquid position mesurement is independent of the sensitivity level of PMT except the output voltges of the mesurement. For sensitivity level of for height of. cm from the se of the flsk, the output voltge exceeds the mximum llowle output voltge which is V red y PMT s shown y the sudden constnt line of the plot. Therefore, ll mesurements of the liquid distriutions were run lower thn PMT sensitivity of. Liquid distriution lso depicts thickness nd it vries t different heights of mesurement. Thickness of liquid t vrying shpes nd positions in shke flsk is not explined here in this pper. However, the thickness of the liquid influences the intensities of oth the fluorescent molecules exposed to the excittion lights nd emission lights. In this cse, PMT sensitivity of.5 ws chosen most pproprite pplying for ll heights of mesurement Fig. 8: The percentge of devitions of different numer of rottions sved for the mesurement for liquid distriution mesurement for 5 um fluorescent solutions, ph 8, mm phosphte uffer, rpm of shking frequency, 5 mm of shking dimeter nd 5 ml of filling volume. Symols: ( ) rottions; ( ) rottions; ( ) 5 rottions; ( ) rottions. 5 - Percentge devition [%] D. Repetility nd reproduciility The repetility nd reproduciility of the results re shown y the influence of the numer of rottions of the liquid in shke flsk in terms of the percentge of devitions. Percentge of devitions of less thn 5% is considered cceptle nd vlid for these evlutions. Fig. 8 shows tht the percentge of devition pproximtely less thn 5 % for different rottions tken s the men dt mesurement. However, there is very slight rupt increse of the percentge devition especilly t the strt of the ulk liquid most likely due to the sudden detection of the ulk liquid coming to the point where the opticl fiers re positioned. All in ll, the mesurement technique is very relile for liquid distriution determintion. VI. CONLUSION The work on the liquid distriutions determintions hs een done for mny conditions nd different prmeters like shking frequency, filling volume nd viscosity which re not ll shown in this pper. This technique hs successfully determined the liquid distriution y the detection of the strt nd end of the ulk liquid. This is very eneficil for the understnding of gs-liquid mss trnsfer of microorgnisms in shke flsk during fermenttions. ACKNOWLEDGMENT The uthors cknowledge the Electricl workshop especilly Mr. Kosfeld for setting the electricl setup for this mesurement, Mechnicl workshop for uilding the setup, Mr. Schemnn for progrmming the LVIEW progrm, nd Mr. Rnom for progrmming the Grphicl User Interfce (GUI) in MATHLAB for utomtic liquid distriution determintions. Acknowledgments re lso given to the sponsors of the scholrships of Higher Ministry of Eduction, Mlysi nd Deutsche Akdemischer Austuch Dienst (DAAD), Germny for the first uthor nd second uthor respectively. REFERENCES [] U. Mier nd J. Büchs. (). Chrcteristion of gs-liquid mss trnsfer in shking iorectors. Biochemicl Engineering Journl. 7. pp [] U. Mier, M. Losen nd J. Büchs. (). Advnces nd understnding nd modeling of gs-liquid mss trnsfer in shke flsk. Biochemicl Engineering Journl. 7. pp [] H.Zhng, W.Willims-Dlson, E. Keshrvrz-Moore nd P.A. Shmlou. (5). Computtionl-fluid-dynmic (CFD) nlysis of mixing nd gs-liquid mss trnsfer in shke flsk. Biotechnol. Appl. Biochem.. pp. -8. [] J. Büchs, U. Mier, S. Lotter nd C.P. Peter. (7). Clculting liquid distriution in shke flsk on rotry shkers in wterlike viscosities. Biochemicl Engineering Journl.. pp. -8. [5] C.P. Peter, S. Lotter, U. Mier nd J. Büchs. (). Impct of out-of-phse on screening results in shking flsk experiments. Biochemicl Engineering Journl. 7. pp ISBN: WCE 9