Flocculation bioreactors engineering based problems

Transcription

1 8th Conference SSCHE, Proceeding on CD ROM, Tatranké Matliare (SK), 5 May, Flocculation bioreactor engineering baed problem António A. Vicente - Joé A. Teixeira Centro de Engenharia Biológica IBQF, niveridade do Minho, Campu de Gualtar, Braga, Portugal, phone: ; fax: ; jateixeira@deb.uminho.pt Keyword: flocculation bioreactor, high cell denity, ma tranfer,, hydrodynamic Abtract The ue of flocculation bioreactor i one of the mot intereting way to provide an increae of the productivity of biotechnological procee. A other high cell denity ytem, flocculation bioreactor allow the achievement of high bioma concentration in each moment inide the bioreactor a well a an improvement of downtream procee. Beide thee advantage, hared by all high cell denity ytem, flocculation bioreactor are clearly more intereting due to the abence of a upport ince it i well known that price i a limiting factor for the implementation of thoe ytem. Thi being o, there are engineering problem that need to be addreed. In firt place, the hydrodynamic of three-phae bioreactor are conidered, followed by the evaluation of the ma tranfer propertie both from ga to liquid and from liquid to olid phae. Finally, application of flocculation bioreactor are dicued and example are preented. Introduction The bioreactor i a veel that hould provide the nutritional and phyiological environment required for microbial owth: it i the core of a bioproce. Immobilied catalyt (cell, enzyme, protoplat, organelle) can be employed in variou type of reactor, depending on the immobiliation technique, a well a on the type of the proce. The aim of uch ytem i to increae catalyt concentration while keeping it inide the bioreactor, in order to increae proce productivity. An example of uch high cell denity ytem i given by the production of ethanol uing alginate-immobilied yeat (Gough and McHale 998, Joeke et al. 998, Nguyen and Shieh 99, Tyagi et al. 99) or flocculating yeat (Abate et al. 996, Soua et al. 994a, Jianfeng et al. 998). Baic bioreactor configuration are uually conidered, namely the tirred tank, the packed bed, the fluidied bed, the bubble column and the. To chooe the bet reactor type to uch high cell denity ytem tudie on mixing, heat and ma tranfer between the different phae, a well a an evaluation of operational and maintenance cot have to be made. With flocculating organim, uitable hydrodynamic condition mut be provided (epecially low hear tre) in order to maintain the adequate floc ize, hape and denity characteritic neceary to retain bioma inide the bioreactor a much a poible, although keeping the maximum poible activity. For thee reaon, tirred veel and packed bed are not recommended a flocculation bioreactor, although they are uitable for other immobilied cell ytem which are mechanically more reitant. The fluidied bed i not very adequate for flocculating culture, either, a the denity difference between floc and medium i rather mall and fluidiation would be achieved at very low air/liquid flow rate. Bubble column and epecially reactor are quite appealing to be ued with three-phae ytem in procee involving flocculating organim (Ganzeveld et al. 995, Kennard and Janekeh 99, Merchuk et al. 994, Onken and Weiland 983, Siegel and Robinon 99). The evaluation of the performance of flocculating culture in reactor depend on the

2 undertanding of the following parameter (Michalki 99): ga hold-up, ga-liquid interfacial area, volumetric phae ditribution, liquid mixing time, liquid circulation velocity, liquid and ga phae axial diperion coefficient, fluid-wall heat tranfer coefficient and cell retention capacity. Epecially for thi lat parameter the flocculation ability of the microorganim i a major help a it contribute ignificantly to the cell retention capacity of the bioreactor (Soua et al. 994a). The overall volumetric ma tranfer coefficient a well a liquid-olid and intra-particle ma tranfer are phenomena of capital importance, particularly in a flocculation bioreactor, that will be dealt with later on in a eparate ection. The relation between the variable in term of their influence in bioreactor hydrodynamic behaviour have been ummaried in Figure. OPERATION VARIABLES DESIGN VARIABLES Top Preure Drop Friction Preure Drop Area Ratio (A r /A d ) Solid Load Liquid Velocity Bottom Preure Drop Reactor Height Ga Input Rier Ga Hold-up Vicoity Bottom Clearance Top Clearance Separator Ga Hold-up Downcomer Ga Hold-up Separator Deign Figure : Relation between variable in reactor (adapted from Merchuk et al. 996). Anyway, independently of their oxygen tranfer capability (which may or may not be needed, depending on the proce), mixing (of both liquid and ga) mut be adequate in order to minimie ma tranfer reitance and to provide the deired homogeneity inide the bioreactor. Mixing time i a parameter directly related with mixing and it i ueful to evaluate the deee of mixing in the bioreactor; ga flow rate i a good control variable a it directly influence not only liquid circulation velocity but alo ga hold-up both in rier and downcomer. Thu, correct regulation of the ga flow rate will be crucial to keep olid circulating a well a to optimie the hydrodynamic condition inide the reactor. All the above-mentioned parameter are, in turn, influenced by the deign of one or everal of the part that compoe an. A one of the main feature of high cell denity ytem i the high hold-up of the olid phae (that can go up to 5-6% v/v of the total bioreactor volume, with the correponding reduction in liquid-phae volume) a ignificant reearch effort ha been devoted to optimie the deign of everal part of three-phae reactor, namely in thoe apect related to their ue a high cell denity ytem. A previouly tated the ue of flocculent microorganim require a urplu of attention when deigning a reactor in order to keep floc with the uitable characteritic for the proce (hape, denity and ize, mainly). Hydrodynamic of three-phae internal- and external-loop bioreactor The effect of the ditributing plate orifice diameter, airflow rate, olid loading and olid denity on the hydrodynamic characteritic ga holdup, circulation time and liquid velocity of a three-phae external-loop reactor wa characterized. Experiment were performed in a gla wall external-loop reactor with a working volume of 6 l (Figure a).

3 (a) (b) Figure : a) external loop bioreactor; b) internal loop bioreactor. Air wa ued a the ga-phae and injected through perforated plate with a contant free plate area ratio φ =.% and hole diameter of.5 mm,. mm and.6 mm. The airflow rate wa varied between l/h and 8 l/h correponding to rier uperficial ga velocitie, baed on the rier cro-ection area, between.3 m/ and.7 m/, repectively. Water wa ued a liquid-phae and Ca-alginate bead, with two different denitie, were ued a olid-phae imulating yeat cell floc. The mean diameter and denity of the low denity olid (LD) were (.3 ±.) mm and (3 ± ) kg/m 3, repectively, and the value for the high denity olid (HD) were (.5 ±.5) mm and (48 ± ) kg/m 3. For each type of olid, olid loading applied wa %, 5%, %, 5%, % and 3% (v/v). It wa found that the ditributing plate orifice diameter doe not have a ignificant influence on the parameter tudied, being only oberved a light change of rier ga holdup and circulation time, for low airflow rate and low olid loading, and on liquid velocity, for % of olid. Thi reduced effect of the ga ditributor in reactor, when compared with bubble column, can be acribed to the influence of uperimpoed liquid flow on the flow pattern and bubble rie velocity in the rier, enhanced due to the preence of the olid phae. On the contrary, airflow rate and olid loading have a eat effect on rier ga holdup, circulation time and downcomer liquid velocity. The increae of airflow rate lead to an increae of rier ga holdup and liquid velocity while the circulation time, conequently, decreae. In oppoition, the increaing introduction of olid produce a decreae of the ga holdup and liquid velocity and an increae of circulation time. There i not a well defined trend of the effect of olid denity, in the range of value tudied, on the tudied hydrodynamic characteritic of the three-phae external-loop reactor. In order to evaluate and compare the performance of three-phae internal- and external-loop reactor with the ame working volume, the experimental reult of rier ga holdup, liquid velocity, circulation and mixing time obtained for the two reactor (working under the ame condition) were compared. The internal-loop reactor ued i of the concentric-tube type with an enlarged degaing zone (Figure b). Both reactor howed imilar repone to the change of the operation parameter. The comparion of the two reactor howed that, in all cae, the external-loop reactor preented higher value of rier ga holdup. However, internal-loop reactor exhibited better circulation and mixing performance. Development of an hydrodynamic model A mathematical model predicting the hydrodynamic behaviour of three-phae reactor, working with low-denity olid and with high olid loading, wa developed. The model allow for the prediction of local ga holdup and liquid velocity in bioreactor and wa validated for the external- and the internal-loop reactor ued in the experimental work. 3

4 The rier ga holdup i given by: = r C + + lr r + t r bt The rier uperficial liquid velocity i obtained by the following equation: () INTERNAL-LOOP AIRLIFT REACTOR: gh ( ) gd = A A r d + k ( ) gd D fb d ( ).5 r +.75 r + lr A A EXTERNAL-LOOP AIRLIFT REACTOR: gh = ( H r+ H t) Dr r d.75 + H b) D ( ) D c D r ( ) gd.5 Hβ.75 d + β lr fr, t ( ) ( ) ( ).5 + Ar Ad A A r t, d.75 ( H d.5 d.75 lr ( ) Ar lr k ft, d + k fgc + ( k + k + k ) fc fd, b fb, r ( ) Ad.75 k lr + () (3) The optimiation of the parameter C, bt, a, b and β, allowing for the prediction of value of rier ga holdup and the rier uperficial liquid velocity, wa done uing a developed computer oftware. The reult obtained how that the model predict the experimental value of rier ga holdup and downcomer linear liquid velocity obtained, for all the experimental cae tudied, with high accuracy (with an error of ±% and ±5%, repectively) (Figure 3 a, b) % +5% (calc).5. -% vld (calc).5-5% (exp) v ld(exp) a b Figure 3. Predicted value v. experimental value of rier ga holdup (a) and downcomer linear liquid velocity (b), for all the experimental condition (.6 mm:! - LD,! - HD;. mm: " - LD, " - HD;.5 mm: - LD, # - HD): LD low denity olid ; HD high denity olid For both reactor, for the etimated parameter, a imilar effect of the olid loading on hydrodynamic wa found. The ditribution parameter (C) preent ome ocillation howing that, depending on the amount of olid, the olid-phae affect the flux in different way. The terminal velocity of a ingle bubble ( bt ) increae with the increae of olid loading, a a conequence of the increae of coalecence deriving from the increae of the interaction between the bubble. The parameter β exhibit different trend for the two reactor, reulting from their ditinct geometrie. 4

5 The effect of the preence of ethanol in ytem hydrodynamic i particularly relevant, ince mot of the application of flocculation bioreactor deal with ethanol fermentation. When water i replaced by an ethanol olution ( kg m -3 ), a reduction in the urface tenion occur and change ignificantly the repone of the reactor in term of the ga and olid hold-up in both rier and downcomer, the circulation and mixing time and the rier and downcomer intertitial velocity (Freita and Teixeira 998). In fact, an increae of the rier and downcomer ga hold-up i regitered, together with the conequent decreae of olid hold-up in thoe ection and the reulting decreae of rier and downcomer intertitial velocity. However, the difference between ga and olid hold-up in the rier and in the downcomer remain practically contant when ethanol i added; the driving force for the circulation i thu maintained and, o, the preence of ethanol while increaing mixing time, ha no meaurable effect on circulation time. Oxygen ma tranfer in a high olid loading three-phae internal-loop reactor For the previouly decribed internal-loop reactor with an enlarged degaing zone, the volumetric ma tranfer coefficient wa determined. Airflow rate, olid loading, olid denity and liquid-phae were the parameter manipulated. It wa hown that the volumetric ma tranfer coefficient diminihe with the increae of olid loading, epecially for high airflow rate, due to an increae in bubble coalecence. Reduction between 4% and 7% were obtained with the introduction of % and 3% of olid. Solid denity alo affect k L a. A ignificant decreae on k L a reulting from a mall increae of olid denity (from 3 to 48 kg/m 3 ) wa oberved, a a conequence of the effect of olid denity on olid ditribution in the reactor. A proeive increae on the concentration of olid in the lower ection of the reactor, a the rier and the downcomer, with the increae of olid denity i reponible for the enhancement of coalecence and the conequent decreae on the pecific interfacial area. Airflow rate and the preence of ethanol were hown to enhance the volumetric ma tranfer coefficient - airflow rate by primarily increaing ga holdup and ethanol by inhibiting coalecence. The increae on k L a by the preence of ethanol depend on the amount and type of olid and on the airflow rate. For rier uperficial ga velocitie up to.75 m/, the augment i very mall, while for higher airflow rate, value of k L a for aqueou ethanol olution are about.5 to time the value obtained for water. The volumetric ma tranfer coefficient wa correlated to the rier uperficial ga velocity (u ) and the olid loading ( ), for the two type of olid and for the two liquid, water and ethanol olution. Aeement between the calculated value and the experimental data of ±%, for water, and of ±%, for aqueou ethanol, were obtained (Figure 4 a,b,c and d). The obtained correlation were: Water/ Low denity olid : k a = ( ) ( ) L u u (4) Water/ High denity olid : k a = ( ) ( ) L u u (5) Ethanol/ Low denity olid : k a = ( ) ( ) L u u (6) Ethanol/ High denity olid : k a = ( ) ( ) L u u (7) where < u ( m / ) <.5 < (% v / v) < 3 5

6 .3.3 kla (calc).. +% -% kla (calc).. +% -% (a)...3 kla (exp) (b)...3 kla (exp) kla (calc).. +% -% kla (calc).. +% -% (c) kla (exp) (d) kla (exp) Figure 4: Comparion between calculated k L a value and experimental data: (a) water/ low denity olid ; (b) water/ high denity olid ; (c) aqueou ethanol olution/ low denity olid ; (d) aqueou ethanol olution/ high denity olid. Ma Tranfer of Solute in the Floc Vicente et al. (998a) tudied ma tranfer characteritic (effective diffuivity, D e, and external ma tranfer coefficient, K c ) of glucoe and oxygen in yeat floc (.9 mm to.4 mm in diameter) of S. cereviiae uing inactivated cell in a modified diffuion cell (Vicente et al. 997) developed in order to avoid floc detruction. D e and K c were calculated uing two method: a claical one, baed on analytical olution of Fick law of diffuion and a numerical one, baed on general ma balance of a component in floc and bulk phae. Diffuion coefficient were found to be, for glucoe, 7% of the diffuivity in water and, for oxygen, between.% to % of the diffuivity in water, which i in aeement with the data from Ananta et al. (995) if the ize i conidered. K c value increaed with the agitation rate, a expected, and have value which range from m - to 5-9 m -. Thee value indicate that not only the ma tranfer inide floc, but alo the one outide them, may be a limiting tep in thi proce. The value obtained were then ued to etimate the penetration depth of the olute in floc by modelling diffuion-reaction phenomena. Concentration profile of glucoe inide agegate of S. cereviiae were imulated and calculation were made for different poible ize of the yeat floc, conidering alo the preence or the abence of a polymeric additive (Vicente et al. 998b) (Figure 5). The preence of a polymeric additive that enlarge the pace between cell inide floc proved to be effective in reducing ma tranfer limitation. 6

7 Dimenionle glucoe concentration Dimenionle floc radial coordinate Figure 5. Comparion of dimenionle glucoe concentration profile for floc own with (dot-dahed line) and without (olid line) a flocculation additive, with a radiu of.8 mm (the parameter near the line identify the value of the bulk concentration to which each line correpond). Application of flocculation bioreactor Beide all the other factor pointed out through the preceding page, the operational condition under which a given bioreactor can be operated are alo largely dependent on the flocculation ability of the microbial train involved in the fermentation. If a highly flocculent train i ued, it continuance inide the bioreactor poe no problem even if tough operational condition are ued, uch a high aeration and dilution rate (Vicente et al. 999). However, when a given train ha weak flocculation abilitie, the reactor deign play a fundamental role, being an one of the mot appropriate, epecially if equipped with a edimentation zone (either at the top of the reactor, for an internal loop, or at the top of the downcomer, for an external loop ). In Figure 6 it i poible to follow the operation of an external loop with a edimentation zone, a volume of. L and fed at different dilution rate, a hown (Teixeira and Mota 99).. Conidering the need to develop new and impler fermentation ytem and the uitability of the bioreactor for culture uing flocculating microorganim, a 5.4 L internal loop bioreactor wa teted and compared with the previou ytem (Soua et al. 994a) uing a highly flocculating train of S. cereviiae owing on glucoe. A comparion wa made in term of tartt (day) Figure 6. Flocculation bioreactor repone to increaing dilution rate (D) with a feed lactoe concentration of 57. g L - ; # - bioma concentration in the bioreactor (X);! - bioma concentration in the effluent (X); " - lactoe concentration (S); $ - ethanol concentration (P). 7

8 up evolution, overall performance and power cot. The bet ethanol productivity wa obtained for the concentric tube reactor (.9 g L - h - ), but both ytem behaved in a imilar way and the productivity value were about even time higher than in commercial ytem. There wa alo a clear indication of a higher cell activity in the concentric tube bioreactor when compared to the external loop, thu compenating for the lower cell retention capacity of the former. The work proceeded, then, with the concentric tube (Soua et al. 994b), by tudying the evolution of fermentation parameter of the ame flocculent train of S. cereviiae during the tart-up of a continuou fermentation. A trong influence of the dilution and aeration rate wa found on both bioma and ethanol concentration and kinetic parameter. The operating parameter, in turn, do not eem to affect glucoe conumption rate but affect, intead, the toichiometry of it converion to either bioma or ethanol, uggeting a hift in the metabolic mechanim a bioma build up. One of the hortcoming of flocculation bioreactor, already mentioned, i the preence of ma tranfer limitation inide the floc and it ha been pointed out in the previou ection that a reduction in floc ize could be expected to bring a reduction in ma tranfer limitation, leading to an increae of productivity. Vicente et al. (999) introduced tatic mixer in the draught tube of the internal loop bioreactor ued by Soua et al. (994a,b), achieving an effective reduction of the floc ize (3 mm to mm in diameter). Steady tate data at different dilution rate were meaured for both ytem (the original and the modified bioreactor) and the reult were compared in term of pecific conumption/production rate and ethanol productivity. A 4% increae wa obtained in the maximum dilution rate at which a glucoe converion higher than 98% could be achieved. The repiratory quotient had a contant value (around 3) at all dilution rate, meaning that the metabolic tate of the cell in floc remained contant, having a trong fermentative metabolim. The floc ize reduction contributed to the higher oberved reaction rate, not only by mean of an increaed dilution rate, but alo becaue of reduced diffuional limitation, leading to a 3% increae of ethanol productivity when compared with the original ytem. One of the firt commercial application of the edimentation characteritic of flocculating microorganim wa made by the brewing indutry, back in 97, in order to facilitate the eparation of the yeat cell from beer at the end of the proce (Greenhield and Smith 97). Still, it i motly in the brewing indutry that flocculation bioreactor are widely ued. However, in thi cae, flocculation i eentially a eparation technique and not a way to immobilie cell in continuou high cell denity ytem. Mot brewing companie and brewing reearch oup have everal reearch work running uing high cell denity bioreactor, motly with configuration, in order to invetigate their potential ue in continuou beer fermentation, with the advantage pointed out throughout the preceding text (Dillenhöffer and Röhn 996, Dömeny et al. 998, Linko et al. 997, Machelein 997, Menour et al. 997, Šmoovicová et al. 997, Tata et al. 999). Neverthele, none of thee work actually deal with flocculating culture, though ome mention them a a poible alternative to the exiting procee, in particular for beer maturation (Linko et al. 997). The advantage of bioma retention in bioreactor make the ue of thee ytem particularly attractive in continuou fermentation. Thi i the cae of flocculation bioreactor. In Table, a ummary of work with flocculation bioreactor i preented, proving an emerging interet for thi type of ytem. A can be een, the majority of the work preented deal with flocculating microorganim for continuou ethanol production, which i not urpriing ince, for the moment, continuou high cell denity ytem are adequate for high volume low added value product. A alo indicated, mot of the tudie on flocculation bioreactor have been done in bench-cale apparatue due to cot and complexity involved in larger cale reearch. Being o, further information on hydrodynamic and ma tranfer needed for reactor cale-up i till miing and it i not urpriing that, o far, indutry till heitate to elect a flocculation-baed proce for commercial purpoe, in pite of the operational advantage of thee ytem. 8

9 Table. Work with flocculation bioreactor. Organim Reactor type Volume Main ubtrate Main product Productivity Ref. [L] [g L - h - ] S. cereviiae bubble column -5 wort beer - Smith and Greenhield 974 S. cereviiae bubble - molae ethanol 5-3 Kida et al. 989 column K. marxianu external loop. lactoe ethanol 4.4 Teixeira et al. 99 S. cereviiae external loop glucoe / ethanol 68 Fontana et al. 99 ucroe S. cereviiae CSTR 3 glucoe ethanol 5 Kida et al. 99 S. cereviiae erie of.5 + molae ethanol 9.3 Kuriyama et al. 993 CSTR.5 S. cereviiae internal loop 5.4 glucoe ethanol - Soua et al. 994b S. cereviiae external +. + glucoe ethanol.9 Soua et al. 994a internal loop 5.4 S. cereviiae fluidied bed molae ethanol 5 - Wieczorek and Michalki, 994 S. cereviiae external loop ucroe ethanol - Roca et al. 995 S. diactaticu external loop Z. mobili + Saccharomyce p. Rhodiola achalineni agitated conical flak internal loop S. cereviiae internal loop Schizoaccharomyce external loop pombe S. cereviiae internal loop Jerualem artichoke extract ethanol + inulin - Schorr-Galindo et al ethanol.5 Abate et al alidroide - Jianfeng et al een beer maturated beer. ape mut deacidified ape mut deproteinied cheee whey - Mafra et al Soua et al. 993 ethanol - npublihed data A particular reference mut be made to the work of Domingue et al. (999), who decribe the ue of a recombinant flocculating train of S. cereviiae expreing the LAC4 (coding for β- galactoidae) and LAC (coding for lactoe permeae) gene of K. marxianu to perform alcoholic fermentation of lactoe with the previouly decribed bioreactor. In continuou operation, an ethanol productivity of g L - h - wa obtained (with a feed lactoe concentration of 5 g L - and a dilution rate of.55 h - ), being even-fold larger than the one in conventional continuou ytem. Depite of the flocculence intability of the recombinant train, a high bioma concentration wa achieved inide the bioreactor a it deign allowed for a election of the mot flocculating cell from a mixed culture, contributing thu with a elective preure for the maintenance of the flocculating cell inide the bioreactor. The mot direct application of thi work i the highproductivity fermentation of the lactoe preent in cheee whey to produce ethanol, not only contributing for the bioremediation of thi by-product of the dairy indutry produced in large amount, but alo allowing for the production of a ueful fuel. There i the need for more reearch on high cell denity ytem, in general, and on flocculation bioreactor, in particular, in order to gather the neceary information to make them an intereting alternative to the procee ued nowaday, which are in mot cae very well etablihed 9

10 and tudied. The potential urely exit in thi new technology, but it ha to be demontrated before the indutry rik inveting largely in it. Reference Abate, C., Callieri, D., Rodríguez, E. and Garro, O., 996. Ethanol Production by a Mixed Culture of Flocculent Strain of Zymomona mobili and Saccharomyce p. Appl. Microbiol. Biotechnol., 45, Ananta, I., Subroto, M. A. and Doran, P. M., 995. Oxygen Tranfer and Culture Characteritic of Self-Immobilized Solanum aviculare Agegate. Biotechnol. Bioeng., 47, Dillenhöffer, W. and Rönn, D., 996. Continuou Maturation of Beer. Beverage World International, Nov./Dec., Dömény, Z., Šmoovicová, D., Gemeiner, P., Šturdík, E., Pátková, J., Malovíková, A., 998. Continuou Secondary Fermentation uing Immobilized Yeat Biotechnol. Lett.,, Domingue, L., Danta, M. M., Lima, N. and Teixeira, J. A., 999. Continuou Ethanol Fermentation of Lactoe by a Recombinant Flocculating Saccharomyce cereviiae Strain. Biotechnol. Bioeng., 64, Fontana, A., Ghommidh, C., Guiraud, J. P. and Navarro, J. M., 99. Continuou Alcoholic Fermentation of Sucroe ing Flocculating Yeat. The Limit of Invertae Activity. Biotechnol. Lett., 4, Freita, C., Teixeira, J. A., 998. Effect of Liquid-Phae Surface Tenion on Hydrodynamic of a Three-Phae Airlift Bioreactor with an Enlarged Degaing Zone. Bioproce Eng., 9, Ganzeveld, K. J., Chity, Y. and Moo-Young, M., 995. Hydrodynamic Behaviour of Animal Cell Microcarrier Supenion in Split-Cylinder Airlift Bioreactor. Bioproce Eng.,, Gough, S. and McHale, A. P., 998. Continuou Ethanol Production from Molae at 45 C uing Alginate- Immobilized Kluyveromyce marxianu IMB3 in a Continuou-Flow Bioreactor. Bioproce Eng., 9, Greenhield, R. N. and Smith, E. L., 97. Tower-Fermentation Sytem and their Application. Chem. Engineer, May, 8-9. Jianfeng, X., Jian, X., Aiming, H., Puun, F. and Zhiguo, S., 998. Kinetic and Technical Studie on Large-Scale Culture of Rhoiola achalineni Compact Callu Agegate with Air-Lift Reactor. J. Chem. Technol. Biotechnol., 7, Joeke, I., Moran, P. J. S., Rodrigue, J. A. R., Wendhauen, R., Tonella, E. and Caiola, F., 998. Characterization of Saccharomyce cereviiae Immobilized onto Chryotile for Ethanol Production. J. Chem. Technol. Biotechnol., 73, Kennard, M. and Janekeh, M., 99. Two- and Three-Phae Mixing in a Concentric Tube Ga-Lift Fermentor. Biotechnol. Bioeng., 38, 6-7. Kida, K., Kume, K., Morimura, S. and Sonoda, Y., 99. Repeated-Batch Fermentation Proce ing a Thermotolerant Flocculating Yeat Contructed by Protoplat Fuion. J. Fermentation Bioeng., 47, Kida, K., Yamadaki, M., Aano, S.-I., Nakata, T. and Sonoda, Y., 989. The Effect of Aeration on Stability of Continuou Ethanol Fermentation by a Flocculating Yeat. J. Ferm. Bioeng., 68, 7-. Kuriyama, H., Ihibahi, H., Miyagawa, H., Kobayahi, H. and Eiichi, M., 993. Optimization of Two-Stage Continuou Ethanol Fermentation ing Flocculating Yeat. Biotechnol. Lett., 5, Linko, M., Virkajärvi, I., Pohjala, N., Lindborg, K., Kronlöf, J., Pajunen, E., 997. Main Fermentation with Immobilized Yeat a Breakthrough?. EBC Cone 997, Mafra, I., Cruz, J. M. M. and Teixeira, J. A., 997. Beer Maturation in a Continuouly Operating Bioreactor ing a Flocculation Brewer Yeat Strain. EBC Cone 997, Machelein, C. A., 997. A Realitic View on the Role of Reearch in the Brewing Indutry Today. J. Int. Brew., 3, 3-3. Menour, N. A., Margariti, A., Brien, C. L., Pilkington, H., Ruel, I., 997. New Development in the Brewing Indutry ing Immobilized Yeat Cell Bioreactor Sytem. J. Int. Brew., 3, Merchuk, J. C., Ladwa, N., Cameron, A., Bulmer, M. and Pickett, A., 994. Concentric-Tube Airlift Reactor: Effect of Geometrical Deign on Performance. AIChE J., 4, 5-7. Merchuk J. C., Ladwa, N., Cameron, A., Burmer, M., Berzin, I. and Pickett, A. M., 996. Liquid Flow and Mixing in Concentric Tube Air-Lift Reactor. J. Chem. Technol. Biotechnol., 66, Michalki, H. J., 99. Air-Lift Bioreactor. In: M. Berovic and T. Koloini, ed. Bioreactor Engineering Coure Workhop Note; Italy, 99. Nguyen, V. T., Shieh, W. K., 99. Continuou Ethanol Fermentation ing Immobilized Yeat in a Fluidized Bed Reactor. J. Chem. Tech. Biotechnol., 55, Onken,. and Weiland, P., 983. Airlift Fermenter: Contruction, Behavior, and e. In: Advance in Biotechnological Procee. New York: Alan R. Li, Inc., 983, Roca, E., Ghommidh, C., Navarro, J. M. and Lema, J. M., 995. Hydraulic Model of a Ga-Lift Bioreactor with Flocculating Yeat. Bioproce Eng.,, Schorr-Galindo, S., Ghommidh, C. and Guiraud, J. P., 995. Inulin Enrichment by Fermentation in a Flocculating Yeat Reactor. Biotechnol. Lett., 7, Siegel, M. H. and Robinon, C. W., 99. Application of Airlift Ga-Liquid-Solid Reactor in Biotechnology. Chem.

11 Eng. Sci., 47, Šmoovicová, D., Dömény, Z., Gemeiner, P., Malovíková, A. and Šturdík, E., 997. Reactor for Continuou Primary Beer Fermentation uing Immobilized Yeat. Biotechnol. Tech.,, Soua, M. J., Teixeira, J. A. and Mota, M., 993. Mut Deacidification with an Induced Flocculant Yeat Strain of Schizoaccharomyce pombe. Appl. Microbiol. Biotechnol., 39, Soua, M. L., Mota, M. and Teixeira, J. A., 994b. Influence of Operational Parameter on the Start-up of a Flocculation Airlift Bioreactor. Col. Surf. B: Biointerface,, Soua, M. L., Teixeira, J. A. and Mota, M., 994a. Comparative Analyi of Ethanolic Fermentation in Two Continuou Flocculation Bioreactor and Effect of Flocculation Additive. Bioproce Eng.,, Tata, M., Bower, P., Bromberg, S., Duncombe, D., Fehring, J., Lau, V., Ryder, D., Stai, P., 999. Immobilized Yeat Bioreactor Sytem for Continuou Beer Fermentation. Biotechnol. Prog., 5, 5-3. Teixeira, J. A. and Mota, M., 99. Flocculation Bioreactor. In: T. G. Villa and J. Abalde, ed. Profile on Biotechnology. Spain: Servicio de Publicacione, niveridade de Santiago, 99, Teixeira, J. A., Mota, M. and Goma, G., 99. Continuou Ethanol Production by Flocculating Strain of Kluyveromyce marxianu: Bioreactor Performance. Bioproce Eng., 5, 3-7. Tyagi, R. D., Gupta, S. K., Chand, S., 99. Proce Engineering Studie on Continuou Ethanol Production by Immobilized S. cereviiae. Proc. Biochem., 7, 3-3. Vicente, A. A., Dluhý, M., Ferreira, E. C., Mota, M. and Teixeira, J. A., 998. Ma Tranfer Propertie of Glucoe and O in Saccharomyce cereviiae Floc. Biochemical Eng. J.,, Vicente, A. A., Dluhý, M., Ferreira, E. C. and Teixeira, J. A., 998. Modelling Diffuion-Reaction Phenomena in Yeat Floc of Saccharomyce cereviiae. Bioproce Eng., 8, Vicente, A. A., Dluhý, M. and Teixeira, J. A., 997. A New Technique for Meauring Kinetic and Ma Tranfer Parameter in Floc of Saccharomyce cereviiae. Biotechnol. Tech.,, 3-6. Vicente, A. A., Dluhý, M. and Teixeira, J. A., 999. Increae of Ethanol Productivity in an Airlift Reactor with a Modified Draught Tube. Can. J. Chem. Eng., 77, Wieczorek, A. and Michalki, H., 994. Continuou Ethanol Production by Flocculating Yeat in the Fluidized Bed Bioreactor. FEMS Microbiol. Review, 4,