Mathematic modeling of Rennin Production by Mucor miehei

Transcription

1 An ASABE Meetng Presentaton Paper Number: Mathematc modelng of Rennn Producton by Mucor mehe Yang Hu Department of Agrcultural and Bologcal Engneerng, Pennsylvana State Unversty, State College, USA, 1680 Center for Nanocelluloscs, Pennsylvana State Unversty, State College, USA, 1680 Gulten Izmrloglu Department of Agrcultural and Bologcal Engneerng, Pennsylvana State Unversty, State College, USA, 1680 Dana Mears-ener Department of Agrcultural and Bologcal Engneerng, Pennsylvana State Unversty, State College, USA, 1680 Center for Nanocelluloscs, Pennsylvana State Unversty, State College, USA, 1680 Evan Rusad Department of Agrcultural and Bologcal Engneerng, Pennsylvana State Unversty, State College, USA, 1680 Wrtten for presentaton at the 010 ASABE Annual Internatonal Meetng Sponsored by ASABE Davd. awrence Conventon Center Pttsburgh, Pennsylvana June 0 June 3, 010 Abstract. Rennn s the most popular enzyme n the food ndustry as a coagulator of mlk to produce cheese. An effcent large-scale fermentaton for rennn producton by Mucor mehe s developed by the mathematc equatons to combat the need for rennn not derved from nfant rumnants. Scalngup of the fermentaton process, nvolvng calculatons to derve ndustral parameters from the laboratory are used extensvely to determne the feasblty of the developed process. mtatons and recommendatons of the desgn are dscussed to pont out the complcatons of assumptons and to grasp engneerng ngenuty by crcumnavgatng such problems. Ultmately we show that usng aerobc lqud fermentaton n a contnuously fed fermenter, 1,580,000 grams of rennn per annum (an estmated 10% of market supply) can be produced wth three 80,000 lter boreactors after noculaton wth 5.% (v/v) M. mehe. After the fermentaton process, mportant rennn separaton and The authors are solely responsble for the content of ths techncal presentaton. The techncal presentaton does not necessarly reflect the offcal poston of the Amercan Socety of Agrcultural and Bologcal Engneers (ASABE), and ts prntng and dstrbuton does not consttute an endorsement of vews whch may be expressed. Techncal presentatons are not subject to the formal peer revew process by ASABE edtoral commttees; therefore, they are not to be presented as refereed publcatons. Ctaton of ths work should state that t s from an ASABE meetng paper. EXAMPE: Author's ast Name, Intals Ttle of Presentaton. ASABE Paper No St. Joseph, Mch.: ASABE. For nformaton about securng permsson to reprnt or reproduce a techncal presentaton, please contact ASABE at rutter@asabe.org or (950 Nles Road, St. Joseph, MI USA).

2 purfcaton processes are dscussed: fltraton, ammonum sulfate precptaton, anon-exchange chromatography and crystallzaton. Keywords. Mucor mehe, Rennn, Fermentaton, Modelng. The authors are solely responsble for the content of ths techncal presentaton. The techncal presentaton does not necessarly reflect the offcal poston of the Amercan Socety of Agrcultural and Bologcal Engneers (ASABE), and ts prntng and dstrbuton does not consttute an endorsement of vews whch may be expressed. Techncal presentatons are not subject to the formal peer revew process by ASABE edtoral commttees; therefore, they are not to be presented as refereed publcatons. Ctaton of ths work should state that t s from an ASABE meetng paper. EXAMPE: Author's ast Name, Intals Ttle of Presentaton. ASABE Paper No St. Joseph, Mch.: ASABE. For nformaton about securng permsson to reprnt or reproduce a techncal presentaton, please contact ASABE at rutter@asabe.org or (950 Nles Road, St. Joseph, MI USA).

3 Introducton Rennn was frst dscovered by the ancent Egyptans 4000 to 5000 years ago. In 1947, Josef Hundsbchler dscovered a way to produce rennn from dred calves stomachs (Fankhauser, 007). Today, rennn s most commonly used commercally for the producton of cheese. It s a proteolytc enzyme that causes the proten component n mlk (casen) to curd and allows the lqud components to separate and run off as whey (Emmons, 1990). Tradtonal cheese-makng (more often found abroad) uses rennn from the stomach of young mlk-fed calf, lamb, or goats. Currently, heghtened vegetaran, ethcal, and kosher concerns renforce the need for a mcrobal producton method as opposed to enzyme extracton from nfant rumnants. Another need stems from the potental shortage nduced by the ncrease n demand for meat n the Unted States and abroad (USDA.gov, 009). It has been shown that mcrobal rennn produced through fermentaton has smlar qualty and proteolytc actvty to tradtonally used calf rennn (Yun et al, 1993). In the Unted States, especally n recent years, mcrobal and genetcally engneered rennn use s on the rse. These rennn types are produced from a varety of bacteral and fungal speces (Neelakantan et al, 005). Mcrobally produced rennn s ganng popularty because of FDA approval and evason of non-specfc proteolyss of casen proten whch can leave a btter taste n the cheese product (Fankhauser, 007). There s varablty n mcrobally produced rennn from mcroorgansm to mcroorgansm, but lterature has shown that Mucor mehe has the greatest potental value because of ts mnmal non-specfc proteolytc actvty, ease of producton, and low toxcty (flamentous fung are consdered safer than other mcroorgansms for use n food-product producton). Rhzomucor puslls, Endotha parastca, Irpex lacts, Bacllus subtls, Fusarum monlforme are some of the other mcroorgansms that are capable of rennn producton. Kluyveromyces lacts, Aspergllus nger and Eschercha col are the leadng recombnant mcroorgansms for rennn producton (Seker et al., 1999; Neelakantan et al., 1999; Rao and Mathur, 1975). Although many organsms have been studed for mlk-clottng enzymes, M. mehe, M. puslls and E. parastca are the most common for ndustral applcatons (Seker et al., 1999). M. mehe s a member of the class of Zygomycetes whch s comprsed of three orders: Mucorales, Entomorphtorales and Zoopagales. The Mucor genus s a thermophlc fungus growng up to 57 C (Maheshwar et al., 000). The colones are fast-growng, yellow to whte, fluffy, and ultmately develop sporanga (UA, 008). The fermentaton by M. mehe to produce rennn wll be carred at a temperature of 37.6 ºC, ntal ph of 6.8, dssolved O and controlled agtaton rate (Seker, et al., 1998; Ayhan et al., 001). The aeraton of the fermentaton broth wll be calculated later to scale up from the lab to the ndustral scale. After the fermentaton, centrfugaton followed by fltraton wll be used to separate the medum broth nto M. mehe cells and rennn. Further purfcaton consstng of: salt precptaton usng ammonum sulfate, chromatography, dehydraton and crystallzaton wll be carred out to separate and purfy the rennn enzyme from ts surroundng components (Subramanan, US Patent ). M. mehe can grow on frut, vegetables, starchy foods, and n sol. Even though M. mehe s a thermophlc mold, the optmum temperature for growth s between 35 and 45 C (Maheshwar et al., 000). M. mehe s an aerobc organsm and requres oxygen for rennn fermentaton. Table 1 shows the requrements of M. mehe for rennn producton. For effectve fermentaton, we chose 37.6 C and a ph range of 4.6 to 5..

4 Table 1. Growth parameters of M. mehe Value Source ph Ayhan et al., 001 Temperature 37.6 C Seker et al., 1998 Water Actvty ~ Table. Propertes of rennn by M. mehe (Ayhan et al., 001) Fungal Rennn Commercal Rennn MCA (SU /cm³) Total Proten (mg/cm³) MCA/ Total Proten (SU/mg) PA/ Total Proten (PU/mg) RMCA/PA (SU/PU) Mlk Clottng Actvty Proteolytc Actvty Wthout purfcaton or concentraton from fermentaton broth Table 3. Knetc parameters of M. mehe Parameters Value Reference Mlk Clottng Actvty SU/ Ayhan et al., 001 Glucose Consumed 1.6 g/ Ayhan et al., 001 Maxmum Growth Rate /h Seker et al., 1998 Producton Rate 16 IU//h Seker et al., 1998 Consumpton Rate g//h Ayhan et al., 001 Yeld (Yproduct/substrate ) SU//g/ Ayhan et al., 001 Yeld (Ybomass/substrate ) 0.01 h Ayhan et al., 001 SU: Soxhlet Unt 3

5 Table 4. Medum components and fermentaton condtons of M. mehe Medum components and fermentaton condtons Concentraton or workng values Carbon sources Molasses 30% (300 ml/, assume about 60 o Brx) Ntrogen sources Trace elements ml mcronutrent soluton Fermentaton parameters Corn steep lquor Skm mlk powder (NH 4 ) SO 4 MgSO 4 7H O K HPO 4 Na HPO 4 H O FeSO 4 7H O KCl Fe(NH 4 )(SO 4 ) ZnSO 4 MnSO 4 CuSO 4 CoSO 4 Temperature ph Agtaton rate Inoculum rato 1% (10 ml/) 1% (10 g/) 4.5 g/ 0.1 g/ 3g/ 5.6 g/ g/ 0.05 g/ 1 g/ 1 g/ 0.5 g/ 0.08 g/ 0.1 g/ C Intal 6.8 and workng ph To be determned 5.% (v/v) of workng medum Because rennn from M. mehe has a hgh R factor (mlk clottng actvty/proteolytc actvty), M. mehe s the most sutable mcroorgansm for the producton of mcrobal rennn (Seker et al., 1999). Table llustrates the characterstcs of the fungal enzyme; hgh mlk clottng actvty (MCA) and proteolytc actvty (PA) show that rennn from M. mehe can be compettve wth present commercal mlk-clottng enzymes. Seker et al. (1999) state that to obtan a good cheese qualty and yeld, the Phe-Met bond n kappa casen must be specfcally cleaved; rennn from M. mehe has acheved ths specfc cleavage makng t an adequate choce for enzyme producton. Fermentaton parameters of M. mehe for rennn producton were calculated based on the observatons of Ayhan et al (001) and Seker et al (1998). Ayhan (001) carred out fermentaton n a chemcal medum showng the effect of fermentaton varables such as temperature, ntal ph, ntal carbon source, noculums sze, and shakng rate. In ths study, M. mehe ATCC340 stran was used. Subcultures were grown on potato dextrose agar at 37 C and stored at 4 C (Ayhan et al., 001). Table 3 shows the fermentaton parameters of M. mehe. Table 4 summarzes the fermentaton medum and condtons of M. mehe. 4

6 Boreactor Desgn To desgn a boreactor system a varety of parameters must be taken nto account. These nclude: maxmum bomass concentraton, total fermentaton tme, boreactor total and workng volumes, pre-fermentor total and workng volumes, and pre- and man reactor dmensons and sterlzaton tmes. The calculatons for the desgn of a boreactor for rennn producton by M. mehe are shown n the proceedng sectons. Maxmum Bomass Concentraton (Xm) X m X o + Y x/s (S o - S) (1) The ntal concentraton of bomass s 5. % (v/v) (Escobar et al., 1993) and bomass s 0.18 g/ for noculums (Seker et al, 1998), whereas the fnal concentraton of substrate s 3. g/ (Seker et al., 1998; Ayhan et al., 001). X o C Bomass concentraton (g/) 0.05x g dry weght cell/ broth X m 0.18 g/ g /g x (0-3.) g/ (3) X m g / Where: X m: Maxmum bomass concentraton (g/) Xo: Intal bomass concentraton (g/) Yx/s: Yeld of bomass (g bomass/g substrate) So: Intal substrate (g/) S: Fnal concentraton of substrate (g/) C: Concentraton rato of ntal noculums () Exponental Growth Tme (t exp ) t exp (1 / µm ) x ln (Xm / Xo) (4) t exp [1 / (1/h)] x ln (13.86/0.18) 330 h The specfc growth rate, µ m (1/h), s assumed to be / h (Ayhan et al., 001). Mcrobal fermentaton can be growth-assocated where the product s produced durng the stage of actve mcrobal growth, non-growth assocated where producton s durng the statonary phase, or a combnaton of the two called mxed growth fermentaton. In ths last type of fermentaton the product s formed durng the growth and statonary phase. Rennn producton by M. mehe s mxed-growth assocated, however, after 500 hours of fermentaton tme producton slows down consderably and becomes nsgnfcant (Ayhan et al., 001). In ths project fermentaton wll be run durng the statonary phase and at 480 hours, rennn producton wll be stopped. 5

7 Batch Cycle (t c ) A batch cycle ncludes the preparaton tme, the summaton of lag phase, harvestng, and sterlzaton of boreactor and medum, exponental tme, and statonary phase. For our fermentaton, the preparaton tme s assumed to be ten hours, and the other tmes are assumed by followng Fgure 1 (Seker et al., 1998) Exponental phase Statonary phase 330 h 470 h Fgure 1. Bacteral growth curve and product producton curve (Seker et al., 1998). In order to acheve our annual producton goal of rennn and to reduce the waste as much as possble, we assume that the exponental phase s 330 h. We also assume that the tme from the exponental phase to the boundary of statonary phase s 140 h. tc t1 + texp + ts (5) tc 10 h h h 480 h tc 0 days tc: tme taken by one batch fermentaton cycle (h) t1: fermentor preparaton tme (h) texp: exponental growth phase (h) ts: Statonary phase (h) Producton of Rennn Enzyme n One Batch Fermentaton Cycle (Pbatch) Pbatch Pyear x tc / Workng Days (6) Workng Days 80 days [ (Sundays + Holdays) 15 (Mantenance Days)] 6

8 Snce our annual goal s 1,580 kg/year: Pbatch (1,580,000 g/year x 0 days) / 80 days 11, g (n one batch cycle) Where: Pbatch : Producton of rennn n one batch cycle (g) Pyear : Producton of rennn n one year (g) Workng Volume of Fermentor (Vw) Vw Pbatch / (Yp/s S) (assume substrate s consumed completely) (7) Vw 11, g / ( g/g 0 g/) 480, Desgnng a boreactor wth a volume of 480, s unreasonable so sx boreactors (480, / 6 80,040 ) wll be used to attan the annual producton goal. The total volume of each boreactor wll be 96,000 [80,000 workng volume + 16,000 headspace (0%)]. Boreactor Dameter and Heght Dameter: D (V/ Π) 1/3 (8) D [( x 96 m³) / 3.14] 1/3 D 3.93 m Heght: H D x 3.93m (9) H 7.86 m A boreactor wll be bult wth of dameter 3.93 m and heght 7.86 m. Although we calculated the mcrobal knetcs of M. mehe for rennn producton for our prevous work, we needed to change some of those parameters such as: yeld of bomass and yeld of product because of unt problems. Accordng to study of Ayhan et al, the product yeld was In our prevous work, the unt of yeld of product was SU/cm³/g/dm³ whch s not convertble to grams, so recalculaton of the product yeld was necessary. After re-calculatng the new value s g /g (Seker, et al 1998). Pre-fermenter Desgn Molds have a desred noculums sze range between 5-10% (w/v). The M. mehe noculums wll be 5.% accordng to the prevous assumpton (Seker et al, 1998). The workng volume of the pre-fermentor (Vsf) wll be 5.% of the total volume of the boreactor. There wll be sx prefermenters used; one for each boreactor. Vsf 5.% nvb m m 3 ~ 5m 3 (10) Note: Vb 96m 3 80m 3 (workng volume) + 16m 3 (0% head space) 7

9 The rato of dameter to heght n each pre-fermentor wll be 1:. Vsf π(d/) D (11) D (Vsf/π)1/3 ( 5m3/π)1/3 1.47m H D 1.47 m.94m (1) The dmensons of the pre-fermentor wll be 1.47m dameter.94m heght. The assumpton that nearly 100% of substrate was consumed (although complete consumpton s mprobable) was ncorporated nto the calculaton for boreactor volume. Because rennn producton by M. mehe s a mxed fermentaton type based on the graphs of bomass and product concentratons over fermentaton tme, we assumed that a neglgble amount of glucose would reman after the ten day fermentaton process (Seker et al., 1998). Sterlzaton Product Fermenter Sterlzaton s an mportant part of fermentaton to avod any contamnaton present n the medum that wll affect the growth of M. mehe. For most large-scale equpment and lquds, thermal sterlzaton s used. Usually, the dependence of the specfc death rate on temperature s gven by the Arrhenus equaton: k Eod / RT d α e (13) Most thermal sterlzatons take place at 11 0 C, and the values of typcally range from 0.5 to 5.0 mn -1. In our desgn, the sterlzaton tme can be calculated by the equaton of the probablty of an unsuccessful fermentaton f we know the probablty of unsuccessful fermentaton ( 1 P0 ( t) ), k d, spore concentraton ( n 0 ) and the volume of fermentaton tank (V ). k d t n0v 1 P0 ( t) 1 [1 e ] (14) Accordng to prevous calculatons, the volume of one fermentaton tank has been decded ( V ). We assume that the ntal spore concentraton present n the fermentaton 4 medum s 10 spores/ and k d s 1 mn -1. In addton, we assume that the probablty of an unsuccessful fermentaton ( 1 P0 ( t) ) equals (10-3 ). Now, all known values are substtuted nto the equaton (14) [1 4 4 k d t e ] (15) Wth the help of the sterlzaton chart (Fgure 4), accordng to the values n the term (15), we get k d t 7.5, where t s sterlzaton tme (mn). Therefore, sterlzaton tme for the producton fermenter, t 7.5 mn. Pre-Fermenter The same assumptons are made for the pre-fermenter as the producton fermenter to calculate the sterlzaton tme, 1 P 0 ( t) 10, n 0 10 spores/, k d 1mn. Referrng to the prevous 8

10 calculaton, the volume of the seed fermenter s as there are three pre-fermenters correspondng to three product fermenters, respectvely. Accordng to the equaton (1) and wth the help of the sterlzaton chart, we get [1 7 k d t 5 10 e ] (16). Therefore, sterlzaton tme for the seed fermenter, t 3.0 mn. Aeraton and Agtaton M. mehe consumes oxygen as the energy source for rennn producton. Durng the producton of rennn bomass s also produced. In order to produce rennn, bomass needs to consume oxygen. Therefore, there s a relatonshp between oxygen consumpton and oxygen supply. Due to ths, oxygen s an mportant parameter that needs to be metculously mantaned n an aerobc fermentaton. Aeraton plays a large role n mantanng the oxygen supply n the boreactor. In addton, agtaton also helps wth mantanng the concentraton of oxygen n the boreactor. Agtaton plays an mportant role n ensurng that the mxture of the fermentaton broth s homogeneous and n ncreasng the bubble resdence tme, mantanng the suspenson of cells, dspersng gases n the lqud, and decreasng the bubble sze (through an ncrease n surface area). Oxygen Uptake Rate (OUR) Oxygen uptake rate s the amount of oxygen used by the mcroorgansms to grow. The equaton that wll be used s as follows: OUR qo X (17) Where, q O : Specfc O consumpton rate, mg O/g cell/h X : Bomass concentraton (g/) From the lterature, we know (Matos et al., 001), from the prevous calculaton. Therefore, OUR qo X 18 mg O / g cells/h g/ mg O / h Standard Saturated Oxygen Concentraton ( C ) Standard saturated oxygen concentraton s the maxmum amount of oxygen that can dssolve n a medum. In order to calculate the value of, we need to determne the chlornty of the medum broth. There s a relatonshp between the salnty and the chlornty of the medum broth. It s gven by the followng equaton: Salnty chlornty (18) C Salnty s the amount of salts that are dssolved n one lter of medum broth. It s calculated by addng all of the salts that are used durng the fermentaton. The followng equaton also correlates salnty wth total salts: std std 9

11 Salnty Cs / ρ (19) Where, C s : Concentraton of salt (g/) ρ : Densty of broth, kg/m 3 Densty of broth s assumed to be the same as the densty of 60 o Brx (190 kg/m 3 ) (Eckart et al., 1910) Cs g/ 1000 /m g/m 3 By substtutng all values nto the equaton: Salnty Cs / ρ 1351 g/m3 / 190 kg/m g/kg and then, Chlornty Salnty / g/kg / g/kg when the broth temperature s 37.6 o C (~38 o C) and the chlornty s 5.69 g/kg (between 5.0 g/kg and 10.0 g/kg), the dssolved oxygen s estmated to be the average of mg/ and 6.05 mg/. Ths average can be calculated as follows to gve the value of C C std 6.17 mg O / 4 std Calculated Saturated Oxygen Concentraton ( C ) Determnaton of Where, Here, C comes from the followng equaton: C C std P P B 760 P P : Barometrc pressure, mm Hg B P WV WV WV : Water vapor pressure, mm Hg T ( C ) + 35 (0) P 10 (1) WV From table 4, we know the fermentaton temperature of M. mehe s 37.6 o C, therefore we can calculate P : WV P WV 10 kewse, ( ) 35 T C mm Hg P 760 mm Hg + Gauge pressure (mm Hg) () B Assumng that the head pressure n the boreactor s 5 ps: 10

12 1atm 760mmHg Gauge pressure 5 ps mm Hg 14.7 ps 1atm Then, Fnally, std P B 760 mm Hg mm Hg C can be calculated by the equaton (0) accordng to prevously known: 6.19 mg/ P mm and P mm Hg. C, Hg B WV C C Std P P B 760 P WV WV mmHg 48.67mmHg 6.17mg / 8.41mg O / 760mmHg 48.67mmHg Actual Oxygen Concentraton n the Broth ( C ) C s the value correspondng to the actual dssolved oxygen concentraton n the broth (mg/). Durng the course of fermentaton, the dssolved oxygen concentraton frst decreased from 7.0 mg/ to 1.5 mg/ and subsequently t s mantaned a value n the range of mg/ after almost 350 h (Seker et al., 1998). It can be assumed that under the crtcal condtons (when oxygen transfer s a rate lmtng step), or at a steady state, C mg / and we choose the average of the range for our desgn gvng: C 3.5mg /. Volumetrc Oxygen Transfer Coeffcent ( k a ) Under crtcal condtons or when oxygen transfer s a rate lmtng step, OTR OUR. OTR s defned as the oxygen transfer rate from the gas to the lqud phase, and OTR s gven by the equaton (3). OTR ka( C C ) (3) If OTR OUR, OUR OTR k a( C C ) (4) From the prevous calculaton, we have known OUR mg O //h, C 3.5mg /, by the equaton (4), we can get C 8.41mg O / and OUR k a ( C mgO / / h h 9.35 mn C ) (8.41mgO / 3.5mgO / ) Speed of Impeller (N) 11

13 Mechancal agtaton s usually requred for fung fermentaton. Research shows that dfferent agtaton rates can affect the producton of mold acd protease of M. mehe (Escobar et al., 1993). Snce the man fermenter s large, excellent agtaton s desred to gve good solublty of oxygen and heat transfer and to ensure the homogenety of cell concentraton and product concentraton. Accordngly, the hydrodynamc state should be set n the turbulent range and not lamnar or transton. However, too much strong turbulent agtaton probably causes the cells to face a hgh sheer force, whch could potentally damage the cell growth. In order to ensure the proper mxng of broth, Reynolds number ( Re ) s determned to be 5000 for our desgn. The value reconcles the fact that our desgned man fermenter s accommodates such a large volume. Accordng to the equaton (5): Re ρnd µ (5) Where, ρ : Densty of broth, kg/m3 N : Speed of mpeller, rpm D : Dameter of mpeller, m µ : Vscosty of broth, Pa sec For our desgn, we assume that the broth vscosty s equal to the vscosty of 60% molasses used as the carbon source. From Fgure 5, vscosty of 60% 60 o Brx molasses s about 80 cp0.08 Pa sec0.08 kg/(m sec). The dameter of mpeller ( D ) s assumed to be 10% of the dameter of the man fermentor accordng to the equaton (6). D (6) D ma ermentor We have know nf 10% D ma ermentor m nf, D m m Now, we have known 3 ρ 190 kg/m for 60 o Brx of molasses, Re 5000, D m, µ kg/m sec, and the speed of mpeller ( N ) can be calculated as follows: µ Re N ρd 0.08kg / m sec kg / m (0.39m) 60sec rpm 10 rpm 1mn Power Requrement (Pg) We know, P no P g (7) 3 5 ρn D Where, P : Power number, W no 1

14 P g : Power requrement ρ : Densty of broth, kg/m3 N : Speed of mpeller, rpm D : Dameter of mpeller, m For vscosty of broth ( µ 80cp ), we decded to choose the mpeller type as Flat-blade turbnes to ensure effcent oxygen transfer and adequate mxng of the medum broth. For Re 5000 and the type of Flat-blade turbnes mpeller, P no 6. Therefore, 3 P P ρn D g no kg / m 3 (10rpm 1mn 3 5 ) (0.393) 60sec W 796HP W 0.73 HP Superfcal Gas Ext Speed ( v ) s Another equaton (8) used to calculate k a k p g ( ) vs N Vw (8) a k s as follows: Where k s constant and s assumed as 1, and from prevous calculatons we can calculate v s as follows: v s ka pg 0.4 k( ) N Vw mn W 1mn 1 (10rpm ) 80 60sec 0.5 1mn 60sec m/s Total Orfce Area ( A ) total We know Q A total (9) v s 13

15 where Q s ar flow rate (m 3 /sec). Snce the value of OUR n our desgn s a lttle large, the arflow rate s assumed to be 1.0 vvm to provde suffcent oxygen for the growth of M. mehe. 3 3 Q 1m O / m / mn 80m Then, A total Q v s 3 1 1mn 60sec.33m 3 O /sec 0.005m / s 1.33 m 3 O 53 m /sec Number of Orfces ( n ) We know A total n (30) Ahole Where, A s the area of orfce (m ) hole We assume that the dameter of hole s 0 cm for each n our desgn, and then A n A total hole 53m 0.0 π Product Recovery and Purfcaton Fltraton Calculaton Removal of product from medum and separaton of bomass s the crtcal fnal step n the fermentaton process. There are a varety of mechansms to acheve adequate separaton. Rennn producton by M. mehe wll employ constant pressure vacuum fltraton as the frst step n separaton and purfcaton. Table 5 shows the parameters used to calculate the surface area of the flter requred for ths process. All assumed parameters were decded upon after vewng varous fermentaton studes from two relable sources (Demrc, 009; Shuler and Karg, 00). Table 5. Parameters used to determne flter area. Known parameters Value Workng volume (V) 40,000 1 Medum vscosty (µ) 1.5e - kg/m/s Assumed parameters Weght of cake/volume of fltrate (C) 1000 kg/m 3 Average specfc resstance of cake (α) 5.0 m/kg Pressure drop ( P) 3.45e 5 N/m 14

16 -1 Flter medum resstance (r m ) 0.0 m 1 The workng volume was reduced snce t was dffcult to manpulate the parameters effcently to flter 80,000. Therefore the workng volume of each reactor wll be pumped to two separate flter systems, each processng 40,000. Medum resstance was assumed to be neglgble snce usng an arbtrarly low value dd not create a large dfference n area and fltraton rate. If a value s chosen for r m the quadratc equaton must be appled to the Ruth Equaton to solve for flter area and ths method was also practced and consdered. Usng the Ruth Equaton (31) t s possble to solve for the necessary area of the flter needed. The ntal volume s a functon of medum resstance (r m ) whch can be assumed neglgble snce the cake resstance (r c ) greatly supersedes the medum resstance durng the fltraton process (Demrc, 009). Wth ths n mnd, equaton (31) smplfes to equaton (34). Wth workng tme and volume known t s possble to solve for k usng equatons (3, 33) and then to employ algebra to solve for the area (A) of the flter. The amount of tme s assumed to be 6 hours to allow one workng day for run and clean-up. Where, V r A m αc 0 (3) A k pg c αcµ r A α C + 0 (31) V VV kt (33) m Snce we assume V 0, therefore 0 V kt Accordng the values from table 6, we can calculate k A pg αcµ (34) 5 A 3.45e e 1000 c kt k (360mn) (35) A Area of flter surface requred m dv / dt To determne the fltraton rate,, t s frst necessary to calculate the resstance of the flter cake, r c (36) and apply to equaton (37). dv dt e m αcv r c A (36) g pa c m / s 70 / m (37) µ ( r + r ) m c 15

17 Wth the above fltraton rate t wll take 56 mnutes to flter the entre 40,000 lters of fermentaton broth. Ths s consderably lower than the desred goal of 360 mnutes whch means that the parameters could be re-assessed for effcency and adjusted to elongate fltraton tme and reduce energy consumpton. Recovery of Pure Rennn The characterzaton of an actve commercal product (n ths case mlk-clottng enzyme) requres a strngent purfcaton process for rennn producton by M. mehe. There are several methods to obtan pure rennn after fermentaton, such as fltraton, evaporaton, ultra fltraton, precptaton (salt or solvent), dalyss, electrophoress, and freeze-dryng (Joyeaux, 198). For our producton, purfcaton of rennn from the fltrate wll use: ammonum sulfate precptaton ( saltng out ), on exchange chromatography, and crystallzaton (Sternberg, 1970). Concluson The objectve of ths project was to desgn a boreactor and determne operaton parameters for the producton of 1,580 kg of rennn annually by Mucor mehe. Rennn was frst dscovered by the ancent Egyptans 4000 to 5000 years ago. Today, mcrobal and genetcally engneered rennn producton has been most commonly used commercally for the producton of cheese nstead of tradtonal cheese-makng usng rennn from the stomach of young mlk-fed calf, lamb or goats. M. mehe has shown the greatest potental value of producng rennn. The fermentaton process of M. mehe for rennn producton can be performed at a temperature 37.6oC, ntal ph 6.8 and workng ph range of 4.6 to 5.. In our desgn, 30% molasses served as an economc carbon source nstead of glucose. Regular fermentaton of M. mehe s executed n sold medum wth gelatn as ts ntrogen source. However, our desgn nvolved growth of M. mehe n lqud medum, so we decded to use 1% corn steep lquor as the ntrogen source, as well as, 1.0% skm mlk powder as another ntrogen source to enhance rennn producton (Slvera et al., 005). The trace elements prmarly came from the salts of ammona, magnesum, potassum, phosphate, sodum, ferrum, and copper. Accordng to the lterature and our assumpton, the bomass concentraton was determned to be 13.9 g/. Accordng the cell growth curve, we predcted the exponental perod (330 h) and statonary perod (140 h). Once the bomass concentraton and the batch cycle (0 days) were obtaned, we calculated the rennn producton on one batch cycle n terms of our annual producton objectve. Snce the calculated rennn producton for one batch cycle was huge we dstrbuted the rennn producton of one batch cycle to 6 dentcal fermentors, and the volumes of producton fermentor and pre-fermentor were subsequently determned to be and 5000 n terms of the above calculated results. Assumng ntal concentraton of spores n the medum to be 104 spores/, the sterlzaton tmes at 11oC for producton fermentor and prefermentor were calculated to be 7.5 mn and 3 mn, respectvely. Dssolved Oxygen (DO) s an mportant parameter n aerobc fermentaton. Oxygen wll be ntroduced to fermentaton broth by spargng ar. Oxygen Uptake Rate (OUR) for M. meher was calculated to be mg O/ accordng the specfc oxygen consumpton rate of 18 mg 16

18 O/g cell/h from the lterature and bomass concentraton of g/. At 5 ps gauge pressure n the fermentor, calculated saturated oxygen concentraton was 8.41 mg O/ and actual oxygen concentraton n the broth was 3.5mg O/. The volumetrc oxygen transfer rate, was calculated as 9.35 mn-1. Agtaton can be acheved mechancally or non-mechancally. The flat blade turbne, wth a calculated dameter of m, was chosen as t s sutable for medum vscosty of 80 cp and provdes less shear rate to bomass. The speed of mpeller was calculated as 10 rpm and would requre 0.73 HP motor to operate. Once the fermentaton was completed, the bomass separaton from the fermentaton broth was suggested by contnuous rotary flter havng surface area of m. Total tme of fltraton was estmated to be 56 mn for half of one product fermentor. The purfcaton of rennn was contnued wth ammonum sulfate precptaton, anon exchange chromatography and crystallzaton. Acknowledgements We would lke to thank Dr. Demrc and Merck for walkng us through the desgn of a boreactor and showng us the complextes of scalng up from the lab to the ndustry. We especally enjoyed seeng what an 80,000 reactor actually looks lke as opposed to just n our desgn proposal; t was nterestng (and exctng) to see our potental desgn n real lfe! References Please do not use hyperlnks-- Ayhan, F, S. C. Serdar, and A., Tanyolac The effect of fermentaton parameters on the producton of Mucor mehe acd protease n a chemcally defned medum. J. Chem. Technol. Botechnol. 76: Chen, J.C.P. and C. C. Chou Cane Sugar Handbood. 1th ed. John Wley and Sons, NJ, USA Emmons, D.B. and M.Bnns. Cheese Yeld Experments and Proteolyss by Mlk-Clottng Enzymes. Journal of Dary Scence Vol. 73, No. 8, Escobar, J., and M. S. Barnett Effect of agtaton speed on the synthess of Mucor mehe acd protease. Enzyme Mcrob. Technol. 15(1): Eckart, C.F., R.C.. Perkns, H.. yon, N. Deerr and S.S. Peck The Hawaan Planters Record. Experment Staton. Unversty of Calforna lbrary. Fankhauser, D.B. Rennet for Makng Cheese. Date accessed: 3 January Hanknson, C.., 194. The preparaton of crystallne rennn. Presented at the 37th Annual Meetng Amercan Dary Scence Assocaton, Mchgan State College, June 5, 194 Joyeaux, A Novel fungus of Mucor mehe and ts use n obtanng a new mlk-clottng enzyme. US Patent eong, S.A. and R.M. Berka Molecular Industral Mycology: systems and applcatons for flamentous fung. New York, NY: Marcel Dekker, Inc. pps evadoux, W., M. Johnson, and G. Andre Montorng the degradaton of commercal mcrobal rennet preparatons. J. Dary Sc. 7: Maheshwar, R., G. Bharadwaj, M.K. Bhat Thermophlc fung: Ther physology and enzymes. Mcrobology and Molecular Bology Revews. 64(3):

19 Marston F., owe P., Doel M., Schoemaker J., Whte S., and Angal S., Purfcaton of Calf Prochymosn (Prorennn) Syntheszed n Eschercha col, Bo/Technology, Pp.800, Chcago, Illnos, 1984 Matosc, S Applcablty of the Three Dmensonal Growth Model n Descrpton of Mucor mehe NCAIM 538 Cultvaton and Renn Bosynthess. Mckelsen J., and C. A. Ernstrom Factors affectng stablty of rennn. J. Dary Sc. 50(5): Neelakantan, S., A.K.Mohanty, J.K. KAushk Producton and use of mcrobal enzymes for dary processng. Current Scence. 77: Rao,.K. and D.K. Mathur Studes on the producton of bacteral rennet n a plot plant fermantor. Botechnology and Boengneerng. XVII: Seker, S., H. Beyenal, and A. Tanyolac Modelng mlk clottng actvty n the contnuous producton of mcrobal rennet from Mucor mehe. J. Food Sc. 64(3): Seker, S., H. Beyenal, F. Ayhan, and A. Tanyolac Producton of mcrobal rennn from Mucor mehe n a contnuously fed fermentor. Enzyme and Mcrobal Technol. 3: Shammet, et al Proteolytc Actvty of Some Mlk-Clottng Enzymes on k-casen. J. Dary Sc.75: Shuler, M.., and F. Karg. 00. Boprocess Engneerng Basc Concepts. nd ed. Prentce-Hall PTR, NJ, USA Slvera, G. G., G. M. Olvera, E. J. Rbero, R. Mont, and J. Contero Mcrobal rennet produced by Mucor mehe n sold-state and submerged fermentaton. Brazlan Arch. Bo. Technol. 48(6): Sternberg, M. Z Crystallne mlk-clottng protease from Mucor mehe and some of ts propertes. Journal of Dary Scence. 54:, Subramanan, S, Purfcaton of mcrobal rennet from Mucor mehe. US Patent Unversty of Adelade, 008. Mycology onlne. Accessed: Feb 4, 009 Avalable at: l Vela, C. and S. Walker, 001. A Commercal Scale Process for the Producton of Chyomsn (Rennet) from Recombnant Eschercha col. ABE 497B/468 Semester Project. The Pennsylvana State Unversty, UP, PA. Wang, N.S Enzyme Purfcaton by salt (ammonum sulfate) Precptaton. Avalable at: Accessed on Aprl 0,