Lab Pilt Fermenter Type LP351, 75 Liters OPERATING INSTRUCTIONS Befre yu start... 1. Autclave antifam and base slutin (in glass bttles) alng with three feeder tubing lines with hypdermic needles attached n ne end. There is n need t sterilize the acid slutin. 2. Secure a dispsable septum, metal cllar and stpper nt fur free prts n the lid f the vessel. 3. Make sure there is enugh lubricant (80% glycerl in water) in the canister at the bttm f the vessel. 4. Cnnect the cables frm the fermenter t the frnt and back panels f the Intelligent Frnt Mdule (IFM) cntrl bx. Turn n the main switch n the IFM. 1
5. Calibrate the ph prbe thrugh the IFM On the ph panel, press "Menu" and use the < > buttns t select CALI. Press OK. Select CALIB. MEAS, press OK. Press OK n passwrd. Enter 1ST REF = 7.00 ph (Always d ph 7 first), and dip the prbe int the apprpriate ph slutin with the white dt facing the current. Wait until the current stabilizes, then select CALIBRATE. Rinse the prbe with deinized water. Enter 2ND REF = 4.00 ph, and repeat the previus step with the apprpriate ph slutin. Select CALIBRATE when the reading stabilizes. Select CALIB DONE and press OK. Munt the ph prbe t the side prt f the fermenter vessel white dt n the prbe facing the flw current. Turning n the energies: 1. In the Autclave rm: Turn the Autclave switch t POWER and wait fr the steam pressure t build up t ~ 4 bars (Open the dr underneath the autclave chamber t check the steam pressure gauge). Turn the yellw steam valve at the right side f the Autclave t align in parallel with the pipeline. CAUTION: The steam pipe is ht! Check that all industrial cld water valves n the back wall are pen (parallel t the pipelines) 2
2. Turn n the cmputer (Username: user; Passwrd: 123456). The BiScadaLab sftware shuld pen autmatically. Make sure that the IFM is n befre turning n the cmputer. 3. Click n Batch Start and enter the fermentatin infrmatin. 4. Turn n the air and xygen supply lines n the wall. 3
Pressure Leak Test 1. Drain residual liquid: Open the harvest valves (H1 and H2) t drain residual liquid frm the vessel. With the SAMPLING valve clsed, pen S1 until all residual cndensate has drained. CAUTION: Ht steam will be released. 2. Open A1, A2, EXH 3. Clse all ther valves: OXY, C1, C2, C3, S1, S2, H1, H2, CW, SAMPLING 4. The air filter (FL) shuld be in a raised psitin (handle pints t the back) 5. In BiScadaLab, click n the Pressure panel and select "Cntrlled". Repeat fr "Universal Air" and "Universal O2" 4
6. Enter the fllwing set pints in the crrespnding white fields: Pressure: 1.2 bar Universal Air: 100% Universal O2: 0% 7. Once the actual pressure (indicated in the gray field) has reached 1.2 bar, clse A1, C2, and EXH. 8. Wait fr 5 10 minutes, and check that there is n significant reductin in the actual pressure. Vessel Sterilizatin 1. Open C1, C3, EXH (fully) 2. Clse all ther valves: A1, OXY, C2, S1, S2, H1, H2, CW, SAMPLING 3. Lwer FL (handle pints t the frnt) 4. Add culture media int the vessel via the Innculatin Prt. 5. In BiScadaLab, click n the Agitatr panel and select "Cntrlled". Enter 250 rpm in the white field. The agitatr shuld nw be activated. 6. In BiScadaLab, click n the Temperature panel and select "Sterilizatin". The sterilizatin cycle will nw start. (The sterilizatin parameters have been pre set t return t 37 C at the end f the cycle.) 5
7. Wait fr the system t heat up. Once 95 C has been reached, the IFM display will read "CLOSE THE VALVE, WHEN DONE PRESS OK". Turn EXH t 90% clsed, then press OK 8. The cntrller will heat up t 121 C, at which pint sterilizatin will cntinue fr 20 minutes. 9. After the sterilizatin time is finished, the system will start cling t 100 C, and the IFM display will read "OPEN THE VALVE, WHEN DONE PRESS OK". Open A1, EXH, CW Clse C1, C3, and all ther valves Lift FL (handle pints t the back), then press OK 10. Temperature will nw decrease and be maintained at 37 O C. Installatin f Antifam, Acid and Base Feeder Slutins 1. Install the tubing leading frm the antifam, acid and base slutins nt the crrespnding rtatry pumps at the side f the IFM. 2. On the lid f the vessel, unscrew a stpper and pur ethanl n tp f the septum. Light a small flame t sterilize. Hld the hypdermic needle frm the tubing abve the flame and remve the needle case. When the flame is almst extinguished, pierce the needle thrugh the septum and screw it dwn in place. Repeat fr each tubing. 6
Fermentatin Parameters Settings On BiScadaLab (PC), click n each cntrl panel and select "Cntrlled". Enter the desired parameters n the crrespnding white fields. The recmmended Agitatr speed is ~250 rpm. Innculatin 1. Open A1, A2, OXY, EXH, C1, and C3 2. Remve the stpper n the Innculatin Prt and flame the muth briefly. Intrduce the innculum directly int the vessel. Maintain sterility as much as pssible. Sampling 1. Open S1 fr 5 10 secnds t sterilize the sampling pipe. CAUTION: Steam and cndensate will be released. 2. Open the SAMPLING valve t cllect sample. Harvesting 1. Open C2 and then S2 fr 5 secnds t sterilize the cndensate line. 2. Open H1 and then S2 t sterilize the harvest line. 3. Clse S2 and pen bth H1 and H2 t harvest the fermentatin batch. Wrapping Up 1. On BiScadaLab, click n each cntrl panel and select "Off" 2. Stp the batch run n BiScadaLab. Username: Admin, Passwrd: admin 3. Clse all valves, and turn ff all energies (air, xygen, steam). 4. Rinse the vessel by filling it with water. On BiScadaLab, right click n Agitatr and select "Cntrlled". Enter 250 rpm as the set pint. Drain the water by repeating the steps described in the Harvesting sectin. Turn the Agitatr ff when dne. 5. Turn ff the PC and the main switch n the IFM. Cntact persn: Widya (510) 717 8816 widyam@stanfrd.edu 7