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Background : Continuous Simultaneous Saccharification and Fermentation (cSSF) system is the preferred configuration to produce ethanol biologically.This system can improve the effectiveness of enzymes, achieve higher volume productivity, reduce labor costs, reduce vessel down time for cleaning and filling, and eliminate the need for seed fermenters. Also the organisms can adapt to inhibitors and other environmental factors. Enzymes are one of the bottlenecks in lowering the price of lignocellulosic enthanol in real industry. Therefore, the theenzyme dose reduction is is much needed for Simultaneous Saccharification and
Fermentation(SSF) process, which has been proved both theoritically and experimentallly supeior than separate hydrolysis and fermentation (SHF) in a lot of aspects, especially in enzyme use.
For continuous system, multistage continuous system is the preferred system comparing to single stage. However, some important challenges lie in the continuous processing of lignocellulosic materials such as contamination, feeding insoluble substrate, and concerns about organism washout .Air bubble feeding system is set up to prevent the biomass settling in the tube.
Continuous enzyme adding system is also set up to make sure enough enzyme is feed during this process. The whole setup should be run under sterile conditions.
1.Fermenter introduction

Three 3 L autoclavable Bio Reactor of the same model are from APPLIKON
BIOTECHNOLOGY. The Applikon autoclavable Bioreactor Systems basically consist the following parts: 1) an autoclavable Bioreactor with the appropriate auxiliaries like a stirrer assembly, baffles, an aeration assembly, etc. 2) an ezControl for measurement and control of process variables (pH, Temperature, DO level, and stirrer speed) and calculates corresponding controller outputs in order to keep process conditions on set-point 3) the ezControl combines and supports actuators like pumps and valves in order to optimize the use of limited bench-space.
Two BIOSTAT-fermenter (3L and 5L) systems are from sartorium stedim biotech. The fermenter system consist of two parts: UniVessel and Touch Panel

2. Substrate Feeding
Magnetic stirring plate and magnetic stir bar (V&P Scientific, San Diego) is used to mix the feed. Air bubble system is set up to prevent the settling in the feeding tube.
The feeding bottle is equipped with a special lid, enabling the feeding of air to the bottlethrough a sterile filter. On the feed tube a T-piece is mounted, which is equipped with a non-return valve and connected to an air supply line. Air flow is switched on periodically to empty the feed line completely to prevent the clogging of the tubes.
1) Corn stover was milled to pass a 1 mm screen by a hammer mill (Wiley). The composition of the corn stover was measured by following NREL LAPs. The optimal pretreatment condition is chosen.
2) We use the Level control antifoam Peristaltic pump on the reactor. The two level sensors on the Applikon reactor must be connected permanently. For the Biostat reactor, the level sensor must be connected to conductor. L/S 16 (Masterex, Cole Parmer) tube is chosen to transport both feedstock and slurry between reactor

3) Control the time of feeding through level control timing. Control the dead time and circle time, then you can control the dilution rate and flow rate. Dead time = cycle time - pulse time.
You should adjust the flow rate every time you use it. The feed flow rate depends on the tubing which is used, the amount of air bubbles which are fed concomitantly and the run time of the feed pump. At the feed tube just above the feeding bottle a special 1/8"T-piece was mounted, which is equipped on the branch end with a 1/8" non-return valve and connected to an air tubing connected to the front of the reactor control unit. the line from the pressurized nitrogen source is split, one part goes to the feed bottle as describe above and the other one is connected to the reactor control unit at its backside.
3.Transport between reactors
1.
Pressurized nitrogen is used to transport the slurry between different stages.
2.
Dip tube is mounted to control the height of stages.
4 Enzyme Feeding
1) 0.01" diameter tubing is chosen to feed the enzyme. It is difficult to cut this kind of tube, the ends should be grinded off to stop its closing.
2) Peristaltic pump (Watson Marlow Pumps Group) is used to feed enzyme. Pump adjustment should be done.
3) Enzymes (a mixture of C-tec and H-tec) were diluted 1 to 10 with 0.05M citric acid buffer
(pH 4.8) and sterile filtered. The dilution is necessary in order to be able to adjust the correct enzyme flow rate; without the dilution the flow rate would be too low to be adjustable. To avoid thermal enzyme deactivation, the enzyme solution is placed in a refrigerator

5. Solution Calculation
1) Tetracycline (10 mg/mL in 70% ethanol) A mL
2) 10× YP was used as nutrient for yeast, which containsyeast extract 100 g/L and Peptone 200 g/L. Autoclave for 30 minutes at 121oC, B mL.
3) Determine the Inoculum Dry Cell Mass Concentration according to the NREL Laboratory
Analytical Procedure (LAP), C mL.
4) If the total volume of the reactor loading is F. Buffer 1M, For D5A is citric acid, M=0.05*F.
And the solid loading is S. So the water you need to add is = F-SF-M-C-B-A*0.8523