Department of Chemical Engineering

MEMORANDUM

Department of Chemical Engineering

Michigan Technological University

TO: CM4125 Students, Weeks 7 Group 1: Microfiltration

Experiment starts Tue. Feb. 21, 2006 in room 205 (CSEB)

FROM: David R. Shonnard

Professor and Faculty Supervisor

Email:

Phone: 487-3468,

Abraham Martin-Garcia

Graduate Student Laboratory Supervisor

Email:

DATE: 21 Feb., 2006 – date of start to experiment

SUBJECT: Microfiltration Objectives

Introduction

Microfiltration is one of many possible bioseparation steps just after fermentation (others include centrifugation and vacuum filtration). The purpose is to separate the cells from the soluble products, in our case separating C. glutamicum from the L-lysine product. To accomplish this, you will be using a cross-flow microfiltration device. The cross-flow arrangement within the device assures that a minimum of cell buildup occurs on the membrane surface, which should keep the volumetric fluxes higher than for simple flow through filtration.

Objectives

You are to;

1. Conduct a batch microfiltration experiment where the retentate stream is recycled backc to the feed tank (see powerpoint slides for background on the set up and theory). The experiment will be conducted at room temperature.

a) Periodically measure the cell concentration of the samples as per the accompanying instructions (about 5 ml at 500 nm wavelength (A500 for cells) using a visible spectrophotometer (Milton Roy 21D). The conversion between absorbance and cell numbers is y = 1.034x109 x, where y is the cell numbers per millileter of solution and x is A500. If you wish to express cell concentration in units of mg dry cell weight, you can convert from cell numbers (y) to mg by knowing that there are 0.5 mg dry cell wt. for each 109 cells. A filtered sample from the bioreactor (0.22 mm pore size) is taken according to the schedule on the computer in the lab to detect L-Lysine using high performance liquid chromatography (HPLC) or enzyme assay. Glucose is also monitored using HPLC, but also by enzyme strips. From the data collected

i)  Calculate and plot the concentration of cells and L-lysine in the feed tank over time.

ii)  Calculate and plot the mass of cells and L-lysine in the feed tank over time.

iii)  Match the model prediction of cell and L-lysine concentration to the data and determine whether the cells are a perfectly retained species and whether L-lysine is a perfectly permeating species.

2. Using the first draft procedures provided to you, conduct the laboratory experiment (in the presence of the faculty advisor and TA) and note improvements to be made. Integrate these improvements into the existing procedures (this statement of objectives and procedures will be attached to email) paying particular attention to the safety and operational aspects. You should submit these improved procedures to me as an email attachment, and mark the changes using the “Track Change” function.

3. Interim written report: Prepare graphs of the data from the microfiltration experiment (pressure, cell and lysine concentrations) vs. time. Discuss your experimental run and results in a report format. Attach appendices as needed.

4. On the day of the experiment, be prepared to discuss the important safety features for this experiment. You will be quizzed on this and must pass the quiz in order to start the experiment. Also, be prepared to describe the data you will collect and the subsequent calculations to address your objectives.

Job Safety Assessment Form

Department of Chemical Engineering

Michigan Technological University

Overview Page

Equipment Name: Crossflow Microfiltration

Room Number: 205

Written By: David Shonnard Date: 02/20/2006

Revision #: Revision Date:

Hazard Checklist:

Toxicity / Mechanical Hazard / Pressure Hazard
Fire/Flammability / Electrical Shock / Biohazard:
Reactivity / Hot Surfaces/ High Temp / Other:

PPE required for Lab:

Long Pants / Safety Glasses / Hard Hat / Apron
Long Sleeves / Splash Goggles / Insulated Gloves / Ear Protection
Non-porous Shoes / Face Shield / Chemical Gloves / Other:

Available Safety Equipment:

Nearest Fire Extinguisher: By the wall near the door / Nearest Eyewash: By the east wall
Nearest Spill Kit: In the glass cabinet by the door / Nearest Safety Shower: By the entrance door
Nearest Telephone: Room 205 / Nearest First Aid Kit: By the east wall
Other: (air monitor, etc.) Laminar hoods / Other:

Chemicals Used:

Chemical Name / Potential Hazards / PPE Required
Health / Flamm. / Reactivity / Other
Tween-80 / Safety goggles
Ethyl Alcohol / 1 / 3 / 0 / 1 / Safety goggles, Vent hood, Proper gloves, Face shield
Sodium Azide / 4 / 0 / 1 / 0 / Safety goggles, Proper gloves

Location of Nearest Required Spill Response Supplies:

Floor-Dri: (or N/A ) / Spill Dikes: (or N/A )
Sodium Bicarbonate: In the cabinet above the work area (or N/A ) / Drain Plugs: (or N/A )
Spill Pillows: (or N/A ) / Additional Supplies: Acids and Bases in the cabinet below the laminar hood (or N/A )

1

1

Safe Operating Procedures Page

Sequence of Steps / Potential Hazards / Recommended Safety Procedure / PPE Required
Emergency Shutdown
1. Turn off the peristaltic pump.
2. Leave the lab. / Safety glasses
Start-up Procedure
1. Attach the fitting kits by following the installation instructions in the manual. (This has been done by the TA).
2. Assemble the Microfilter. Place a clear silicone intercassette gasket (supplied with the filter assembly) on the bottom manifold plate. Now place the minicassette (from the refrigerator in 205) and carefully install the top acrylic plate. Tighten each nut on the Microfilter holder. (This has been done by the TA).
3. The pump is connected via silicone tubing to the feed inlet port of the holder. Tubing then is connected as follows: from the feed container (4 liters) to the pump inlet, continuing to the feed port of the holder, from the retentate port of the holder back to the feed container. The permeate will be collected in a separate tank (1 liter). (This has been done by the TA).
4. Set the pump adjacent to the Microfilter holder on the lab bench top. Load the tubing into EASY-LOAD pump head (consult the pump instruction manual). Wrap the threads of the pressure gauges three times around with Teflon tape and screw the gauges into the ports on the side of the lower acrylic plate (only if not already attached). (This has been done by the TA).
5. Check pump operation prior to assembling the Pellicon System. Plug the system in and turn the pump switch to the counterclockwise position. Check to make sure that the tubing is not crimped and that it exhibits peristaltic movement inside the pump head. (This has been done by the TA).
6 Tubing pump operation: Set the speed control knob in the tubing pump to “zero” position and turn the pump toggle switch to “off” position.
7. The preservative solution (sodium azide 0.05%) in the Microfilter cassette is purged out in a flushing step before starting the sample collection. 500 ml graduated cylinders can be used to collect the purged solutions. After starting the pump, adjust the permeate valve until the permeate flowrate is approximately 200 ml/min. Continue purging until cells appear in the retentate stream, then turn off the pump and route the retentate stream back in the feet tank. Make sure that the volume collected from the permeate tube is sufficient to displace the preservative in the tube. Then stop the pump. Dispose the preservative solution down the sink with a stream of tap water. / Minicassette is stored in Sodium Azide (toxic!)
Abrasion to skin from pump inlet and outlet
Electric Shock
Sodium Azide (toxic!) / Avoid exposure to skin, by ingestion, and inhalation. Wash off any contact immediately.
Do not turn on pump unless fingers and cloths are away from pump inlet and outlet
Make sure no water is spilled on the bench or near the electrical outlet.
Avoid exposure to skin, by ingestion, and inhalation. Wash off any contact immediately. / Safety Glasses at all times.
Wear gloves.
Wear gloves.
Run Time Procedure
The retentate stream contains the cells (C. glutamicum), which is re-circulated to the feed. Lysine and any other soluble fermentation products pass through the membrane and into the permeate stream. Conduct experiment at room temperature.
1. Set the control knob so that the feed flow rate is 3 lpm using the chart in Appendix A.1. The permeate flow rate should be 200 ml / min. You are to collect a total of 1000 ml of permeate solution over about a 5 minute period.
2. Before the start of the pumping, take a 5-ml sample from the feed tank. Measure cell concentration using the visible spectrophoto-meter at 500 nm wavelength as in the fermentation experiment. Note the initial volume in the feed tank. You can calculate the initial mass of cells in the feed tank from these measurements.
3. Assure that the tubes are in the proper configuration for recycle mode of microfiltration. Start the pump. Take 5 ml samples from the feed tank every 30 seconds. Take 5 ml samples from the permeate stream going into the permeate tank every 30 seconds. Note the pressures on the inlet and outlet ports on the microfilter. Cell concentration will be measured using the visible spectrophotometer. There should be no cells in the permeate stream unless the membrane is ripped and needs replacing. / Biohazard: C. glutamicum will be treated as a level 2 pathogen. / Avoid ingestion and contact with skin. Wash with anti-microbial soap before leaving the lab. / Safety glasses at all times.
Shutdown Procedure
1. The membrane filters should be flushed, cleaned and sanitized after each experiment.
Membranes are flushed with warm water and a solution of 1 ml of Tween-80 per liter of solution. Do this until all cells are gone from the retentate stream.
2. The membrane cassette is stored in sodium azide (0.05%) and then ready for the next run. A solution of sodium azide is pumped through the microfilter until all traces of Tween-80 are gone. Clamp the ends of the tubes to retain the preservative solution. / Spill / Transportation buckets / Rubber gloves

End of procedures and safety assessment.

CM4125 Bioprocess Engineering Lab. 6 Microfiltration; D. Shonnard

I. READING THE ABSORBANCE (SPECTROMETER OPERATION)

1. Turn the power switch on.

2. Use the mode button to change to absorbance mode.

3. Change the desired wavelength by using the knob next to the wavelength screen.

4. To calibrate the spectrophotometer, use sterile media as your calibration media. Insert the clean cuvette in the sample reader filled with sterile media up to the fill line. Use the “INCREASE KNOB” to calibrate to .000.

5. Take the sample in a clean cuvette, once more filling the cuvette up to the fill line. Discard the first sample to prevent contamination from previous samples. Put the cuvette in the sample reader and wait a few seconds until it is stable, take the reading from the screen.

6. After finishing taking the samples, turn the power switch off.

7. Unplug the machine.

II. LYSINE ENZYME ASSAY PROCEDURE (Use only if HPLC is not functional)

As this is a lengthy procedure that involves many chemicals and steps, it is suggested that you gather and clean hardware and locate chemicals at the beginning of the procedure. If you are unsure how to use the Mettler H80 balance to measure the minute quantities required by this procedure, need assistance in calibrating the pH meter, or have any other questions ask your TA or Dr. Shonnard. When performing this procedure plan ahead and be meticulous, but most of all be patient. Your results will be as accurate as you make them.

Hardware needed:

2 125mL Erlenmeyer flask 100mL graduated cylinder

8 test tubes 4-5 specs (spectrophotometer tubes)

2mL volumetric pipet 40-200mL Oxford BenchMate

5mL volumetric pipet 100-1000mL Oxford BenchMate

disposable pipet w/ bulb Tips for both Oxford Benchmates

pH meter w/ calibration samples spectrophotometer

Chemicals needed:

KH2PO4 (Potassium Phosphate Monobasic)

EDTA (Disodium Ethylenediamine tetraacetate)

L-lysine

HCl or NaOH(concentrated)

NADH (b-Nicotinamide Adenine Dinucleotide, Reduced Form)*

a-Ketoglutaric Acid*

Saccharopine Dehydrogenase*

* chemical stored in freezer, remove only as needed

Procedure

A.  Preparation of Solutions for 20 Assays (10 Calibration Standards and 10 Experimental Samples)

1.  Reagent A (KH2PO4 (FW=136.09 g/mole) and EDTA (FW = 372.24 g/mole))

a.  Measure 1.3609 g KH2PO4 (100 mM) and .0372 g EDTA (1 mM) on the analytical balance and transfer to a 125-mL flask.

b.  Add 100 mL of distilled water to these reagents to make a solution of 100 mM of KH2PO4 and 1 mM of EDTA using a 100-ml graduated cylinder.

c.  Mix thoroughly by using the Fisher Vortex Genie 2.

d.  Calibrate the pH meter using calibration standards. Carefully add concentrated HCL or NaOH (about 6 N), using a disposable pipet while swirling, until the pH of the resulting solution is 6.8. Cap this solution with parafilm.

2.  Reagent B (NADH, FW = 709.4 g/mole)

a.  Measure .0098 g b-NADH (.23 mM) on the analytical balance.

b.  Add 60 mL of Reagent A to the b-NADH using 100 mL graduated cylinder