MEMORANDUM - Michigan Technological University



MEMORANDUM

Department of Chemical Engineering

Michigan Technological University

TO: CM4125 Students, Week 8 Group 2: Microfiltration

Experiment starts Tue. Feb. 28, 2006 in room 205 (CSEB)

FROM: David R. Shonnard

Professor and Faculty Supervisor

Email: drshonna@mtu.edu

Phone: 487-3468,

Abraham Martin-Garcia

Graduate Student Laboratory Supervisor

Email: armartin@mtu.edu

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 back 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. You may need to dilute samples taken for cell concentrations. From the data collected

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

ii) Calculate and plot the concentration (and total mass) of cells and L-lysine in the permeate 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. Before preparing the interim report, schedule a data debriefing to present and discuss your graphs and tables.

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 ) |

Safe Operating Procedures Page

|Sequence of Steps |Potential Hazards |Recommended Safety Procedure |PPE Required |

|Emergency Shutdown | | | |

|1. Turn off the peristaltic pump. | | |Safety glasses |

|2. Leave the lab. | | | |

|Start-up Procedure | | | |

|1. Attach the fitting kits by following the installation | | |Safety Glasses at all times. |

|instructions in the manual. (This has been done by the TA). | | | |

|2. Assemble the Microfilter. Place a clear silicone intercassette | | |Wear gloves. |

|gasket (supplied with the filter assembly) on the bottom manifold |Minicassette is stored in Sodium|Avoid exposure to skin, by ingestion, and | |

|plate. Now place the minicassette (from the refrigerator in 205) |Azide (toxic!) |inhalation. Wash off any contact immediately. | |

|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 |Abrasion to skin from pump inlet|Do not turn on pump unless fingers and cloths are| |

|feed container. The permeate will be collected in a separate tank |and outlet |away from pump inlet and outlet | |

|(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 |Electric Shock |Make sure no water is spilled on the bench or | |

|Microfilter cassette is purged out in a flushing step before | |near the electrical outlet. | |

|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 100 ml/min. Continue purging until cells appear in | | | |

|the retentate stream, then turn off the pump and route the | | | |

|retentate stream back in the feed 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. | |Avoid exposure to skin, by ingestion, and | |

| |Sodium Azide (toxic!) |inhalation. Wash off any contact immediately. | |

| | | |Wear gloves. |

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|Run Time Procedure | | | |

|The retentate stream contains the cells (C. glutamicum), which is | | |Safety glasses at all times. |

|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 2 lpm using | | | |

|the chart in Appendix A.1. The permeate flow rate should be 100 | | | |

|ml/min. You are to collect a total of 1000 ml of permeate solution| | | |

|over about a 10 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 |Biohazard: C. glutamicum will be|Avoid ingestion and contact with skin. Wash with| |

|measurements. |treated as a level 2 pathogen. |anti-microbial soap before leaving the lab. | |

|3. Assure that the tubes are in the proper configuration for | | | |

|recycle mode of microfiltration. Start the pump. Take 5-8 ml | | | |

|samples from the feed tank after 200 ml of permeate is collected. | | | |

|Take 5-8 ml samples from the permeate tank at the same time. Note | | | |

|the pressures on the inlet and outlet ports on the microfilter. | | | |

|Cell concentration will be measured using the visible | | | |

|spectrophotometer after suitable dilution. There should be no | | | |

|cells in the permeate stream unless the membrane is ripped and | | | |

|needs replacing. | | | |

|Shutdown Procedure | | | |

|1. The membrane filters should be flushed, cleaned and sanitized |Spill |Transportation buckets |Rubber gloves |

|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. | | | |

End of procedures and safety assessment.

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

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 40-200(L Oxford BenchMate

5mL volumetric pipet 100-1000(L 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 ((-Nicotinamide Adenine Dinucleotide, Reduced Form)*

(-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 (-NADH (.23 mM) on the analytical balance.

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

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

3. Reagent C ((-Ketoglutaric Acid, FW = 146.1 g/mole)

a. Measure .0233 g (-Ketoglutaric Acid (79.8 mM) on the analytical balance.

b. In test tube add 2 mL of Reagent A to the (-Ketoglutarate using a 2 mL pipet.

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

4. Reagent D (L-Lysine, FW = 182.65 g/mole)

a. Measure .0.050 g L-Lysine (54.75 mM) on the analytical balance.

b. In test tube add 5 mL Reagent A to the L-Lysine using a 5 mL pipet to make a 10 g Lysine / L solution.

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

5. Reagent E (Saccharopine Dehydrogenase, mix right before using)**

a. Measure .0004 g of Saccharopine Dehydrogenase (2 units of enzyme) on the analytical balance and add 2 mL of Reagent A using a 1 mL pipet in a test tube.

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

** 1.0 unit of enzyme per ml of Reagent E.

B. Dilutions of the Calibration Standards for Lysine

1. Starting with the 10 g/L (54.75 mM) lysine solution already prepared, dilute in order to get several lysine concentrations distributed fairly even from 0 to 10 g/L.

2. Approximately 5 different concentrations will be sufficient to achieve an

effective calibration curve.

3. Dilute from the 10 g/L lysine solution every time.

4. For example, if you wanted to prepare a 5 g/L solution from the 10 g/L solution, you could add 1 ml of Reagent A to 1 ml of the 10 g/L solution in a new labeled vial, making sure to use separate 2 ml pipets.

C. Preparation of the Spectrophotometer

1. Clean 4 or 5 spectrophotometer tubes thoroughly.

2. Set the spectrophotometer to a wavelength of 340 nm.

3. Add distilled water to the white line of a clean spec and use this throughout the

experiment as an equipment blank spec.

4. Use KimWipes to clean this spec, place it in the spectrophotometer, and zero out the absorbance.

D. Preparation of the Enzyme Spectrophotometer Tubes for Lysine Calibration and Sample Analysis

1. Add 2.75 mL of Reagent B to a spec using a 2 mL pipet.

2. Add .1 mL of Reagent C to the same spec using a 40-200(L Oxford BenchMate.

3. Add .1 mL of Lysine Calibration Standard or experimental sample to the same spec using a 40-200(L Oxford BenchMate.

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

5. Clean the equipment blank spec with a KimWipe, place it in the spectrophotometer, and re-zero.

6. Remove the equipment blank spec, clean the enzyme test spec with a KimWipe, place it in the spectrophotometer, and record the absorbance (wait until constant).

7. Remove the enzyme test spec and add .1 mL of Reagent E using a 40-200(L

Oxford BenchMate.

8. Immediately, mix thoroughly by using the Fisher Vortex Genie 2.

Clean the enzyme test spec with a KimWipe, place it in the spectrophotometer, and record the decrease in absorbance every 20 seconds for 5 minutes. Consistency in time between mixing and beginning of recording absorbance is crucial.

APPENDIX A

A.1 Correlation of speed with flow rate of distilled water:

Table A.1.1 Pump Speed vs. Flow Rate

|Speed |Flow Rate (lpm) |

|1 |0.9 |

|2 |2.3 |

|3 |3.3 |

|4 |4.7 |

|5 |6.0 |

|6 |7.4 |

|7 |8.7 |

|8 |9.9 |

|9 |10.7 |

|10 |11.1 |

A.2 Calculation of tangential flow rate:

Membrane Specifications:

Width of the channel = 6 inches

Height of the channel = 0.0021 inches

Cross sectional area perpendicular to the flow direction = 6 * 0.0021 in2

= 0.0126 in2

= 0.0126 x (0.0245m)2

= 8.1x10-6 m2

Fluid velocity in the channel is in the range between 3m/s and 10m/s

Therefore, tangential flow rate = Cross sectional area * Fluid velocity

= 8.1x10-6 m2 x 3 m/s = 2.43x10-5 m3/s

= 2.43x10-2 l/s

= 1.46 lpm

Lower range = 1.5 lpm

Higher range = 4.9 lpm

Recommended Retentate Crossflow for a 5 sq.ft cassette is 3 lpm +/- 50%

Safety Guidelines

CM4120 chemical plant operations Laboratory

Bioprocess Experiments: Fermentation

Biological Safety Guidelines

The microorganism being used in this course is a pure culture composed of the bacterium Corynebacterium glutamicum. Although this strain of bacteria is not pathogenic (harmful to human health), in this laboratory we will treat them as if they may be and therefore, aseptic techniques will be used in handling the microorganism.

The Centers for Disease Control/National Institutes of Health (CDC/NIH) have developed guidelines for proper handling and disposal practices of potentially disease causing microorganisms in the laboratory. They were developed to reduce exposure of laboratory personnel to hazardous agents and prevent their release to the atmosphere. The CDC/NIH guidelines describe four levels of biosafety practice, or levels of containment, which govern standard laboratory practice and facility features and operation. Primary containment concerns control of exposure to personnel within the laboratory and is accomplished through the use of proper laboratory techniques and safety equipment. Secondary containment has the objective of controlling the release of biohazardous agents to the environment and involves the design of the facility itself and it's operation.

The biosafety levels are designated in ascending order by the degree of protection provided. Biosafety level 1 is appropriate for use with microorganisms not known to cause harm to adult human health and therefore has the lowest level of protection. Biosafety level 2 is suitable for agents with moderate impacts to human health and the environment. Biosafety level 3 is used for infections agents which cause serious disease or are potentially lethal through exposure by inhalation or ingestion. Biosafety level 4 is required for work with agents which have a high individual risk of life-threatening disease. In this laboratory, standard laboratory practice consistent with biosafety level 1 will be used with additional precautions to limit environmental exposure.

Laboratory Practice and Techniques

There are standard laboratory practice which apply to all biosafety levels.

• limit access to the laboratory

• decontamination of work surfaces daily or after a spill of viable material

• prohibit mouth pipetting

• prohibit eating, drinking, smoking, and applying cosmetics in the laboratory.

• ban the storage of food in cabinets and refrigerators used to store laboratory agents

• wash hands after handling viable materials before leaving the laboratory

• minimize the creation of aerosols

• if possible, all handling of viable microorganisms will occur in the laminar flow hood.

In addition to these standard practices of laboratory operation, there are several important steps which will be taken to limit exposure to the outside environment. These precautions affect the proper disposal of viable materials.

• all disposable pipettes which contact viable microbes will be disposed in biohazard bags.

• all solutions containing viable microorganisms will be steam autoclaved or chlorinated.

• all biohazard bags will be steam autoclaved before disposal in the trash.

Safety Equipment

Except for the steam autoclave, there is no additional safety equipment required for this laboratory other than strict adherence to proper aseptic techniques as described above.

Specific Interim Report Requirements

There is no page limitation to the interim report, but the more concise the better given that you will be submitting several interim reports this semester. It should have the following sections; title page (with report title, course number, dates of experiment, group members, and faculty/TA supervisors), short introduction (summary of objectives), overview of experimental methods used, results (use tables and/or figures but don’t present the same data in both formats), discussion of results (address your objectives), conclusions, references (if any is necessary), appendices (calibration curves, derivations, etc.).

The grading is based primarily on completeness and technical quality. However, having stated that, I do look for clear presentation of the material, brevity and conciseness, and ease in reading. Ease in reading means that there are no misspelled words, no grammatical errors, no errors in punctuation, no run-on sentences, no shifting verb tense within paragraphs, no shifting from active to passive or the other way around within paragraphs, and neatness in presentation of graphs and tables. I prefer past tense because you are describing what your group did during the experiment and subsequently in calculations. I also prefer a passive voice in the report, though this is not a requirement and I will not deduct points if an active voice is used correctly. However, using an active voice in certain sections of the report may be a mistake. For instance, in the background sections and the introduction of the report, an active voice doesn't seem to be the best.

I hope these comments will help you in preparing your interim report. If you have additional questions, please contact me either in person or through e-mail.

David R. Shonnard, Professor, Department of Chemical Engineering, MTU

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