Job Safety Assessment Form

Department of Chemical Engineering

Michigan Technological University

Equipment Name: Enzymatic Hydrolysis JSA Author: Andy Michaelson

Room Number/Building: 205/CM Faculty Supervisor: Dr. Shonnard

Revision #: 4 Revision Date: June 2014

Purpose of Experiment / Equipment: Briefly describe what this experiment is designed to achieve and the types of data collected.

The enzymatic saccharification of cellulose from pretreated lignocellulosic biomass to glucose in order to determine the maximum extent of digestibility possible. Data collected includes glucose measurement via a blood glucose meter to determine glucose concentration throughout the hydrolysis.

Personal Protective Equipment (PPE) – Check all PPE worn during the entire experiment. Do not list these in the procedure section.

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

Hazard Summary – Check all general hazards that are likely to be encountered during this experiment and list the major source of the hazard.

Hazard / Major Source of Hazard
Toxicity / Cycloheximide, Tetracycline
Fire/Flammability
Reactivity
Pressure Hazard
Electrical Shock / Pressure cooker
Mechanical Hazard / Pressure cooker
Hot Surfaces/ High Temp
> 150 F
Biohazard
Laser Radiation
Ionizing radiation
Other:
Other:

Expected Operating Conditions –

Temperature / Pressure
Normal: 50 degrees Celsius / Normal: atmospheric pressure
Minimum: N/A / Minimum: N/A
Maximum: N/A / Maximum: N/A


Special Operating Conditions - Check all that apply and consult department Safety Manual.

Unattended Operation: / Drying Oven:
Regulated Chemicals: / Class 3b or 4 Lasers:
Pressures Exceeding 35 atm (515 psia) or Equipment Specifications:
Temperatures Exceeding 1000oC or Equipment Specifications:

Available Safety Equipment – Provide the location of each item shown below. Show the location of this equipment on the attached floor plan. If not available, type “NA” in the field.

Item / Location
Fire Extinguisher: / Locate in the lab
Eyewash: / Locate in the lab
Safety Shower: / Locate in the lab if available
Telephone: / Locate in the lab
First Aid Kit: / Locate in the lab
Other: MSDS / Locate in the lab - provided with kits
Other: Fume Hood / Locate in the lab

Spill Response Supplies - Provide the location of each item shown below. Show the location of this equipment on the attached floor plan. If not available, type “NA” in the field.

Item / Location
Spill Kit: / Locate in the lab
Floor-Dri: / N/A
Spill Dikes: / N/A
Sodium Bicarbonate: / Locate in the lab
Drain Plugs: / N/A
Spill Pillows: / Included in Spill Kit - Locate in the lab
Mercury Spill Kit: / N/A
Other: / N/A
Other: / N/A

Required Attachments:

Diagram of process or equipment
Label all valves and identify all equipment for reference in procedure.
Laboratory Floor Plan
Identify the location of your experiment and all safety and spill response equipment.
Equipment Specifications
Include materials of construction, maximum temperature and pressure, standard operating values, and any other specifications important to the safe operation.
Material Safety Data Sheets (MSDS)
Include for all reactants, products and any intermediate or other chemicals which may occur.

Additional Attachments: As necessary.

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August, 2004 Rev. 1

Chemical Information Page

Fill in as much data below as available. If data are not available, leave the field blank.

List all chemicals, including reactants, products, intermediates, solvents, and any others used.

Chemical Properties and Hazards

Chemical Name / Physical State
S, L, G / NFPA Ratings* / Incompatible Chemicals
List chemicals present within the laboratory, and any others that may come in contact. / Flash Point
Temp. / Flammability Limits
H / F / S / Sp. / LFL / UFL
Accellerase / L / 2 / 1 / 0 / HMIS Ratings, not NFPA Ratings)
No incompatible chemicals / N/A / N/A / N/A
N/A / N/A / N/A
Tetracycline / S / 3 / 0 / 1 / Strong oxidizing agents / N/A / N/A / N/A
Cycloheximide / S / 4 / 1 / 0 / Strong oxidizing agents, strong bases, acid
chlorides, acid anhydrides / N/A / N/A / N/A
Wood chips / S / 0 / 1 / 0 / HMIS Ratings, not NFPA Ratings)
No incompatible chemicals / N/A / N/A / N/A
Citric Acid, 1 M / S / 3 / 1 / 0 / Metals, strong oxidizing agents, strong reducing
agents, strong bases, metal nitrates / N/A / N/A / N/A
N/A / N/A / N/A
Ethanol / L / 2 / 3 / 1 / Oxidizing agents, peroxides, acids, acid chlorides,
acid anhydrides, alkali metals, ammonia / 48F / N/A / N/A

*NFPA Ratings: H – Health, F – Flammability, S – Stability, Sp. – Special

Chemical Toxicology, Regulation and Disposal: List the same chemicals that appear above, in the same order.

Chemical Name / Toxicology / Hazardous
Waste Number# / Regulated?
See Safety Manual / Personal Protective Equipment
Specific to this Chemical
TWA / PEL / Other
Accelerase, 1500 / N/A / N/A / Chemical-resistant laboratory gloves
Accellerase BG / N/A / N/A
Tetracycline / N/A / N/A / Chemical-resistant laboratory gloves
Cycloheximide / N/A / N/A / Chemical-resistant laboratory gloves
Wood chips
Citric Acid, 1 M / N/A / N/A / Chemical-resistant laboratory gloves
Ethanol / N/A / N/A / Chemical-resistant laboratory gloves

#See Chemical Engineering Hazardous Waste Manual.

Chemical Reactions: Provide details below on any chemical reaction(s) that occur in your process. Please show the species involved, the stoichiometry and the heat of reaction, if available. Also list side reactions and any other reactions that may impact safety. You cannot type subscripts in the form field provided – use the names for the species and the stoichiometric coefficients.

Spezyme Cp
Cellulose)n + nH2O ------> nGlucose
Spezyme Cp
(Cellulose)n +mH2O ------> mGlucose Oligomer
Spezyme Cp
(Cellulose)n + 0.5nH2O ------> 0.5 n Cellobiose
Cellobiose
Cellobiose + 2H2O ------> 2 Glucose
The heat of reaction for all of these reactions is approximately zero.
All of these reactions take place in dilute conditions.


Job Safety Assessment Form

Safe Operating Procedures Page

Sequence of Steps / Potential Hazards / Procedure to Control Hazard / PPE or Equipment Required
Emergency Shutdown
Turn off main power to equipment and unplug. / Electrical Shock / Ensure that the area around the equipment and your
hands are dry and unplug correctly - do not tug on
cord.
Start-up Procedure
Hot Plate. Turn on hot plate
Note: The sample may be frozen wet for long periods.
Solutions provided including the citric acid solution, cycloheximide, and tetracycline are not to be injested. / Electrical Shock / Ensure that the area around the equipment and your
hands are dry.
Do not swallow. If this occurs, consult physician
immediately. Drink 1-2 glasses of water. Induce
vomiting if person is conscious.
Transport the flasks from the hood over to the
counter by the sink where the distilled water is
obtained. Use 8-inch plastic transport container. If
graduated cylindar is broken, clean up glass and
dispose of in the broken glass container.
Wear chemical resistant gloves and the other PPE
listed above for the entire experiment.
Wear safety glasses. If hazard occurs rinse
immediately with water for at least 15 minutes.
Work in shallow plastic container. If spill occurs,
sponge immediately wearing purple nitrile gloves
and rise down the drain. / Splash googles, latex gloves
Run Time Procedure
(1) Transfer 1 gram of the Aspen wood chips to a 50 mL Erlenmeyer flask.
(2) One control should be made . The control will contain
all of the same compounds as the flasks with wood samples, but no biomass. Follow the remainder of this procedure for the controls as well as the flasks with biomass.
(3) Add distilled water to each flask
Wood chips or control - 19 mL
(4) Add 1 mL of sodim citrate buffer 1 M pH 4.5 to all
flasks (controls and flasks with wood samples)
(5) Add 80 microliters tetracycline (10 mg/mL in 70%
EtOH) to all flasks
(6) Add 60 microliters cycloheximide (10 mg/mL in dH2O)
to all flasks
(7) Stopper flasks with a rubber stopper, cover with tin foil,
and parafilm
(8) Let flasks equilibrate to 50C (1 hour in incubator) or pressure cooker if incubator is unavailable
(9) Determine dry weight of biomass:
a. Determine the % saturated the current biomass is by reading it off of the bag the biomass was taken from
b. Dry Mass = Wet Mass*(1 - % Saturated)
i. % Saturated in decimal format
(10) Remove flasks from shaker and add enzymes in the
following loadings:
Accellerase 1500: 250 µL per 1 gram of dry biomass
Accellerase BG: 90 µL per 1 gram of dry biomass
(11) One sample is to be withdrawn from each flask every 24 hours of incubation, starting at time zero. Withdraw 10 microliters per sample using the hand-held pipetter. Cover and return each flask to the incubator.
(12) Measure glucose concentration using the glucose meter and test strips according to manufacturers instructions. Record these glucose meter readings in your laboratory notebook or on a piece of paper at different time points according to instructions in (10). In order to have readable measurements throughout 72 hours it may be necessary to dilute the withdrawn samples with 0.05 M sodium citrate buffer.
(13) Glucose Meter Calibration: The glucose meter readings must be calibrated against known concentrations of glucose dissolved in the same solution as the controls. After the last samples are taken, the control solution can be used for the calibration. Take 10 mL of the control solution and transfer into a large test tube or closed cap 20 mL vial. Add enough glucose powder to make a 5 g/L solution of glucose in the control solution (0.05 g glucose). Mix well. This is the maximum concentration of glucose that you will use for calibrating the meter. Then, use this 5 g/L solution and dilute with control solution to create 1, 2, and 4 g glucose/L solutions, each in 1 mL total volume.
/ Broken Glass
Ingestion/skin Contact
Ingesttion/skin Contact
Electrical Shock
Broken Glass
Electric Shock / Work on counter top and handle with care
Wear gloves
Wear gloves
Make sure area around incubator is dry and that your
hands are dry
Transport test tubes in plastic container with high
walls (approx 6 inches) to avoid dropping them. If
there is a break, clean up glass pieces and dispose of
in the broken glass disposal. Make sure area around incubator is dry and that hands
are dry / **For all work with
chemicals, wear latex gloves.
Shutdown Procedure
Shut off hot plate. . / Electrical Shock / Ensure that the area around the equipment and your
hands are dry and unplug correctly - do not tug on
cord.
Cleanup / Waste Disposal
Liquids are dilute and can be disposed of down the drain.
Solids (biomass) can be disposed of in the trash. / Spills / Transport all test tubes in plastic transport container.
Clean up spills immediately with a sponge and rinse
down the drain. / Latex gloves

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