Overall presentation
100 75 50 25
Overall presentation
5% Professional layout style. Figures, titles, and legends organized logically neatly and clearly Professional looking presentation. Figures, titles, and legends organized logically neatly and clearly Less care taken in presentation. Figures, titles and legends organized logically but less neat and clear Insufficient care taken in presentation. Figures, titles and legends not organized logically and are not neat or clear
Abstract 10% Clear, concise summary of aims results and conclusion Clear, but less concise summary of aims results and conclusions Summary of aims results and conclusions are less clear Abstract does not link aims, results, and conclusions in any way
Introduction
10 % Information is relevant, well researched and comprehensive.
Information is relevant, with appropriate referencing
Information is less relevant, with some references
Information provided is random with no link to the study objective
Material and Methods
10% Summarized the methods in journal style. Converted the proposed methods into a concise section that is professionally written in past tense. Methods must enable a reader to confidently repeat the experiment based on these instructions. Methods referenced where necessary Summarised the methods less concisely. Or summarized the methods with less detail than needed to repeat experiment. Methods referenced where necessary Summarised the methods less concisely and with less detail than needed to repeat experiment.
Methods referenced where necessary Simply copied the lab manual methods and no references used
Results and discussion
(figures and tables )27.5 % All results figures are included and presented clearly and correctly. Figures have logical titles and figure legends are correctly written. Axis and scales are correct. Discussion is comprehensive and relevant to the results All results figures are included and presented clearly and correctly. Figures have logical titles and figure legends have been attempted. Axis and scales are correct.
Discussion is less comprehensive and but still relevant to the results All results figures are included and presented. Figures have logical titles, but figure legends have not been attempted. Axis and scales may need improvement.
Discussion less comprehensive or less relevant and possibly erroneous. In-complete set of results presented Figures have logical titles, but figure legends have not been attempted. Axis and scales may need improvement.
Discussion not comprehensive or relevant.
Incorporating an industry relevant recommendation on a sustainability perspective
7.5 %)
Expected to be covered in 1-2 paragraphs A logical and concise recommendation to improve the sustainability of the process quoting published scientific data
Thoughtful comments/
suggestions offered with less evidence to justify comments Less thoughtful comments/
suggestions offered with no evidence Suggestions not relevant
Conclusion
5% 3 detailed logical and concise conclusions are reached from the results offered and relate the aims.
2 detailed logical and concise conclusions are reached from the evidence offered and relate to the aims
Only 1 logical conclusion is reached from the evidence offered that relate to the aims
No conclusions offered or conclusions are not understandable and /or not relevant.
References
5% Information gathered from a wide variety of sources (at least 8 primary refs).
Provide the references in numerical order throughout the paper, and at the end of the article ie: Endnote or reference managing program used correctly Information gathered from a wide variety of sources (at least 6 primary refs). Provide the references in numerical order throughout the paper, and at the end of the article ie: Endnote or reference managing program used correctly Information gathered from limited sources (at least 4 primary refs). Provide the references in numerical order throughout the paper, and at the end of the article ie: Endnote or reference managing program used Information gathered from a variety of sources (3 or less primary refs). References not in correct style. No reference management program used.
Attendance in laboratory, meticulously entered data into folder: group assessed
20 %
Practical 2: Effect of Glucose Concentration on the Kinetics of a Batch Culture (Week 3, 4 and 5)
Objective
To examine the effect of glucose on sugar consumption, growth rate, biomass accumulation and product formation in aerobic batch culture of bakers yeast (Saccharomyces cerevisiae).
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Introduction
The most popular and best-known bakers yeast Saccharomyces cerevisiae is used for alcohol production through anaerobic fermentation. The baker yeast is used for brewing beer, making bread, making wine, ethanol and distilled beverages. The yeasts appear to be more tolerant of ethanol than other strains of yeasts. The concentration of sugars can influence the physical and biochemical aspects of cell growth and can be metabolized using several different biochemical pathways. The yield of alcohol from the fermentation depends on the amount of substrate (sugars) that is being utilized during the fermentation process. In this experiment you will monitor the cell growth and ethanol production in bioreactors with relative to the sugar concentration in the growth media. Students will measure the optical density, cell count, biomass weight, glucose concentration and ethanol concentration at different timepoints.
Two BioFlo 320 bioreactors with glucose concentration of 1% and 2% (10g/L and 20g/L) will be set up during the morning session of lab 2. Technical Officer will demonstrate the inoculation of Saccharomyces cerevisiae in the bioreactor. The bioreactors will operate at 1 vvm, 800 rpm, 30C and pH 5. Foam is controlled but dissolved oxygen concentration is not controlled.
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Materials
Per Class
Saccharomyces cerevisiae (UTS - S9.1) culture in Basal Medium grown in two bioreactors.
Spectrophotometer
Disposable spectrophotometer cuvettes
Analytical balance
50 mL Falcon tubes
Serological pipettes (25 mL, 10 mL)
Centrifuge
Large teat for the serological sterile pipettes
Labels, Permanent marker
Methylene blue
Glucose testing kit
Per Group
15 mL Falcon tubes
Syringe (5 ml)
Syringe filters (0.22 m)
Waste containers
KOVA Glass counting slide
Pasteur pipettes
Maximum revival medium
Wash buffer
1 ml pipette
1.5 mL Eppendorf tubes
Sterile water
Clickers-counters
Expected timeline of the experiment
Week 3
Demonstration of bioreactors
Collection of samples and appropriate labeling
Filter 5 mL of each time point sample (Ethanol and Glucose analysis)
Start cell count, dry weight, optical density for cultures
Week 4
Demonstration of glucose analysis
Collection of T9 (24hr) samples and appropriate labelling
Preform Glucose analysis for all samples
Gas Chromatography demonstrations (Tour of Research Labs)
Finish cell count, dry weight, optical density for cultures
Week 5
Catch up week finish any remaining tests
Industry report workshop (ask any burning questions about the assignment)
Endnote demonstration
Methodology
Groupwork
Each Pod is divided into two teams of 3 students and one team will work with Bioreactor 1 and the other with Bioreactor 2. Each class will have four time points to analyze, then all the results will be collected on Canvas and shared so everyone can have data from T0 T9 for the industry report assignment.
Collecting your samples
Sample Numbers Who will analyse Approximate Time
T0, T1, T2 AM Class 9:30 AM, 10:30 AM, 11:30 AM
T3, T4, T5 PM Class 1:30 PM, 2:30 PM, 3:30 PM
T6, T7, T8 Evening Class 5:30 PM, 6:30 PM, 7:30 PM
T9 All 9:30 AM Day 2
Technical staff will collect samples at 24h next day (T9). These samples will be handed over to each group during the start of Week 4 for analysis.
580072539624000Samples (2 x 15ml) should be taken at regular intervals specified as above, it is very important that you record the EXACT time that you took the sample (to the minute). You should also record the following: Agitation speed, air flow rate, temperature, pH and dissolved oxygen concentration (% saturation) just prior to inoculation (time = - hours). All bioreactors are inoculated with a 20% (V/V) inoculum. The final volume is 3 liters (600 ml of inoculum in 2.4 liters of fresh medium.)
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Please note that accurate labelling of sample is extremely important. Use codes to identify pod number, group name, team, bioreactor, time point or any other parameters as required.
e.g. if you are in POD 19, use something like A2_BR1_T4 (To represent Group A (Pod-19), Team 2, Bioreactor 1, Time Point 4)
Between weeks you will be able to freeze your culture and have it rethawed for the next practical session. For best results, you should filter 4mL of the culture for ethanol and glucose analysis before freezing.
The over the next few weeks the will be assayed for:
Analysis Volume of Culture Used
Ethanol (GC) (n=2) 1mL x 2 (Filtered)
Optical density (n=2) 1mL x 2
Dry weight (n=2) 10mL x 2
Total cell count (Kova method) (n=2) 0.5mL x 2
Glucose (n=1) 50L (Filtered)
Methods in Detail
62960253619500Biological experiments are carried out in replicates. You will perform experiments in duplicates (n=2) except for glucose. For your team calculate the average value and standard deviation for each value. If one team has a very different value, review and discuss the calculation. This is peer learning, and a normal part of research team activity. Do not assume that the majority view must be correct. Provide the averaged values to the class with standard deviation.
Filter 4mL of sample using 0.22 m syringe filters. The volume will go down so filter into a new centrifuge tube then transfer 1mL into two GC Vial for ethanol analysis, and use 50L for glucose concentration. You should have ~1.95 mL left over as spare. Please note that after you collect the last time point just before the class finishes, you will have to filter before leaving class.
All other analysis uses the whole culture, be sure to swirl to mix before all of the methods. You will need to ration your culture as there will not be much left over.
OD600: 2 x 1mL, Dry weight 2 x 10mL, cell count 2 x 0.5mL = 23mL
Ethanol Analysis (n = 2)
1 ml of filtered sample is transferred into the provided GC vials for ethanol determination using Gas Chromatography analysis. The GC results will be provided at the following practical class. Please be sure to collect the data from the other groups. The appendix has the full method.
There will be a short demonstration of GC operation and analysis during week 4.
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Optical Density (OD600) (n = 2)
Place your disposable spectrophotometer cuvettes in the cuvette rack: 1 for each sample and 1 for the blank
Write down which sample is in which position of the cuvette rack
Add approximately 1 mL of media to the blank cuvette
Swirl your flask to mix the culture
Transfer approximately 1 mL of each sample into the respective sample cuvette
Place the blank cuvette in the spectrophotometer set the absorption wavelength to 600 nm
Blank the spectrophotometer and remove the cuvette
Place each sample cuvette in the spectrophotometer, measuring and recording the 600nm absorption for each as the OD600 for that sample
If the OD600 is greater than 1.0, then dilute the sample 5-fold and measure again (200 L sample and 800 L water in an Eppendorf tube, then mix). Remember to multiple the measured value by the dilution factor when you record the data.
Discard the samples, pipette tips, and cuvettes in the appropriate waste bins
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Dry Weight (n = 2)
Swirl your flask to mix the culture
Label and weigh a new 15mL falcon tube without its lid
Measure 10mL aliquot of the culture into the tube
Cap the tube well
Centrifuge the tubes at a speed of 8000 rpm for 10 mins.
Transfer the tubes gently and remove the supernatant using a Pasteur pipette.
Add 1mL of wash buffer to the cell pellet in the falcon tube
Shake the sample to mix well
Centrifuge for another 5 minutes 8000 rpm
Transfer the tubes gently and remove all visible supernatant using a Pasteur pipette.
Overnight, oven dry the cell pellet
Calculate the dry weight by weighing the tube with the pellet and subtracting the tubes mass
Cell count KOVA Slides (n = 2)
(based on Kova International directions https://www.kovaintl.com/kova-prod0180.html
https://www.whitelabs.com/news-update-detail?id=53
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Swirl your flask to mix the culture
Withdraw a 500 L (0.5mL) sample from the culture flask and dispense the sample into an Eppendorf tube
With a fresh micropipette tip, add 500 L of methylene blue to the Eppendorf tube
Seal the Eppendorf tube and mix by inverting 3-5 times.
Wait for one minute
Pipette 1 drop onto the notch of a Kova slide cell (capillary action should draw the sample into the counting grid
Look at the counting grid under the microscope and use the clicker(s) to count the number of yeast cells in 5 small squares (11.111 nL volume per square, aka 90 squares per 1 L). If there are too many cells to accurately count (more than 100 per small square), dilute the sample 5-fold with sterile water (200 L sample and 800 L water in an Eppendorf tube, then mix) and try again.
You can differentiate the dead cells (dark blue) and live cells (non-colored)
Calculate the average number of cells per small square, then calculate the cells per mL:cellsmL culture= CellsSquare 90squaresuL 1000uLmL 1mL fixed sample0.75mL culture dilution factorDiscard the samples, pipette tips, and tubes in the appropriate waste bins
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436129040033Yeast cells can appear as a chain from a mother cell.
Cells that are half the size of (or smaller than) the mother cell should be excluded from viable/non-viable counts.
Cells that are touching the top and left sides of the square should be counted
0Yeast cells can appear as a chain from a mother cell.
Cells that are half the size of (or smaller than) the mother cell should be excluded from viable/non-viable counts.
Cells that are touching the top and left sides of the square should be counted
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Glucose Concentration Assay
Glucose concentration is determined using enzymatic assay kit from Enzytec. 1 mL of sample is filtered using a 0.22 m filters. Please take care to use only what you need from the kit, we only have a few for the whole cohort of students.
Glucose assay
Preparation of solutions
Use the Reagent 1 and Reagent 2 and standard undiluted. Note the concentration of the std
Ensure that the reagents are at the laboratory temperature before use (20 - 25 C)
Performance of assay
555307595694500The assay is carried out in cuvettes. For each assay you should have at least one blank (distilled water) and at least one known glucose concentration (standard). Ideally all the glucose determinations for one experiment should be carried out at the same time although time constraints may prevent this. The accuracy of this assay is dependent on the accuracy of your pipetting, mixing and your time keeping. The amount of glucose in the cuvette should range between 1.0 to 50 g of D-glucose / cuvette.
D-glucose + ATP HK Glucose-6-P + ADP
1. Set up 6 cuvettes as shown and add appropriate amounts as per table below
Pipette into cuvette Blank Sample (all 4) Standard
Distilled water 50 L 50 L 50 L
Reagent 1 1000 L 1000 L 1000 L
Blank, Sample or Standard 50 L (water) 50 L (supernatant) 50 L (glucose)
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2. Mix with a pipette, incubate for 1 min. at 37 C or 3 min. at 20 - 25 C, read absorbance A1 as below.
3. The spectrophotometer is set to 340 nm (for a tungsten halogen lamp spectrophotometer) and zeroed with distilled water. After 1 minutes the absorbance should be read, this absorbance is labeled A1 and represents the absorbance at the start of the reaction. This absorbance is affected by the supernatant color and the amount of NADPH present at the start.
G-6-P + NAD+ G6P-DH Gluconate-6-P + NADH + H+
4. Start second reaction by addition of 250 l of Reagent 2 (enzymes) into every cuvette (blank, samples and standard) as below.
Pipette into cuvette Blank Sample (all 4) Standard
Reagent 2 250 l 250 l 250 l
5. Mix, wait till the end of the reaction (incubation for approx. 10 min. at 37C or approx. 15 min. at 20 - 25 C), then read absorbance at 340 nm (A2). The amount of NADPH formed as is proportional to the amount of glucose initially present.
Notes about Glucose Assay
If A1 or A2 have an absorbance reading greater than 0.8 then the concentration of the glucose is too high for this assay (>0.5 g/l) and the glucose sample should be diluted.
If A2-A1 has an absorbance reading less than 0.100 the concentration of glucose is too low for this assay (< 0.08 g/L). Where possible a lower dilution should be used. In some instances, a higher volume can be used (ask the demonstrator).
Typically, a fresh 20g/l glucose solution will have to be diluted 1:50 to get it in the 0.1 to 0.05 g/l range. Use a 50-liter volumetric. A stationary phase culture will not need dilution as the glucose concentration should be close to zero. An intermediate sample could be any glucose concentration so it is recommended that you make up at 1:50 1:25, 1:10 and neat (no dilution) sample. Discard the any with an A2 of >1 or < 0.0.5. Average the remaining final glucose concentrations.
Calculations
The amount of NADPH can be measured by measuring the absorbance at 340 nm. A1 is a measurement of NADPH present in the mixture and other absorbances at 340 nm. A2-A1 is a measure of the amount of NADPH formed after the addition of glucose-6-phosphate dehydrogenase. This is the same as the initial glucose concentration.
Sample solution
396042183218Where:
V (Total volume) = 2.600 [ml]
MW (Molecular weight) = 180.16 [g/mol]
d (Optical path) = 1.00 [cm]
v (Sample volume) = 0.100 [ml]
(Extinction coefficient NADH) [l x mmol-1 x cm-1]: 340 nm = 6.3, 334 nm = 6.18, 365 nm = 3.4
df = (sample volume + R1) / (sample volume + R1 + R2) = 0.808.
00Where:
V (Total volume) = 2.600 [ml]
MW (Molecular weight) = 180.16 [g/mol]
d (Optical path) = 1.00 [cm]
v (Sample volume) = 0.100 [ml]
(Extinction coefficient NADH) [l x mmol-1 x cm-1]: 340 nm = 6.3, 334 nm = 6.18, 365 nm = 3.4
df = (sample volume + R1) / (sample volume + R1 + R2) = 0.808.
CD-GlucosegL=V MW A d v 1000
CD-GlucosegL= 2.600 180.16 A6.3 1 0.1 1000
CD-GlucosegL= 468.416 630 AA=(A2-df A1)samplestandard-(A2-df A1)blank
After the calculation is preformed apply your own df as the one in the equation accounts only for the dilution within the cuvette, not any dilution that was necessary because the absorbance was too high. If you have several valid dilutions average the final glucose concentration. See next page for assay details for your report.
C1 V1= C2 V2C2= C1 V1 V2Remember to adjust your calculated glucose concentration to compensate for any dilution that was necessary.
Print out from the Enzytec kit
Bioreactors and Bioprocessing - Results
Bio Reactor Information (feel free to write down anything else you find useful for your report)
Bio Reactor number Agitation (rpm) Air flow rate (L/min) Temp (C) Glucose (%) pH Notes
1 2 Bioreactor 1 Sample Information Bioreactor 2 Sample Information
Sample Name Exact time taken (hh:mm) Time since T0 (hours) Sample Name Exact time taken (hh:mm) Time since T0 (hours)
T9 T9 Make sure you note any dilution factors you have used
Cell Count Optical Density
Sample Name Total viable /
square Total non-viable / square Total live cell count (Cells / mL) Sample Name Absorbance Average OD at 600 nm
Bioreactor 1 Bioreactor 1 T9 T9 Bioreactor 2
Bioreactor 2 T9 T9 Make sure you note any dilution factors you have used
Dry Weight Ethanol Concentration
Sample Name Vial weight (g) Biomass after drying (g) Dry weight (g/L) Sample Name Concentration (g/L) Average concentration (g/L)
Bioreactor 1 Bioreactor 1 T9 T9 Bioreactor 2 Bioreactor 2 T9 T9 Glucose Concentration
Sample Name A1 A2 Concentration (g/L)
Bioreactor 1 Blank T9 Standard Bioreactor 2 Blank T9 Standard
THE APPENDICESAppendix A: Media CompositionUsed for S.cerevisiae bioreactor culture in complex media.
Component
Amount (g/L)
Peptone
20
Yeast Extract
10
Glucose
20 OR 10 g/L
Dow-Corning antifoam 10% (V/V) 0.5 mL
(pH controlled with NaOH and HCl) pH 5
Gas Chromatography- Barrier Ion Discharge Chromatography method for analysis of ethanol
(Remember do not copy this directly into your report)
Gas chromatography-Barrier Ion Discharge Chromatography (GC-BID) was used to determine the ethanol concentration. The filtered supernatant from the bioreactors was transferred into GC glass vial with a septum lid and 1 l or 2 l is auto-injected onto the GC connected with a BID detector to determine the concentration of Ethanol.
The Samples were run on a GCMS-QP2020 (Shimadzu Corporation, Kyoto, Japan) equipped with an AOC-20is autosampler (Shimadzu Corporation). The column used was an CP-Wax 57 CB (25.0 m0.25 mm0.25 m). The temperature of the BID was 200C, and the injector temperature was set at 220C. The temperature gradient of the oven was 40C for 2 min, then 5C per minute to 200C. Helium was used as the carrier gas at a constant flow of 1.0 mL min1 and the injection volume was 1 L. Standard ethanol solutions at concentrations of 1, 2, 5 and 10%w/w were used to plot a calibration curve.
Reference: https://pubmed.ncbi.nlm.nih.gov/19406012/Acknowledgements
All figures were created with BioRender.com from their list of icons and templates. Biorender retains all rights, title and interest in and to the assets of the library of icons and templates
Information for the Industry Report
Attendance at practical sessions and timely entry into class excel spreadsheet accounts for 15% of the marks of the assessment.
You are tasked with writing the report in the form of a scientific paper as if it was to be published in the Journal of Industrial Microbiology and Biotechnology (https://www.jmbfs.org/instructions-for-authors/) as a short communication article. Overall presentation marks (5%) will be awarded for sticking to this style. After writing your scientific paper, copy it into the journals article template (found at the URL above), be sure to follow the rules for citing, display of figures and main sections.
Article type
Title of Manuscript
Abstract (10%)
Should be a very concise summary of methods, background, results and conclusions of the paper. Word limit of 150 words.
Introduction (10%)
Roughly 350 words, should describe the background of the experiment and what is known already within the scientific community (references). Discuss the knowledge gap you aim to fill and aims of the experiment.
Materials and Methods (10%)
You will need to rewrite the methods and materials into the style of a scientific paper (in paragraphs and materials listed within the methods not as a bullet point list). No marks will be awarded for copying this manual into your report.
Results and Discussion (30% + 10% for industry recommendation, 40% in total)
Results
This whole section should be written in past tense.
Figures and tables should have captions and should be in the style of the Journal.
Display the error in your results, either use error bars, standard deviation or %RSD where appropriate. Include the number of trials
Any data you choose to leave out as outliers must be addressed and have scientific merit for its exclusion e.g. Grubbs test to remove a single outlier
Adjust the scales of your figures and units to the most readable and easily understood. E.g. cell count (millions of cells / mL)
Discussion
Discuss any differences observed in the growth of Saccharomyces cerevisiae (UTS - S9.1). Comment on the effect of glucose. In your report you should discuss the growth curves and the calculated values.
You should calculate the following values for both bioreactors and discuss what is happening to the culture as a result (backed up by scientific literature):
Yield of biomass per amount of glucose (Ybiomass/glucose)
Yield of ethanol per amount of glucose (Yethanol/glucose)
Ratio of Yethanol/glucose to Ybiomass/glucose
Specific carbon consumption rate (qs) in the exponential phase by calculation
Specific ethanol production (qethanol) in the exponential phase by calculation
Comment on the difference between the total cell count and the intact cell count. Was there a difference? Why might there be a difference? How does a difference, affect calculated kinetic values?
Comment on what you would have expected if E. coli had been used instead of Saccharomyces cerevisiae.
Anything else that you would like to comment on, that would add value to the report
Discuss how knowledge gained in this practical and fieldtrips can impact the bioreactor and bioprocessing industries to make them more sustainable. Suggestions should be backed up with published scientific literature. (10%)
Conclusion (5%)
A summary of the paper that brings together the main findings, solutions you have brought forward and recommendations for further research.
References (5%)
All references should be APA 7th style and should be professionally inserted (not manually). This should be done with a referencing software of your choosing (i.e. EndNote, RefWords or Mendeley) If you need help with this please approach UTS Library on their website or in-person, they have lots of resources. The University provides full licenses for all of the above-mentioned software.
Gas Chromatography- Barrier Ion Discharge Chromatography method for analysis if ethanol
Gas chromatography-Barrier Ion Discharge Chromatography (GC-BID) was used to determine the ethanol concentration. The filtered supernatant from the bioreactors was transferred into GC glass vial with a septum lid and 1 l or 2 l is auto-injected onto the GC connected with an BID detector to determine the concentration of Ethanol.
The Samples were run on a GCMS-QP2020 (Shimadzu Corporation, Kyoto, Japan) equipped with an AOC-20is autosampler (Shimadzu Corporation). The column used was an CP-Wax 57 CB (25.0 m0.25 mm0.25 m). The temperature of the BID was 200C, and the injector temperature was set at 220C. The temperature gradient of the oven was 40C for 2 min, then 5C per minute to 200C. Helium was used as the carrier gas at a constant flow of 1.0 mL min1 and the injection volume was 1 L. Standard ethanol solutions at concentrations of 1, 2, 5 and 10%w/w were used to plot a calibration curve.
Reference: https://pubmed.ncbi.nlm.nih.gov/19406012/
