Fermentation Updates

Welcome!!

2007-01-22T03:22:00.000+08:00

Hi all~!

All of us would like to give a big welcome to anyone or anyone who is reading this blog now! This blog will serve to document the whole fermentation process of our Bioprocess Technology practical, as well as to share what we have learnt with everyone! Fermentation is a process that requires intricate planning as well as understanding of the subject at hand- and this practical handling of a real fermentation gives us a clearer idea of Bioprocess Tech! We hope you will learn something too.

Here's a small introduction to the group members and their assigned tasks:
Evans: Supreme Blog Designer & Sephadex Column Holder & Group Entertainer & The One Who Lives In The Cold Room (hahahhaha flat hair).
Stephanie: Blog Updater & Image Manipulator & Editor & The Person Who Yells At Evans/Alaric (ahaaha!) & The Irritating Commenter In The Video & Day 1 Rep.
Siew Ying: Official Photographer & Diagram Labeler Part 1 & Sephadex Column Washer & Video Narrator & Spectrophotomer Expert (sup, 467nm) & Day 1 Rep.
Li Qiu: Official Model & Public Relations Officer & Captain of Absorbance Readings & Diagram Labeler Part 2 & Notice Board & Dr. Gila (siao one) & Shake Flask Inoculator & Day 1 Rep.
Jason: Sub-Editor & The One Who Gets A Lot Of Shit From Stephanie (thanks ah Jason) & Group Counsellor & Ruler Of The Green Fluorescent Protein & Day 2 Rep.
Alaric: Graph Artist & Habitual Latecomer (it's okay we forgive you AHAHHAA O:) ) & Public Relations Officer 2 & Graph Scanner.
Yoges: Assistant Photographer & Long-suffering Class Rep (lol!) & GFP Isolater Part 1 & GFP Purification Commander & Day 3/5 Rep.
Chu Ying: GFP Isolater Part 2 & Summarizer Part 1 & Cell Lysis Expert Part 1 & Lysozyme Handler & The One Who Risk Her DNA By Operating The UV Lamp (heehee) & Day 3/5 Rep .
Queenie: GFP Isolater Part 3 & Cell Lysis Expert Part 2 & Her Majesty Queen of Ultimate Centrifugation Plus Sonification (*bows*) & Day 3/5 Rep.
Thivyaa: Expert Media Pourer (no foam!) & Assistant Assistant Photographer & Keeper of The Lab Apparatus & Day 4/5 Rep
Dhiviya: Incredible Note-Taker & Assistant Assistant Assistant (*pant) Assistant Photographer & Purification Coordinator & Day 4/5 Rep
Saajida: Very Helpful Purification Member & The One Who Kena Sabo-ed By Stephanie To Streak The LB Plates & Information Logger & Day 4/5 Rep

Love our blog? Tag! Spread the word ;)

Cheers,
The Crew

Day 1!

2007-01-21T21:03:00.000+08:00

On a dreary Monday morning, group 1A mysteriously (not) found themselves in the labs of level 6 to start off their fermentation practical series, the objective of which was to familiarize themselves with every aspect of the fermentation process.

Putting on our labcoats, Mr Ong brought us to the lab where the fermentors sat in glassy silence, and where the air carried the faint pong of fermentations past. We then had to fill in the labels in our practical manuals and the functions of the fermentor parts were explained.

"What is this part ah???"
"I don't know leh... Wait for Mr Ong to tell us the answer hahahahhaa."

This is Li Qiu. Remember her. She will be performing a lot of tricks.

Le fermentor in all its glory, with all the computer monitor/probes, etc.

The fermentor... Labelled!

CLOSE UP!

Control unit labels.

The important functions of some fermentor parts were discussed:
Motor: supplies power to rotate the impeller
Impeller: stirs the fermentation broth, circulate bubbles (wheee) and keeps cells suspended.
Sparger: provides bubbles for aeration
Baffles: prevents formation of vortex and encourages randomized mixing
Inlet Air Filter: sterilize air that enters fermentor
Exhaust Air Filter: sterilize exhaust air before it is released into environment
Condenser: cool exhaust air to retain moisture
Rotameter: measure air flow rate
Pressure Gauge: measure pressure
Temperature Probe: measure temp. of broth
Cooling Jacket: cool broth
pH Probe: measure pH
Dissolved Oxygen Probe: measure amount of oxygen that dissolved
Level Probe: measure liquid level
Foam Probe: detect presence of foam
Acid: lower pH when it is too high
Base: raise pH when it is too low
Antifoam: prevent foaming
Sampling Tube: to draw samples during fermentation
Control Panel: set parameters

The group was also shown how to draw samples from the fermentor! View the crappy video demonstration! Please be warned that you might not be able to view it in school because the school network likes to block stuff :|

We learnt that the pH electrode of the fermentor has to be calibrated, and the pH, dissolved oxygen, foam and level probe have to be installed onto the top plate, while the addition agent lines for acid, base and antifoam need to be connected. The exhaust condensers, air inlet and exhaust filers and manual sampler unit also need to be installed, and the water jacket had to be filled. All this needs to be done before autoclaving.

We learnt that the fermentor is sterilized whole in the autoclave, using foil to cover the sockets and filters to protect them from condensing moisture. All the silicone tubings are clamped except for exhaust air filter and female STT coupling of sampling unity (allow pressure build-up to escape). Later, the fermentor would be autoclaved at 121 degrees C with the growth media in it. This is called in-situ sterilization, but before it can be done, we need our media!

So, the group went next door to prepare our media for the fermentation! But first, we had to read the instructions:

Li Qiu is an avid reader.

We needed 2L of LB broth, and using a pre-mixed LB powder, we measured out 50g of it, and dissolved it on a magnetic stirrer in some distilled water.

And then pouring the dissolved product in a measuring cylinder, we topped up the contents so we had 2L in total.

Measuring and pouring gently to prevent foaming.

Lunch is served!

Partl of this broth will be used to grow our organism in a shake flask for the next day, while the other remainder was placed into the fermentor and sterilized by our helpful Technical Support Officer later on :)

Now that we have our equipment AND media ready, we still need....

*drumroll*

Our organism lor! -__-

First, we will streak the organism of interest on a LB/Amp/Arabinose agar plate. Why is it LB/Amp/Arabinose? We will explain that tomorrow!

That's all for Day 1! Stay tuned for Day 2 ;)

Day 2!

2007-01-19T14:48:00.000+08:00

Day 2. Group 1A sacrificed their precious *cough* time to come into the labs for the next part of their experiment. Today, we shall select positive colonies from our streak plate by viewing under UV light, and transfer the pGLO transformed E.coli from LB/Amp/Ara plate to shake flask (containing 100ml LB medium with ampicillin).

When we reached the lab, the TSO took out our LB/Amp/Ara plate that was cultured and incubated from yesterday. We viewed the agar under UV light to select the positive colony of pGLO transformed E. coli, which will glow under the UV light.

2 positive colonies were selected and transferred to the flask containing 100mL LB medium with ampicillin (using aseptic technique). The flask was then placed in shaking incubator at 32ºC for 24 hours. To ensure everything was carried out using sterile techniques, we used gloves, working in a laminar flow hood, and when transferring the cells to the shake flask, disposable pipette are used.

Scrape 2 colonies...

Dip inoculating loop into flask...

Shake it around!!

Cap it and label!

OKAY that's just about all we did on Day 2! Short day, so we will take this time to explain the rationale behind some procedures, as well as cover the properties of our experiment....
WHY DO WE USE LB/Amp/Arabinose agar plate!!!?

AMP: The pGLO plasmid has an antibiotic resistant gene that confers resistance of the organism to ampicillin, so bacteria that has successfully taken up the desired gene will also have ampicillin resistance, hence they are able to grow on ampicillin plate!

Arabinose: The purpose of having arabinose on the agar is to turn on the pGLO genes, because there is a molecular switch on the plasmid, and it is activated by arabinose sugar.

LB: There is LB in the plate because LB provides the nutrient necessary for bacteria growth!

Let's next get into specifics on what we're isolating from our experiment: Green fluorescent protein (GFP), that comes from the bioluminescent jellyfish Aequorea Victoria.

GFP consists of 11 antiparallel strands of amino acid monomers forming a b-barrel which contains an aromatic chromophore which are form intramolecular reactions between Serine-65 and adjacent residues. The chromophore itself, a phenol in wild-type GFP (GFP-wt), strongly absorbs ultraviolet light of wavelength 395 nm to reach an excited state from which it emits a distinct green light of wavelength 508 nm. GFP is widely used as reporter protein in biological and bioengineering applications. This is because it can be detected non-invasively, continuously, and without the addition of any additional substrate or cofactor. It also provides clear visual representation of any protein within a cell.

Let's get to how we get the GFP gene in a bacteria!:

First, the GFP gene is isolated via PCR. The cloning vectors chosen have to have the following features: accept foreign DNA, replicates both itself and foreign DNA in a host cell and must not be rejected by host cell after each replication. We can then transform bacterial cells using either the CaCl₂ treatment or by electroporation, so that the plasmid can enter. Lastly, we can do what we have covered for Day 2, which is to select for the bacteria- by antibiotic resistance! Alternatively, a PCR diagnostic test can be performed, and after that, gene insert can be sequenced completely to determine if gene sequence is correct.

You must be wondering something!

Why do we perform step-wise scale-up instead of transferring directly to the fermentor?
This is because there is difficulty in maintaining homogeneity, as well as a drastic change in surface to volume ratio, causing the cells to be unable to adapt to the sudden and large change of physical environment. There is also a change in control regime.

Phew, what a dry day! Till tomorrow!

Day 3

2007-01-18T20:40:00.000+08:00

It is Day 3! Carrying on from yesterday... Today we will carry out scale-up fermentation process to increase the yield of desired protein product, and to monitor cell-growth and product formation through manual sampling and computer data logging.

First, we took a sample from the fermentor for a blank reading (0 hours) and then set the acid and base control unit, and then inoculated the fermentor with 100ml of culture from the shake flask. The set pH of 7.5 is optimal for GFP folding. This pH varies based on the different strains of E-coli used. Set temperature of 32°C is optimal for GFP folding and fluorescence takes place at a temperature of 32°C or below. About 20% dissolved oxygen is maintained using Cascade- which controls 2 other parameters: air flow and stirrer speed. If the cascade is below 20%, the stirrer speed will increase which might be detrimental to the cells.

Samples were then taken every hour until 5.30pm to record the culture absorbance for the next day. As we had classes, we only managed to take samples for 12.30pm and 5.30pm, and the rest of the samples were taken by the TSO.

Here's a summation of the theory before we call it a day!

A fermentor is usually used to grown genetically aerobic or anaerobic engineered bacteria on industrial scale to produce desirable product. Fermentation is termed in two different circumstances: metabolism (growth in the absence of an external electron receptor) and industrial microbiology (growth of large quantities of cells).

In the beginning, microbes with desirable products are selected from natural samples or from a culture collection. Following which, the strain has to be modified to improve production yield. However, modified stain unlikely to survive well in nature as the selection process has changed the regulatory controls in the cell to create metabolic imbalances.

The aim of microbiologists is to produce microbial biomass, specific enzymes or metabolites in large quantities. Because those products have medically benefits/important, for commercial purpose or industrial use.

The most important industrial metabolites are secondary metabolites which are produced in the stationary phase of the culture. These compounds are not necessary for growth of the microbe. Their synthesis is usually tightly regulated by the cell. Therefore to obtain high yields, environmental conditions that draw out regulatory mechanisms must be avoided. In secondary metabolism two phases are noticeable: trophophase (growth phase of the culture) and idiophase (the time when the secondary metabolites are formed).

Strain might not be as efficient in large fermentor as in small-scale due to the different conditions hence scaling up is another problem. Fermentor can either be aerobic (hard to aerate a large vessel with high biomass concentrations, hence more difficult to run) or anaerobic, in which sufficient aeration and mixing (by using spargers and impellers respectively) throughout the large fermentor has to be constantly maintained. Because it may result in high biomass content (desirable for increased product, but oxygen demands are high).

By subjecting the organisms to frozen storage in liquid nitrogen or lyophilization, their carefully selected attributes can be maintained. The inoculum for the fermentor has to be built up from a working strain. In a culture volume, the inocula has to make up 5-10% of it, therefore, the inoculum for a production fermentor may be 10,000L.

Day 4

2007-01-17T21:05:00.000+08:00

Day 4! We're ALMOST done!!!

Today's task is to monitor cell growth and product formation through manual sampling and computer data logging!

Basically, for this day, 40ml of the sample was taken for further purification purposes and for the extraction of the product-- GFP! The technique used is different from the normal sample extraction steps performed, since a larger and specific volume had to be taken out. A long transparent plastic tubing was connected to a pump, and then the pump was switched to on to extract the exact amount of culture broth required.

Just look at how turbid our fermentor broth is!

Extracted!!

AND THEN!! It is time to start to clean up!!!

Firstly, all the functions and power supply of the fermentor and then all the tubings and wires were disconnected from it. Secondly, the first autoclave was conducted in order to kill off everything inside the culture used. Thirdly, the culture was thrown down the sink! Lastly, the second autoclave was conducted in order to sterilize the fermenter for the next use!

Let's now take a look at our cell-growth curve!
Calculation of the log (X/Xo) value for the cell growth curve below:

Sample	Hours	OD₆₀₀	Log (X/X₀)
Control	0.0	0.001	-
1	1.0	0.072	1.86
2	2.0	0.135	2.13
3	3.0	0.183	2.26
4	4.0	0.408	2.61
5	5.0	0.762	2.88
6	6.0	1.224	3.09
7	7.0	1.233	3.09
8	8.0	1.330	3.12
9	9.0	1.374	3.14
10	10.0	1.958	3.29

The graph above shows the accumulated amount of bacterial cells grown in the fermentor over 10 hours. It was found that the amount of the bacterial cells in the fermentor grew larger every hour when the sample was taken. There was also no decrease in the amount of bacterial cells grown at any hour, showing that the cells were having sufficient nutrients and oxygen therefore growing normally. However, there was no increase in the growth of bacterial cells at the 7^th hour; it could be that the cells are absorbing nutrients needed to grow due to depletion however the amount of bacterial cells increased the next hour (8^th hour). This indicates that the cells have not reached the stationary phase yet. Thus, for the time-period these 10 samples were taken, it can be concluded that the cells were in the exponential phase!

The image above depicts the computer generated monitoring History graph

The major parameters discussed in the history plot were the temperature values, stirrer speed values, dissolved oxygen values and the pH values.

According to the graph, the temperature was maintained just close to the optimal temperature of 32’C. Whenever the temperature fluctuates further than the optimum temperature by more than 0.5’C, the temperature probe will adjust the temperature back to optimum levels.

The pH was also maintained within the desired values therefore it indicated that the pH control by the pH probe was very good.

From this graph, the stirrer control and the pO2 control are the values that would indicate the progress of the growth of the bacterial cells. When the PO₂ value goes down, the stirrer speed goes up. This occurs in order to ensure that the oxygen can reach the cells faster. By looking at the stirrer control, it was found that the bacterial cells had gone through 2 growth curves. The purpose of the stirrer is to circulate more oxygen to the cells and mix the culture broth. The first curve showed the initial growth curve and the second curve showed the secondary metabolite curve. The GFP protein was expressed in the initial growth phase itself. Although the bacterial cell reached death phase at around 28 hrs, the GFP protein does not become inactive as when the bacterial cell dies the protein is still functional. This is unlike in mammalian cells where the protein would die if the cells reach death phase. Therefore when the culture was extracted from the fermentor on the 4^th day, it could still be active.

OH NO! There was a power failure around the 7.0hr to 7.6hr during the fermentation process therefore you can see a white area. This could have been the cause for part of the bacterial culture dying but soon after the power came back on, the cells took some time to recover.

The reason for the increased fluctuation in the first growth curve but lesser in the second growth curve is due to the insensitivity and the inaccuracy of the stirrer probe which could be due to the power failure. According to the PO₂values, it was found that there was a lag phase at the start of the fermentation before the initial growth curve. This showed that the cells in the bacterial culture were getting used to the environment of nutrients and oxygen in the culture broth therefore using a lot of oxygen in the process and growing very fast. Before the secondary metabolite curve starts, the PO₂ value should actually increase and then decrease gradually to indicate the start of another growth curve. But in this graph, it is not shown that could be due to the power failure around that time.

At the end of the secondary metabolite curve, it was found that the PO₂ value increased steeply, this showed that the cells are reaching stationary phase and then death phase meaning that the cells are not absorbing the oxygen in the fermentor anymore as they are dead.

....Coming up next is Day 5!!!

Day 5!

2007-01-16T21:28:00.000+08:00

Yay! We're coming to the end of the fermentation practical experiment, and today we are finally isolating the GFP from the broth we have retrieved from Day 5 (we have put the absorbance readings for the samples we have taken from the fermentation broth during fermentation above in Day 4 to prevent confusion). So, 10 ml of culture broth each was collected in two falcon tubes.

These were then spun down at 10, 000 rpm for 5 mins to obtain the cells.

Sitting in the centrifuge!

Regardez! When we observed the falcon tubes under UV lamp, the pellet (which = cells) are GLOWING! This means that thhe GFP protein are in the pellet after centrifugation!

Wowee!

The supernatant was then decanted into another tube. 3 methods were used to disrupt the cells to release the GFP!

FIRST. ENZYMES were used. The cells were resuspended in the 500 ul of TE buffer at pH 7.5 using a micropipetter. Two drops of lysozyme was then added to resuspend the cells and the tube was left to stand for approximately 5 minutes to allow the enzymes to break down the cell wall of the bacteria.

NEXT, Freezing and Thawing! The tube was placed in liquid nitrogen until the contents are frozen. It was then placed in warm water to thaw. This process was repeated another two times. This procedure is done because the cell will expand when freezed and contract when thawed, and this will break it down further..

Brrrr!

Aaaaah...

Lastly, we have Sonication! Ultrasonic waves will cause the cell wall to implode under vibration pressure. This process was done on ice for 4 cycles of 25 seconds with 10 seconds rest in between each cycle.

The contents of the tube was then centrifuged again for 20 mins and at 10 000 rpm. The pellet and the supernatant was then separated. The pellet was then resuspended again using 400ul of TE buffer and the tubes are viewed with the UV lamp!

Now the supernatant is glowing! This shows that the GFP has be succesfully released from the cells!

We will now carry out purification of the GFP that was isolated. 8 falcon tubes were labeled from 1 to 8 and another tube tube was labeled as Blank. The blank was filled with 2.0 ml ammonium bicarbonate. Polymer Gel resins, Sephadex G75 is used in purification. These resins have small pores, thus when extract is poured through the column, larger molecules tend to flow through the column faster while leaving behind the smaller molecules which will interact and diffuse into the small pores of resins. This method allows the separation of products by size. Best fit graphs are drawn to show how the amount of eluted GFP decreases. It also shows when large amount of GFP are eluted.

The column drained carefully until the buffer is just even with the top of the gel bed so as to prevent the gel from drying up. A dropper was used to transfer the supernatent to the top of the gel bed by gently swirling the pipette around the edge of the column.

Fractions of 2.0 ml each was taken for all the 8 tubes, after which, they were then placed into cuvettes and then analyzed using spectrophotometry (absorbance at 476nm). Results were recorded, and the column was then cleaned up by rinsing thoroughly with 50 ml ammonium bicarbonate before storing.

Absorbance Value for graph A

Fraction	OD₄₇₆
Blank	0.0
1	0.002
2	0.170
3	0.041
4	0.011
5	0.012
6	0.004
7	0.004
8	0.000

(graph A = line of best fit)

Graph A shows that fraction 2 has the highest OD reading as compared to the rest, follow by fraction 3. This shows that there is a high concentration of GFP present in fraction 2, followed by 3. When it was viewed under UV light, fraction 2 also showed the most glow intensity than fraction 3. There are points at fraction 4 as well as fraction 7 to show that the absorbance reading is slightly off the decreasing scale. Amount of GFP should decrease as more buffer pass through the column and elute GFP. Most of the amount of GFP is eluted in fraction 2, followed by 3. The absorbance reading of fraction 8 is 0.000; this determines that all GFP have been eluted from the column.

Absorbance Value for graph B

Fraction	OD₄₇₆
Blank	0.0
1	0.005
2	0.061
3	0.026
4	0.020
5	0.015
6	0.002
7	0.000
8	0.010

Graph B also shows that fraction 2 has the highest OD reading as compared to the rest. This indicates the same theory with Graph A. The only difference are the points that are slightly out of the graph at the sixth and seventh fractions. This might be due to experimental error or deviation.

Exploring Further Question number 2!

A protein with Mr of 50,000KD would elute before the elution of 27,000KD GFP. This is because; Sephadex G-75 contains small pores resins which large molecules cannot interact with. Since the protein is larger than the GFP, it will be able to pass through the column faster than GFP and thus get to elute first.

Learning Points :)

2007-01-15T23:26:00.000+08:00

We have learnt that the process of fermentation undergoes many steps before the final desired end-product is achieved. Learning about he fermenter design, the probes attached to it and how all of these work together to produce the desired product was an important part of the experiment. The steps following the understanding of how the fermenter works are relatively easy to grasp.

For the fermentation to take place, the three important preparations done were media, equipment and seed preparations. Each of these required specific steps.

The isolation and purification procedures had to be tailored to suit the product gotten (intracellular product). Different methods used in a combination would finally isolate the desired product. We must also be clear of where the product would be found. Is the product in the supernatant or in the pellet?

The purification procedure also serves as a stepping point for the analysis of the product to begin. The extract obtained has to be purified using a pre-determined method which suits the experimental conditions. Once this is done, the purification procedure can proceed flawlessly. Analysis is conducted using spectrophotometry.

In conclusion, we now have a better idea of how scale-up and fermentation works, thanks to this experiment! We also hope you had fun reading this blog :)