PBR 1000L - INSTRUCTION MANUAL 1.92

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WARNINGS

1. NEVER clean the bioreactor with Windex,Acetone, Ammonia, or other solventbased cleaners. This will destroy theacrylic tank. If cleaning the outside of thetank is required, dilute soap and water isrecommended.

2. Only use cotton to clean the acrylic tank.Most sponges and paper towel (cellulosebased) will scratch the tank, if manualcleaning is required, use cotton rags. If youare unsure, test on a small patch first andlook for scratching, or send us an e-mail.

3. Ensure that the pallet is well supported ona flat sturdy floor. A cement pad is ideal. When full of water the system weighs 2600lbs(1180kg), and can sink into soft ground, which could break the tank as the supportbecomes uneven.

4. Do not run the main water pump with less than 20L of liquid in the tank.

5. If moving the tank without a pallet jack after adding the wooden support blocksunderneath, be careful - these blocks can pinch and damage your fingers.

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UNPACKING & INSTALLATION

Unpacking

Unscrew and remove the top panels of thebig and small crate.

Remove the screws along the bottom ofthe big crate.

Remove the bolts along the edges of thebig crate. It will come apart as 4 panels

Remove all plastic wrap and tape

Installation

1) Bolt the blue metal brackets for the CO2tank, and the water cistern (if using thechiller upgrade) into place onto the blackpallet.

2) Mount the control system on the reactor bysliding it into the sockets and attaching theback white bracket to the reactor and thecontrol system.

3) Loosen the bolts securing the pump andslide it back to create space for the shorttriclamp pipe. Ensure that a triclampgasket is clean and in place at each end ofthis pipe, and secure this pipe in place withthe clamps.

4) Lock the pump in place by tightening the 4bolts to the pump shelf. The shelf holdingthe spray pump is tilted, this is intentional.

5) Connect the outlet of the pump to the longtriclamp pipe, and connect the far end ofthis pipe to the Optical Density sensor, andfinally connect the optical density sensor tothe bottom of the tank.

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6) Connect the drain line to the drainsolenoid. Bolt down the tube to theedge of the machine using the strainrelief bracket.

7) Connect the electrical lines to thesensors and actuators. Everyconnection should be labeled. Thereare 2 rectangular holes below theelectrical box for cords. The right holeis for sensor cords, the left hole is forpower cords. Separating the power andsensor cords reduces signal noise.There are 7 cords that do not use these2 square holes: spray pump, mainpower, top LED, UV, Stir Motors(2) andair pump.

The 4 cords for the T5 fluorescent lights (7A, 11A, 7B, 11B) connect to the 4 lights at the front of the machine (2 on each side of the viewing window).

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8) Connect the CO2 from a regulator to theCO2 solenoid using john guest fittings andtubing provided.

9) Connect the inlet watersolenoid, pre-filters, and UVsterilizer: Connect the blackline from the blue prefilter(10-1um Hydronix, SDC-45-1001) to the prefilter white(0.2um Shellco, MAS0.2-10S3S-B). Ensure there arefilters in the housings thenconnect white prefilter to thepressure gauge, then thepressure gauge to the bottomof the UV.

10) Attach the three Pall micron filters and corresponding tubing.

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11) Install the chiller. Water moves from thechiller's outlet (embossed lettering on the chillerlabeled OUT) to the inlet of the reactor's heatexchanger (labeled “In”). The outlet of thereactor's heat exchanger (labeled “Out”)connects to the top of the transparent watercistern. The bottom of the water cisternconnects to the inlet of the pump, and the outletof the pump connects to the inlet of the chiller(embossed lettering labeled IN). Dipping the endof the black tubing in hot water for 10 secondsmakes it easier to push onto the barbs.

Ensure that the Temperature probe from the chiller is fully inserted into the Temperature probe housing. The housing is located above the pump to the right on the bottom dome of the PBR. There are two holes: one for the PBR’s Probe and one for the chiller’s.

Before plugging in the chiller, fill the cistern with water to the level of the blue metal plate. The cap on the cistern slides off, but fits tightly to avoid evaporation, so may need to be tapped upwards with a block of wood to loosen it. When the pump turns on the 1st time, air pockets will move to the cistern, and it requires topping up.

12) If the chiller option was declined, a 3 way ball valve will be supplied. Connect yourfacility’s cold water supply to the “AB” barb of the valve. Connect the “A” barb to theinlet of the heat exchanger and its outlet to return to the reservoir/drain. Connect the“B” barb to the reservoir/drain. When the machine’s temperature increases beyond theset point in culture settings, it will open and allow cool water to chill the reactor.

13) Install the 8 black wooden blocks underneath thereactor's pallet. Spacing is show in blue on the pictureto the right. These are used to providing support to thepallet to avoid sagging. If the floor is not level,additional shims should be used to level the pallet andwooden blocks and provide even support for the tank.

14) Install the UV bulb into the UV sterilizer by plugging inthe 4 prong bulb to the UV ballast’s receptacle. Do nottouch the bulb with bare hands. Tighten the nut on topof the housing, then the cord grip. Do not drop the bulbinto the UV sterilizer, as it will break the quartz sleeve.

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15) Electrical requirements are on the side of the control box. For North American unit, thecord is wired to North American Standards: Black is live, White is Neutral, and Green isGround. Reactors in North America require a 120V AC 30 amp plug (L5-30).

Plug in the reactor, it will automatically boot into the program. If the reactor does notstart after being plugged in, unplug it, then open the control box to ensure the breakerslocated at the bottom left of the box are flipped to on (L).

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CLEANING & STERILIZING

Cleaning and sterilizing the system between cultures is done in 2 steps: biofilm removal, followed by a bleach pressure wash.

Preparation

A. Disconnect the air inlet quick disconnect fitting(QDC), located below the tank. Leave all otherQDCs` intact and filters in place. Do not run thespray pump without disconnecting this QDC, orwater can be pushed back into the air pump.

B. Turn the handle of the Brass 3-way valve to pushair through the upper branch of air tubing.

Pre-Rinse

C. If the tank isn't empty, drain it from the Manual Controls screen bypressing stop, then drain valve. If your software is up to date thetank will automatically drain before starting a cleaning cycle.

D. On the Cleaning and Sterilization screen, Press “Rinse X2”. Confirmthe air inlet is disconnected (see Step A) and air lever turned todirect air through the upper branch of the tubing (Step B), beforeproceeding. The program will then execute 2 cycles of filling,rinsing, and draining the tank.

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E. Gently, remove the pH probe on the outlet side of the spray pump by holding down theplastic ring of the John-Guest fitting and pulling the probestraight out. Replace the probe with the red (or gray) plasticpH-Blank. This is to protect the probe from the cleaningagents. Do not insert the blank or the probe all the way downto the flange as it makes it very hard to remove. If the pH-Blank is not in place, water will spray out during thecleaning cycle and could damage personnel orequipment.

F. Place the pH probe in pH 7.01 buffer and 4.01. If thetransmitter (Eutech Instruments panel) is reading off of theexpected values, calibrate the probe following the eutechalpha 500 manual:(http://www.eutechinst.com/manuals/english/ts_alpha_controllers/ts_alpha_ph500.pdf).

Biofilm Removal

Warnings:

CIP 100 is a caustic cleaner, once diluted it is harmless and can be added directly to waterways, but DO NOT add CIP 100 laden water directly to your recirc system or holding tank. Ensure the water draining from your reactor is leaving your facility.

Use appropriate protection when handling CIP 100, it can damage eyes and skin.

G. On the Cleaning & Sterilization page,change wash time to 2 hours, and pressthe Chemical Clean button. Water willdrain if the tank is not already empty,then a prompt to: remove pH probe,replace with the red plug, disconnectlower air QDC and direct air flowupwards will appear. The program willask which water is hooked up to the PBR:salt or fresh. If Freshwater is selectedwater will automatically be added forcleaning. If Saltwater is selected theuser will need to add 30-40L offreshwater manually through thetriclamp port in the lid (pictured to theright), because CIP 100 does not work insaltwater. Hit okay on this screen toreturn to the Cleaning and Sterilization page and fill through the port . The progress of thewater addition is displayed in the text box at the bottom of the screen.

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H. A prompt to check that the dome is mostly full of water and to Add XYZml of Steris CIP 100will appear on the screen, where XYZ is calculated for the set rinse volume. Visually confirmthe water level, then add CIP 100 through the triclamp port in the lid. Ensure not to spill it.Hit Okay after adding the cleaning agent and a prompt to replace the triclamp plug in the lidand check other connections will appear. Hit okay and the pump will run for the set time.We recommend 27ml of Steris' CIP 100 per liter of water. Consult Industrial Planktonbefore using any other cleaning agents.

I. After running for the specified time, the PBR will automatically drain, refill with 30-40L's ofnew water, rinse itself, and drain again. The tank is now ready for bleach sterilization. If abiofilm remains, you can try running the biofilm removal cycle again. For heavily soiled PBRsthe cleaning time can be increased and/or up to 40ml/L of CIP 100 is safe to use. If thetemperature in the tank increases past 40OC, stop the routine. Higher temperatures thecaustic can be damaging to the PBR.

If biofilm can be seen on the acrylic, repeat biofilm removal steps before proceeding to autoclaving & bleach sterilization.

Autoclaving

A. Remove the three 0.1 micron filters (air inlet, air vent, water inlet) with associated 1/2"(12.7mm) tubing and QDCs. Leave the 3-way brass valve attached to the air inlet filter duringautoclaving. The QDCs should be left connected together.

Only tubing that attaches to barbs on the tank needs to be autoclaved, but all tubing between the UV and the bioreactor can be autoclaved at the operator's discretion. This includes the peristaltic pump tubing, and the white rod which connects to the peristaltic pump tubing. Do not autoclave the peristaltic pump faces, just the tubing.

If a the bypass barb (for adding nutrients that precipitate and clog the filter) is used, it is also completely autoclavable.

B. Pour out any residual liquids in any of the 3filters or tubing. Do not force water throughfilters using air pressure.

C. Remove the pinch valve tubing (1/4" [6mm]silicone, underneath the machine). This iseasier if you first open the pinch valve by goingto the Manual Controls screen, pressing Stop,then Harvest Valve.

D. If tubes have visible biofilm, or deposits from

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nutrient media, disassemble and scrub with fresh water. Re-assemble tubes with their respective QDCs and filters before autoclaving.

E. Before autoclaving, place tinfoil or autoclavable covers around: the QDC assemblies open ends of 1/2" (12.7mm) tube barbed fittings on the filters

F. Unscrew the 2 small barb vent fittings on all Pall filters to allow steam to enter.

G. Autoclave for a 30 minutes cycle for plastics (1210C).

H. After the cycle finishes, through the foil, disconnect the QDC assemblies wrapping the foilaround the open ends of the QDC. Disconnecting the QDCs blocks airflow through them.

I. Close the 2 small barb vent fittings on each filter by twisting each barb clockwise.

J. Before attaching tubes onto the machine, carefully wipe the inside and outside of the barbs with95% ethanol. Do not allow the ethanol to contact the tank's clear plastic (acrylic), it cancause chemical stress in the material.

K. Attach all tubes to their respective barbs on the machine. Make sure to attach the short pieceof thick walled 3/8" (9.5mm) tubing to the air inlet port (the barb in the lowestplumbing), underneath the machine. Do not connect this tube's QDC yet.

L. When connecting QDCs together, clean the connecting faces with 95% ethanol (making surenot to spray the acrylic). Once all the tubing with male QDCs is attached to the tank's barbs,attach the respective female QDCs attached to the filters, except for the air inlet QDC at thebottom.

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M. Turn the 3 way ball valve to direct air through the upper branch.

N. Remove the pH blank and carefully replace the pH probe. The tip should terminate halfwayinto the plumbing. Do not force it all the way in.

Bleach Sterilization

A. On the Cleaning & Sterilization page, adjust the volume of sodium thiosulphate based on thevolume of bleach that will be used. Based on a stock solution of 167g sodium thiosulphate in 1Lof Distilled water, use 2ml of stock solution per 1ml of 10% bleach to neutralize. Once set,select bleach sterilization. Water is automatically added to the tank then a prompt will appearto add bleach. Per liter of wash water in the tank, manually add 2ml of standard householdbleach (~5-12% sodium hypochlorite). Reduce dose accordingly for higher concentrationindustrial bleaches. For example, 60 ml of (10%) household bleach is recommended for 30L ofwater.

B. The machine will automatically rinse with Sodium thiosulphate PBR will automatically drain,refill with 30-40L's of new water, rinse, and drain again.

C. After the spray cycles are finished, connect the last QDC for air inlet below the machine, androtate the handle of the brass 3-way valve to deliver air to the bottom plumbing. You're readyto inoculate.

INOCULATION

1) Carboys used to inoculate the PBR will ideally be equipped with QDCs to make connectingto the PBR more biosecure. Using 3/8" (9.5mm) tubing, a female QDC should be attachedto a glass tube that runs to the bottom the carboy. The female QDC must have enoughtube to reach the PBR's male QDC inoculation port (upper branch of air inlet tubing) whenthe carboy is sitting on the bioreactor's carboy shelf (left side of the control system).Another piece of 3/8" (9.5mm) tubing should be connected to a male QDC connected tothe airspace above the culture.

2) Check that the inoculation settings are correct. When inoculating with a 20L carboy, werecommend starting with 200L starting volume and setting the Nutrients forInoculation to 0.23 ml of nutrients/L of water if you're using Guillards F/2. This is 2Xthe recommended concentration. Some operators prefer 10X the recommendedconcentration. The optimum concentration will vary depending on your algae and goals.For algae species that are more prone to settling, more water (i.e. 250 or 350L) may bebeneficial to develop adequate circulation from the bottom air sparging.

3) Press Prep for inoculation. The set amount of water and nutrients will be added.

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4) Break the QDC connection for the upper air branch.

5) After cleaning the faces of the QDC with 95% ethanol, attach the female QDC from thecarboy to the male QDC from the air blanket tube (the short tube coming off the reactor).Then attach the male QDC of the carboy to the female QDC coming from the ball valve.

6) Close the black needle valve labeled Air + CO2, then turn the 3-way brass valve to directair towards the top of the machine, then slowly and carefully start to open the Air + CO2

valve. This air will pressurize your carboy and push the culture into the reactor. If air isadded too quickly, the stopper will be pushed out of the carboy, breaking sterility.

7) After inoculating, rotate the brass 3-way valve back to bottom sparging.

8) Adjust the airflow using the black Air + CO2 valve to achieve optimal aeration.

9) Disconnect the two QDCs attached to the carboy.

10) For a standard dilution (1 in 5 or 1 in 10), 4 lights is generally sufficient for the first fewdays of growth. A photoperiod of 16 hours on and 8 hours off can help avoid photoinhibition on the first day. This photoperiod is generally decreased gradually over thecourse of several days. The optimal amount of light used will be species specific anddepend on how they are grown in the carboys.

DENSITY CALIBRATION

This is where the optical density (OD) sensor is calibrated for different species. Multiple calibrations can be saved in the drop down menu, accessed by pressing the box under "Species".

The first time a new species is used, cell counts should be taken over a range of densities as the culture scales up. The cell count, along with the voltage reading of the optical density sensor (displayed to the right of the table) can be input into the table on this page.

The program automatically

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generates a calibration curve based on the numbers you input, correlating the Raw OD voltage with the cell count. A graph of the data is plotted beside the table. It should be a smooth curve.

Use data points that spans the range of growth and voltage from 0-9.99V. The calibration works best if the final data point is a higher density than you will be achieving. The curve on the screen is helpful in estimating a value for this final data point.

Calibrating the OD Sensor

The OD sensor is a custom triclamp fitting attached beneath the manifold. It passes light through the algae, measuring how much light is blocked, which can be correlated to the algae density.

The reactors are factory calibrated with relative density. This setting is used to allow the reactor to function on a 0-10 scale of density, where 0 is water and 10 is extremely dense algae.

By running the PBR in this mode, the density settings on the "Culture Settings" screen can be set to 11. Since the reactor will never exceed 10 (if using the Relative Density settings from the drop down menu), the automated routines such as scale-up, harvest, etc. will never be triggered. With Relative Density, you don't need to calibrate the machine to the specific algae you are growing, however you will not have the cell density in a live readout.

There are two options for calibrating the OD sensor for different algae species. It can be calibrated in-situ (while growing algae) or done ahead of time by taking the OD sensor off and using serial dilutions starting with a dense algae culture.

In-Situ (While the reactor is growing algae)

1) Open the "Culture Density" page and select the specific algae from the dropdown menuunder Species, if not available, add it here.

2) In the data table, enter the "filtered real time sensor voltage" (displayed in a box on thispage), and enter your cell count (in millions of cell/ml). The first point will be for water,so cell count will be 0 million cells/ml.

3) After inoculation or some growth harvest an algae sample from the reactor.

4) Take a cell count (usually a manual count using a microscope & haemocytometer, butdry weight or OD from a meter can also work).

5) Return to the previous culture setting using the dropdown menu.

6) Repeat steps 1-5 over a number of days, at a variety of concentrations spread across thefull density range for the first 8 points.

7) For the 9th point, pick a number that is a higher density (ie 50% more) than will beachieved by your algae. A good guess can be made for this point by trying differentnumbers until a smooth curve is displayed on this page, as shown in the picture. Do notworry that the last point increases vertically up. The logic in the code will work, but itwill not display correct density values past the last point entered.

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Ex-situ - Serial Dilution

1) When the PBR is empty, carefully unclamps the triclamp fittings and remove the ODsensor from below the reactor.

2) Remove the chemical addition port cap from the lid, and attach it to the OD sensor usinga triclamp and gasket, creating a cup that will hold water.

3) Open the "Culture Density" page and select the specific algae from the dropdown menuunder Species, if not available, use “Species 1-4”, or contact Industrial Plankton if thoseare already used.

4) Use a graduated cylinder (or syringe, etc.) to measure 30ml of very dense algae (algaefrom smaller flasks is best, as the PBR 1000L can exceed most 20L carboy densities).

5) Take a cell count (generally a manual count using a microscope and haemocytometer,although weight or OD from a third party meter can also work).

6) With the OD sensor wired to the reactor, pour 30 ml of the sample into the OD sensorcup from step 2.

7) Ensure sample is homogenized and wait for the filtered reading to stabilize (approx. 60seconds). Most algae settles over time, so do not wait much longer than 60 seconds.

8) Input the cell density (from step 5) and the "filtered sensor voltage" into the table, startwith point #8 (second from the last)

9) Pour the sample from the OD sensor cup to a graduated cylinder, and rinse the ODsensor with algae free water. Dry afterwards to keepdilutions accurate (do not usepaper towel).

10) Double the volume of the sample (50% dilution) using normal, algae-free water (use amatching salinity to the culture media so cells don't burst or shrivel).

11) Mix this new 50% dilution, and repeat steps 6-12, filling in points #7, #6, etc. By theend your culture should be barely recognizable from water. For point #1, use plainwater.

12) For the 9th point, pick a number that is a higher density (ie 50% more) than will beachieved by your algae. A good guess can be made for this point by trying differentnumbers until a smooth curve is displayed on this page, as shown in the picture.

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ADVANCED SETTINGS

Sensors and peristaltic pumps can be calibrated here. Feel free to contact Industrial Plankton before changing any settings, or at least record the values before changing anything. Changing these settings can have drastic effects.

Peristaltic Pump Calibration If nutrient bottles are emptying at different rates, check that lines are clear, and only after verifying that they are, re-calibrate them. The peristaltic pumps can be calibrated by pressing Calibrate Pumps on this screen. They will run for 1000 seconds. The user must have at least a 3.5L vessel to draw from and 3 separate containers to dispense into. The tubing should be disconnected from the white bar for this. After it runs, measure the volume dispensed by each and put these values into their respective boxes

pH and Temperature These values are from the slope (m) and y intercept (b) of the lines that are plotted to interpret the voltage sent by the sensor to a meaningful value for the program. If the program is not agreeing with the transmitter for the pH values or with measurements taken for temperature, these values can be changed after a new line is plotted from new data.

OD sensor type and USB OD intensity This section is here to accommodate different generations of the OD sensor. The newer sensor use the USB type, while the older are Analogue. If it has a screw on electrical connector it is a USB type, otherwise it is Analogue. The USB OD Intensity is set for the calibration curves sent with machine. If the species grown are not registering on the OD sensor (always reading 0V or very close, even at high densities), change the USB OD Intensity to the next lower value than it is currently set to. The calibration curves will need to be repopulated to use the new intensity. Similarly if the algae grown immediately saturates the sensor (readings close to 10V), change the USB OD Intensity to the next higher increment.

Tank Volume Calibration The volume of the tank can be corrected for potential drift in the pressure sensor, or changes from altitude. These can potentially cause the sensor to read a few liters when it is empty, which confuses the automated cleaning cycles.

The volume is determined using a pressure sensor located in the manifold.

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If the tank volume is reading higher than the actual volume inside the tank, check that the air vent filter is not filled with water (it can be drained using the threaded barbed connections. As the filter ages and becomes clogged, the volume will also begin to creep upwards.

To recalibrate the volume, 3 fill points are needed. Empty, top of dome (just touching the flat areas), and 3" from top. Fill the tank to these 3 points, and input the "Real Time Voltage" for each point (displayed above the variables).

Ensure that the air flow through the bottom is consistent with what you intend to use while culturing algae and that all filters and seals are in place on the reactor. Air pressure changes inside the tank will affect the volume reading.

Cleaning Variables

These variables are used in the cleaning routines. The first, Cleaning/Rinsing Volume is the

volume of water added during these procedures. The factory sets these at a convervative value

so that the pump does not run dry. The second, Plumbing Drain Time, is the time that the

drain stays open past the time that the sensor thinks the tank is empty. This is to allow the

lower plumbing to drain fully. If there is a significant volume of water remaining in the

plumbing during the cleaning routines, when it should be empty, this value can be increased.

Ensure that the drain hose is not kinked or running up hill causing water to remain in the line,

before changing this value.

Write Log Files to USB

This button will transfer the log files kept by the computer to a USB stick inserted into the

front USB Bulkhead port. It will import a .txt file that can be pasted into a spreadsheet. The

file will be YYMMDD.txt or current.

Exit Program

This button will exit the Industrial Plankton Bioreactor Program to a windows login screen.

While in Windows, only 4 lights will remain on, and no automation will occur (ie. no pH, no

temperature nor volume control). The machine will stay in windows until either Operator is

logged into (password: op) or until a power cycle has occurred either from restarting windows

or unplugging the PBR. From the windows login screen you can access the Admin user

(password: dansarki)

Power Loss

If there is a power outage the program will automatically restart into the Industrial Plankton

Bioreactor Program upon reenergizing. After 5 minutes of inactivity will jump back into life

support.

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CULTURE SETTINGS

Nutrient Boosts

These settings determine when to add extra nutrients required to bring the culture to a higher density. These will avoid it becoming nutrient starved.

These start when the culture is ~80L less than full volume, which gives it the space required to flush nutrients into the system.

These boosts add extra nutrients to keep the culture growing between scale-up and harvest mode.

Once harvesting starts, it takes over as the means of introducing new nutrients, since the harvested algae is automatically replaced with nutrients and water. Any time the tank is missing more than 20L from full it will add the corresponding Harvest Nutrients for the volume missing.

A good starting point is to add 1.5X the recommended concentration of Guillard’s F/2 for every liter of tank water, which is 0.13ml/L x 1.5 = 0.2ml/L. So for a 1000L culture, the Nutrients to Add could be set to 200ml at each boost. Spread the boosts out across the densities you expect to go through. If a boost density is set too high, the culture will stagnate and never reach harvest.

At any time you can change Density Setpoints to lower than the culture's current density to trigger a boost or scale-up to happen.

Light Boosts

Similar to the nutrient boosts, lights can be set to turn on at certain densities.

Until the optimal number of lights to have on at certain densities is determined, the density setpoint for 11, 15, and 22 light should be set to a higher value than it will hit. This keeps them off and prevents accidentally photo inhibiting or killing the culture. In this case, the lights can be turned on when the operator decides it's time, by setting the Density Setpoint to a value of 0, or any value below the current culture density.

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Density Setpoints, Nutrients, & Volumes

When the culture state is Scale Up, which is the state after inoculating, but before reaching maximum volume, there are 3 variables (all labeled scale up) that effect the performance of the scale up.

Under Density Setpoints, this variable tells the PBR when to start a scale up if the culture density exceeds this setpoint.

Under Volumes, this variable sets hows how much water to add (minimum is 20L) each scale up. We recommend 100L to 250L.

Under Nutrients, this variable sets how many milliliters of stock nutrient solution to add with each liter of new water. For example, if the scale up is set to 100L, and the variable is set to 0.26ml/L, then 26ml's of nutrients will be added by each nutrient pump. The amount of nutrients added is not based on the current tank volume, only on the amount of new water being added.

The 3 Harvest variables on this page work in much the same way. Exceeding the Harvest Density Setpoint will harvest the amount of water that's set under Volumes, and replace it that volume with new water and corresponding Harvest Nutrients. Again, the amount of nutrients added is not based on the current tank volume, only on the amount of new water being added.

A good starting point is to set the harvest volume to 1/3 of the tank's volume, and set the Harvest Nutrients variable to add 3x the recommended guillard F/2 concentration. So 3x 0.13ml/L = 0.39ml/L.

pH

This is where the pH control for the culture is set. Closed Loop Control is On is where the program checks the tank ‘s pH against the pH Setpoint at intervals of pH Period and opens the CO2 valve for the CO2 On Time if the value in the tank is higher than the set point. A good starting point is to set the CO2 On Time to 0.5s, and the pH Period to 0.2 minute. We recommend using the closed loop control.

Timed Control can be toggled on by pressing the Closed Loop Control is On . The same button will display that Timed Control is On when it is. In this mode it will open for the CO2 On Time at every pH Period, regardless of pH in the tank. Use this mode only if the pH probe is missing or damaged.

Temperature

This box is the temperature control and over temperature setting for the reactor. The fans turn on when temperature exceeds the Temperature Setpoint (and cooling ball valve, if chiller option was declined). If the High Temp Alarm Temperature is exceeded, the lights turn off to cool the tank.

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If using the chiller option ,the temperature is control is by setting the value on the chiller. It is set in Fahrenheit and can heat and cool the culture. For advanced settings on the Chiller please refer to the TECO TK500/10000/2000 User Manual (https://www.tecous.com/files/TANK_TK-500_1000_2000_Manual.pdf)

MACHINE SETTINGS

This page has three functions : it is used to reset the machine state, to set the max tank volume, and to display the code version running. Reset the machine state if the program runs into an error state or if the scale up mode is desired. Investigate any error states, before resetting the program and consult Industrial Plankton if necessary. The Max Tank Volume setting is used to run less than 1000L in the reactor, but still allow it to get to Harvest mode, as that is volume dependant. For example if only 150L of algae/day is required, Max Tank Volume can be set to 450L while still harvesting 1/3rd of the total tank volume per day.

MANUAL CONTROLS

This page is used to harvest samples, add extra nutrients, etc. Life Support will always turn on after 5 minutes of inactivity, so this page is not meant as a place to run the reactor from. To access most buttons on this page, life support should be stopped. When the main program (life support) starts, any buttons on this page will be reset to whatever they should be doing, based on the settings in your "Culture Settings" page.

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GENERAL CULTURING INFORMATION

Biosecurity

Ideally, the bioreactor should be fed with water from cleanest source available. For example, a header tank being recirculated through a UV sterilizer, or a pasteurized water system. The bioreactor does have a built in prefilter, UV sterilizer, and bacterial control filters down to 0.1µm absolute, but their performance, and life, will be improved by providing a water source with low turbidity and bacterial counts, resulting in less incidences of culture contamination.

Lights

Twenty-Two (22) T5 lights and an LED top light are used to light the culture. Cultures can become photo inhibited or killed by turning too many lights on before the culture is dense enough to handle the intensity. (For a list of available spectrums for replacement bulbs visit http://sunblasterlighting.com/lamp-only.php)

For a 1 in 10 inoculation, a good starting point is 4 lights, with a photoperiod of 16 hours ON, 8 OFF. If may take several days before the culture is dense enough to add more lights.

As a rough rule, increase the light levels when you notice the bottom of the tank becoming dark from the algae culture absorbing the light.

Even at high densities, max light may be too bright for some species. However, if you inoculate with a dense culture that is in exponential growth, some clients have achieved their fastest growth using full lights from day 1. This is highly dependent on your algae species, and whether your inoculum has been "trained" to handle bright light before adding it to the reactor.

pH

The CO2 tank's regulator should be set to 10-15 PSI (69-103KPa).

With Closed Loop Control On the optimal pH for the species grown can be set in the Culture Settings page. When the pH of the culture exceeds this setpoint, CO2 will be added.

If Timed Control is turned on instead of Closed Loop Control, the CO2 will be added at timed intervals based on your settings and will not check the pH before adding more CO2. This setting should only be used if your pH probe is broken or has malfunctioned.

Nutrients

Various liquid nutrients can be used, which are dispensed by the 2 peristaltic pumps (A and B). For simplicity, we often use a 2 part premix, Guillard's F/2 from Pentair Aquatic Ecosystems. A good starting point is 0.26ml/L when inoculating and during scale up, and 0.39ml/L when harvesting. Respectively, this is equivalent to 2X and 3X the concentrations recommended by the manufacturer.

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A magnetic stir bar should be inside each of the nutrient jugs - these will be stirred automatically before being added.

Sodium Metasilicate is necessary for growing Diatoms, and should be diluted into the macronutrient part (N & P source) according to manufacturer’s specification. If Sodium Metasilicate (or any other nutrient that precipitates) is used, it must be added after the final 0.1um filter or the filter will clog almost instantly. The PBRs ship with a small barbed fitting for bypassing the final 0.1 um filter. This bypass barb is completely autoclavable and if required it should be inserted between final 0.1um media filter and the final QDC.

Circulation

The air pump is used to sparge air into the bottom plumbing, causing circulation in the tank as it rises. The amount of air is controlled using a needle valve located below and to the left of the touchscreen, below the inoculation shelf. Too much aeration can damage sensitive cells, while not enough can limit gas exchange and circulation. Too much air will also cause the bubbles to rise through the spray post. All air should rise up the plumbing towards the spay-pump and out around the bubble plate. We suggest aiming for discrete ~4” (10cm) long bubbles in the tubing above the injection point to start with. Do not use the main water pump for circulation, it is only for cleaning and will kill live algae.

SOFTWARE UPDATES

Updating the Program

1) Check if config file is compatible with new code.

2) Open machine settings page and record the code version from the corner of the screen.

3) Send the code version along with your contact info to info@industrialplankton.com oranother contact at Industrial Plankton.

4) They may require your config file. If so, follow the "Getting the config file instructions"below, and send this to Industrial Plankton.

5) Receive new code and revised config file (if required) from Industrial Plankton.

6) Log out of the Culture program by selecting: Menu > Advanced Setting > Okay > Exitprogram > Yes. This opens a windows login screen.

7) Open the on-screen keyboard (bottom left of touchscreen).

8) Select "Admin" and type the password: dansarki

9) Drag the new code (and revised config) to the desktop and unzip the program bydouble-clicking and selecting unzip from the top bar.

10) Run the Setup.exe file from the volume folder inside the newly extract program folder.

11) Follow the prompts and when installed drag the new config file, if needed, into the datafolder within the Photo Bioreactor – Shortcut on the desktop. Select “copy and replace”when prompted.

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12) Double click” Lock computer” on the desktop to restart the computer into the newprogram.

Getting the Config File

1) Log out of the Culture program by using: Menu > Advanced Setting > Okay > Exitprogram > Yes. This opens a windows login screen.

2) Open the on-screen keyboard (bottom left of touchscreen).

3) Select "Admin" and type the password: dansarki

4) Open the “photobioreactor” folder shortcut on the desktop

5) Open the “data” folder

6) Insert a USB jump drive

7) Copy the config notepad file from the data folder to the USB drive, which can now beemailed to Industrial Plankton from another computer.

Connecting to the Internet

Plug in Ethernet cord to reactor or use the following instructions to connect to WIFI.

1) Log out of the Culture program by using: Menu > Advanced Setting > Okay > Exitprogram > Yes. This opens a windows login screen.

2) Open the on-screen keyboard (bottom left of touchscreen).

3) Select "Admin" and type the password: dansarki

4) Unlock the computer

5) Double click unlock computer from desktop

6) The computer will restart and open the culture program

7) Follow step 1-6 again

8) Open the on screen keyboard (will be on desktop, taskbar, or in programs)

9) Select the WIFI icon from the bottom right system tray

10) Select desired network and enter the password.

11) Lock the computer

12) Double click lock computer from the desktop.

13) Windows will restart and open the culture program