The effect of different dilution rates on the production of ethanol in a chemostat
Edwin Chiu, Gemma Fitzpatrick, Philipp Guthrod, Julius Kuah, Bastian Piltz, and Ewe Xjin Lim.
Industrial Bioprocessing and Bioremediation, Murdoch University
Abstract
A chemostat was run with Saccharomyces cerevisiae under anaerobic conditions using “Pure Super Turbo” yeast in order to produce ethanol. The conditions of the bioreactor, which were kept constant, was the temperature at 30oC, and a stirring speed of 4 rpm. The main constituent in the feed vessel was D-glucose at 75g/L, a much higher amount tested than in previous studies. Two different dilution rates (D) of 0.015h-1 and 0.075h-1 were also tested over 4 days and 7 days respectively. The lower dilution rate produced an ethanol concentration of 26.36g/L in 4 days, and the higher dilution rate almost doubled the ethanol concentration produced to 52.62g/L. Previous reports have shown the optimal dilution rate to be 0.0625h-1, but our increased dilution rate gave an even larger concentration of ethanol produced by the yeast. The maximum productivity at D=0.075 was 0.02175g/L/h. The maximum yield at D=0.075h-1 was 0.0065g/L/h. Both the yield and productivity were higher at this dilution rate. One of the better written abstracts this year.
Introduction
The art of using microbes to produce various fermentation products has been known for many centuries. The first evidence for this use is over 9000 years old, where Chinese used alcoholic beverages as intoxicating ingredients(Roach, 2005). The further development of this technique led to the process of beer brewing and wine production as well as bread and cheese manufacturing. All processes have in common that they use a carbohydrate source (like wheat, rye, corn, potatoes) which is converted into ethanol and carbon dioxide under anaerobic conditions. Traditionally these fermentation products are made in batch culture and are not run continuously. But the enlarged industrial interest in bio ethanol as a alternative energy source led to large scale production preferred in batch culture systems because they have until now greater advantages compared to continuous culture systems like a chemostat (good, but do you have reference for the last statement or can list the actual advantages).
The main problem is the maintenance of sterility during a chemostat run. Contamination from outside as well as from inside could destroy a whole culture. Outside contamination is the result of hardly achievable sterilisation of the continuous system, while inside contamination arises from the growth of respiratory deficient mutants (RDM). (Reference where this info is from) The time needed for a chemostat to reach a steady-state of biomass, makes it even more uneconomic for industrial large scale purposes. But after all chemostats have advantages over batch systems because they will have higher rates of productivity, constant product formation, continuous output, the ability to monitor and change several parameters that increase productivity and reduce labour force (Cord-Ruwisch, 1997).
Ethanol is a fuel superior to gasoline as it is a more clean combuster, and can be produced from renewable sources. Today’s technological expertise at producing bioethanol as fuel is not advanced enough as to make it an economically and ecologically feasible process, mainly because of the large amount of energy needed for the distillation and thereby purification of the ethanol.
The aim of industrial bioethanol production is to have a positive energy yield under conditions that can maintained continuously and that do not affect the environment or food supply in a negative way. Therefore substrates that are no longer used for food must be used.
We used a special brewing strain of Saccharomyces cerevisae called Super Turbo Yeast which was selected for increased ethanol production. We fed them with glucose which was converted anaerobically according to the following biochemical reaction (Becker and Deamer, 1991):
Glucose + 2ADP + 2Pi ® 2 Ethanol + 2ATP + 2H2O +2CO2
The electron-carbon flow and the steps of conversion can be seen in the following figure:
Figure 1 Electron/Carbon flow in ethanol formation (ãRalf Cord-Ruwisch)
The aim of our experiment was the continuous anaerobic fermentation of D-glucose into ethanol using the yeast S. cerevisiae under two different dilution rates in order to optimise productivity. (Intro is pretty good, could have use more references or give justification for the aims (explain why they were chosen)
Materials and Methods
Equipment / QuantitySchott Bottle (2L)-for feed vessel, harvest vessel and Reactor / 3
Retort Stand(+ clamps) / 1
Automatic Aquarium Heater (Type RS 25), Blutherm set at 30oC / 1
Thermometer / 1
Stainless Steel Pot (as water bath) / 1
Styrofoam Boxes (holding ice which was packed around the feed vessel and harvest vessel) / 2
Gas Trap for CO2 measurements (attached to reactor)-involved a 1L cylinder with a 250mL cylinder turned upside down inside the 1L one, filled with water / 1
10mL Syringe (one to measure product each day-attached to harvest vessel tubing, and one connected directly to reactor) / 2
Pressure outlet (attached to reactor) / 1
Tubing clamps(plastic or wire)-in order to keep system sealed + anaerobic / several
Terumo needle 25G x 3/4” (0.5 x 19mm)-part of dripper system leading into reactor. / 2
Flea / 1
Flea catcher / 1
IEC magnetic stirring plate (that reactor is placed upon) / 1
Timer (LT48W) / 1
Chemaster Pump (DEMA Australia), 240V 50HZ / 1
0.5L plastic container (to raise pump off bench to help shorten tubing) / 1
Silicone tubing (approx 3mm wide) / Approx 1m
Rubber bungs with holes for tubing / 4
Parafilm(to wrap around the mouth of the reactor vessel where the rubber bung sits with the tubing flowing in and around the needle)
3 way connector(for tubing flowing around the harvest vessel, the collection syringe, and the outflow of pump) / 1
List could be useful, but more important is to show how it fits together.
Figure 2. Chemostat design
Media / g/LD-glucose / 75
Yeast extract / 8.5
NH4Cl / 1.3
MgSO4.H20 / 0.11
CaCl2 / 0.66
H20 / Make up to 1L
The quantities of constituents in the media were chosen based on previous reports(Gibbs et al., 1998) due to their results of a good ethanol production rate. Well done and well justified. Previous groups did not add as much glucose to their media due to their reactor vessel already containing glucose in the form of apple juice for example. The amount of glucose used was based on trying to increase the EtOH production further.
Inoculum
Was prepared using Alcotec “24 hour Pure Turbo Super Yeast” (S.cerevisiae) obtained from Murdoch University. 7.031g was provided (wet or dry?) , all of which was added to the reactor. This yeast was chosen due to it being a high alcohol and temperature-tolerant dual-function yeast, complete with needed nutrients, and the ability to yield up to 14% alcohol in 24 hours.
Media
The media was always autoclaved as well as the tubing leading from the feed vessel to the pump to prevent contamination. The media was replaced daily once the dilution rate was increased to 0.075h-1 but initially at 0.015h-1 it was replaced approximately twice weekly. Ice was replaced daily in Styrofoam boxes.
Batch Culture
Initially a batch culture was set up with 7.031g of the super turbo yeast and 14.22g of glucose made up to 500mL with water. This was run for 4 days to make sure the yeast grew and then the setup was changed to a chemostat.
When any problems resulted with the chemostat, such as contamination, we always reverted to a batch culture before setting up a new chemostat. Good.
Chemostat setup
Silicon tubing was inserted as opposed to plastic tubing so it can be autoclaved, and is more sturdy. The tubing was made as short as possible so there was less chance of contamination and the pumping was more effective. Tubing was autoclaved wrapped in aluminium foil.
Reactor Vessel
The vessel was maintained at 30oC through heating the water it was sitting in with an aquarium heater. A magnetic stirrer and a magnetic flea were used to ensure that the reactor was stirred continuously and evenly. The rpm was 4 units(or the 4th notch on the control). (Could have measured the rounds per minute or the flow rate, which is more useful for the next years group)
After growth in batch mode for 4 days, the reactor vessel was connected to the feed vessel, the sample syringe, and the gas cylinder for CO2 measurements. All attachments to all vessels were made as airtight as possible with wire or plastic clamps in order to keep the system anaerobic. This was also the reasoning behind the rubber bungs and the parafilm around the mouth of the reactor vessel. (Wrapping parafilm around something does not normally help to seal but hides the seal).
Harvest vessel
A pressure outlet was fitted to the rubber bung in the harvest vessel to allow air to enter and leave the vessel, keeping air pressure constant. It also aided in preventing contaminants from the air entering the vessel.
Ethanol:
Ethanol Concentration was analysed via the All Tech All scribe 3300 Gas Chromatography (GC) machine. The samples, collected as often as possible, at approximately the same time each day(12.30pm) from the harvest vessel with a 10mL syringe were stored in the freezer in GC bottles until needed, to prevent further fermentation and ethanol evaporation. When the samples have been defrosted ready for use, it is imperative to be as quick as possible with the analysis for this reason. A syringe designed to be inserted into the GC was used to take a sample from the head space of the GC bottle, and this was inserted into the machine. The amount of EtOH produced can be quantified by comparing the peak heights obtained with peaks belonging to standards of known ethanol concentration.
Biomass:
Biomass was calculated by measuring Optical Density (OD) at 540nm using UV mini 1240: UV-Vis spectrophotometer (Shimadzu).
Calibration of Pump and Determining Medium Flow Rate
After the chemostat was assembled, the flow rate was determined by pumping water through the chemostat. The reactor was filled with 500mL of water, and the feed bottle was filled with 1 L of water. The outflow of the chemostat was sent to a measuring cylinder. This process was timed using a digital stopwatch.
After 1 minute the chemostat was switched off, and the volume of water collected in the measuring cylinder indicated the amount of liquid flowing through the chemostat in 1 minute. The process was repeated three times, and the average of the flow rate per minute was calculated. The trial was repeated to test all the pump settings to determine the flow rates.
Pump Setting / Flow Rate (mL / min) / Flow Rate (L / h)1 / 4.5 / 0.270
2 / 7 / 0.420
3 / 10.5 / 0.630
4 / 15 / 0.900
5 / 21 / 1.26
6 / 22.5 / 1.35
7 / 26 / 1.56
8 / 31 / 1.86
9 / 32 / 1.92
10 / 32 / 1.92
Connecting Timer to the pump:
The timer was connected to the pump to ensure a continuous feed to the reactor. It also assisted in slowing the flow rate down much lower than would be possible by pump settings alone. This allows the medium flow rate of a pump setting to be altered by a fraction, based on the on / off intervals that can be programmed. The fractional on / off interval can be calculated by dividing the desired (D) by the (D) of the pump setting used. For example, if a dilution rate half of the lowest pump setting is desired, the timer can be set to be on for 30 seconds and off for 30 seconds. You do provide nice simple and clear examples to explain to the next years students how it was done and why.
For D=0.015 the timer was set to an on-interval of 1.2s/min and correspondingly for 7.2s/min to reach D=0.075.
Setting up of Chemostat:
Ice was packed around the harvest vessel to prevent further fermentation(which would effect GC and biomass readings), and the feed vessel was also surrounded by ice to prevent bacterial growth. The dripper system seen flowing into the main reactor was designed to prevent back contamination into the substrate and partially controlling the flow rate.
Changes in setup
Initially the setup running into the reactor vessel was 2 syringes connected by 2 needles and rubber bungs( as was the setup in Pittman et al., 2000), but this was found to be unnecessary, and when it came to testing the second dilution rate, this was changed to 1 syringe, 1 rubber bung, and 1 needle
Results
In the results you need to refer to each table and figure by introducing them with the text, explaining what their purpose is and what they show.
Ethanol production
Table 1: Concentrations of ethanol standards used to determine the ethanol produced in the bioreactor over a period of time.
Concentration of ethanol / GC / Baseline: 20g/L / Peaks / Peaks-20
2 / 58.75 / 38.75
5 / 74.50 / 54.50
10 / 90.00 / 70.00
20 / 132.50 / 112.50
30 / 215.00 / 195.00
40 / 341.50 / 321.50