BIO301 Environmental & Industrial Microbiology

BIO301 Environmental & Industrial Microbiology

Chemostat Laboratory Report

Production of Acetic Acid from Ethanol by Commercially-Obtained Acetobacter Using the Trickle-Bed Technique

Raymond Chan, Tone Kenji, Yon Hui Yi, Lau Weng Ho,Dut

Abstract

This report describes our experiment to produce acetic acid from ethanol with the Acetobacter species of bacteria obtained from commercial source, using the trickle-bed technique. A combination of oak pine needles, woodchips and sponge was used for chemostat and we calculated a productivity rate of 0.38g/L/h and a highest concentration of approximately 5.9% m/v of acetic acid in chemostat mode. Our report will also involve the chemistry behind vinegar production as well as a study of the bacterial kinetics of Acetobacter in this experiment.

Introduction

Acetic acid, known as vinegar, can be used as an effective cleaning agent in stain and grease removal, unclog drains and even utilized as herbicide for weed control. It is also commonly used to enhance to flavor of the food we eat, not to mention using it as a preservative to store our food as well. It is used in the production of wood glue, photographic film, as well as many synthetic fibres and fabrics.Uses of vinegar are extensive throughout our society; which also implies the need to produce bulk amounts of acetic acid commercially for industrial and domestic purposes.

Acetic acid is one of the simplest form of carboxylic acid. Formation is by oxidation of ethanol to acetaldehyde, and then further oxidationinto acetic acid. Acetobacter, a genus of the acetic-acid bacteria is involved in this mechanism to intentionally convert ethanol into the desired product, acetic acid.This oxidation takes place according to the following basic equation:

C2H5OH + O2 --> CH3COOH + H2O

Acetobacterare strict aerobes; it is therefore important to realize that all vinegar making processes needs sufficient oxygen for metabolism of the bacteria. Optimum growth temperature in a discontinuous culture was found to be 30.9oC. (I. de Ory et al. 1998), and survives well at pH 5.4-6.3 (Krieg, et al.1989).

During the early days, chemists were developing methods for oxidizing alcohol for acetic acid production via an open-air process. Amuch less complicated method; they opened the barrel of cider and let bacteria do the rest.

Owing to advances in technology, the process of producing vinegar has become much more sophisticated. Modern day methods employ reactors to do the work. There is the submerged fermentation method,CSTR (continuous flow stirred tank reactor) and the trickle-bed method. Close attention is paid to amount of aeration, feed, temperature and pH levels to elevate efficiency and to be more cost-effective.

Submerged fermentationis a continuous process, where the rate of feed into the system equals to the rate of feed out. As mentioned, ample aeration is a critical factor to yield positive results, the product can then be recovered by filtering to remove bacterial sludge, high efficiency can be achieved in this system.

CSTR (continuous flow stirred tank reactor) are stirred tank reactors with the basis to supplyoptimal oxygen airflow and limit the substrate within the reactor. The idea is to make sure there is no growth inhibition of the bacteria as well as continuous conversion of substrate into product, a very feasible way to obtain high amounts of acetic acid.

The trickle-bed reactor method involves immobilizing the bacteria on suitable packing material in a column and tricklingmedia from the top of the column, whilst air is being supplied from the bottom. The flow through is collected and recycled through the system and run in a continuous fashion.It has been shown that high productivity has been achieved with this particular method.

In the course of this experiment, our group will be going further in-depth into analyzing the trickle-bed method for producing acetic acid with ethanol as substrate. Observations, results and comments will be documented.

Materials & Methods

Diagram 1: Schematic Representation of the Batch reactor

Reactor Design

As shown in the above diagram, we had a batch reactor set up initially. It was designed in such a way that it will be easy to change it from batch to chemostat state so as to prevent unnecessary construction. A diagrammatic representation of the chemostat reactor is shown in diagram 2. It is the batch reactor with feed and harvest lines and the addition/removal is controlled by a dual peristaltic pump.

The batch reactor was designed to run continuously by utilizing pump force and gravity. Medium from the reservoir is pumped into the column, where the medium will pass through the wood and sponge packing materials that is in the column and return to the reservoir via the outlet (Gravitational).

Oak pine needles, wood chips and sponge bits were used as the packing materials. The adherence of the bacteria to these materials allowed the bacteria to grow.

The reservoir was placed in a waterbath with constant temperature of 32°C to provide optimum temperature for the growth of the bacteria. Besides that, a filter funnel was modified and used as a trickler to allow even flow of the medium into the column.

A Chemaster dual-line peristaltic pump was used to provide equal flow rates of feed into the reservoir and removal of the harvest.

Diagram 2: Schematic Representation of the Chemostat reactor

Bacteria

Bacteria that were used in this experiment were a mix of Acetobacterculture. The Acetobacterculture was obtained from Anchor Foods Pty Ltd. The culture was aerated all the way from Anchor Foods Pty Ltd to MurdochUniversity with a portable air pump and the culture was kept in a waterbath with constant air supply upon arrival in the laboratory.

Medium

Medium was also obtained from Anchor Foods Pty Ltd. The medium was kept in the cold room at 4°C and allowed to reach room temperature before use. Because the actual recipe for the medium was not provided by Anchor Foods Pty Ltd, we assumed that the contents of the medium are similar as in previous reports.

Ingredient / Amount
Dextrose / 24 kg
Diammonium Phosphate / 12 kg
Malt Beer / 400 L
Raw Vinegar / 3000 L
Ethanol / 2800 L
Water / To 24000 L

Sampling Techniques

GC

1 mL of sample was obtained daily from the reservoir during the batch culture and they were later obtained from the harvest vessel during the chemostat stage. All the samples were stored in 1.5mL eppendorf tubes and stored inside a freezer for subsequent GC analysis.

OUR

By not allowing the medium that was flowing into the column from flowing out, medium was allowed to fill the whole column. After that, the oxygen probe was placed into the column, the column covered up and DO reading was taken over time.

pH

pH of the reservoir was taken during the batch culture and were later obtained from the harvest vessel daily by using an electronic pH probe.

To further confirm the concentration of the reservoir and harvest, 5 mL of samples were taken from reservoir and harvest and were titrated against 0.1M NaOH. The indicator - 0.04% Phenolpthalein was used. \

OD

OD for reservoir was taken periodically after new bacteria were added. The medium was used as blank for calibration with the frequency of the spectrometer being 540nm.

RESULTS

OD measurement studies to show binding efficiency of bacteria to different packing materials

Figure.1. Comparison of bacterial uptake by two different materials; plastic and wood chips + oak pine needles obtained from MurdochUniversity campus.

We stopped recording readings after this as the bacteria were not attaching themselves to the column.The OD should drop at a more significant rate if the bacteria were attaching themselves.The reactor dried up on the following Monday, and were not able to obtain another OD reading for comparison.

The higher initial OD reading can be attributed to the cell density of the inoculated bacterial culture.The gradient of the lines clearly shows a better rate of adsorption (about 2 times) of the bacteria towood as compared to plastic. Rate of medium flowing through the column was kept constant.

Oxygen uptake rate by bacteria

Figure 2. Bacterial oxygen uptake in medium with plastic material, at 30oC

Figure 3. Bacterial oxygen uptake in medium with wood material, at 30oC

The above two graphs indicated a higher gradient of oxygen uptake rate by the bacteria for wood material.

Microbial activity of the bacteria cells was more evident and the likelihood of better attachment to the wood packing material was clearly demonstrated. It was obvious that the Acetobacterbacteria preferred the wood material to the plastic materials for the maintenance of viability and facilitation of its own metabolism.

Titration assay for acetic acid

Figure 4. shows acetate concentration using ethanol concentrations of 3-5% for batch and 10% for chemostat

Titration results of the culture in batch conditions showed that acetic acid concentrations were generally decreasing over time. When our group started the chemostat, the bacteria gradually produced more acetic acid reaching a high of about 5.90% m/v. When the batch was converted to a chemostat, there was a lag phase in the production while eventually led up to an exponential production phase.

Highest productivity was during the exponential phase, we recorded approximately 27.1g/L of acetic acid formed during a span of 72 hours. It gave us a productivity rate of about 0.38g/L/h.

Analysis of Acetate and Ethanol concentrations by GC

Figure 5 & 6. shows relationship of ethanol and acetic acid concentration in batch and chemostat via GC analysis

Batch Culture:

The spike of the ethanol and decrease of the acetate peaks was due to the addition of 200ml medium and water respectively. The peaks show the expected decline of ethanol and simultaneous increase of acetate levels. A prolonged batch is seen to be inefficient in maintaining high levels of acetate production.

Chemostat:

Data shows evidence of conversion of ethanol to acetic acid, perhaps more unusual is the subsequent increase in ethanol and acetate levels during the latter phase of the chemostat system. Additional results from Day 8 onwards yielded exponential increase of acetate amounts from titration assay performed. We were unable to obtain GC results due to time constraints

pH and temperature

Figure 7. Data of pH readings over the course of running chemostat reactor

Figure 8. temperature readings for chemostat process

Maximum pH was experienced at 3.2 for early running of the chemostat reactor. pH decreaseobserved during Day 1-5 initial starting phase of chemostat, but no significant amount of acid produced.Uniform decreasing pattern was noted from Day 7 which coincided with higher amounts of acetic produced. Lowest pH of 2.48 was recorded before end of the chemostat experiment. A final concentration of about 5.9% m/v of acetic acid was established prior to the end of the chemostat process

Temperature fluctuated between 29oC and 30oC. Constant temperature conditions were not met in this experiment.

(Readings not taken during weekends, and therefore excluded in Figures 4,5,6,7,8.)

Discussion

Selection of packing materials for reactor column

OD measurements gave us an indication how effective the adhesion of bacteria was to different materials, and also from the oxygen uptake studies we carried out; we concluded that the wood packing material was more effective in aiding us to produce vinegar using ethanol as substrate. Research also proved that bacteria generally bind better to organic materials that are hydrophilic in nature.

There are numerous variables which affect the binding ability of bacteria to the support. Among these are electrostatic forces, hydrophobicity and the presence of capsules on the bacteria. Research has proved that Acetobacter aceti generally bind better to supports that are hydrophobic or been pre-treated with ferric ions (Hermesse et al). The treatment works by reducing the negative charge of the support. The presence of capsules plays a large role in aiding the bacteria by producing polysaccharides which acts as a ‘glue’ between the cells and the support.

Biofilm formation

Tests should have been done to determine presence of Acetobacter in medium flow-through during post-attachment period. Although there is substantial evidence of bacteria being immobilized in the column, concerted effort could have been taken to maximize binding efficiency to the column. Wash-out of bacteria may have occurred as the medium flow rate through the column was rather high.

We switched the flow of medium pumping through the column from 82.8L/h to 28.8L/h and maintained the same rate before starting the chemostat phase. Kraigsley et al. carried out experiments regarding hydrodynamics of biofilm formation; comparing low, intermediate and high flow-speeds of media running through immobilized cells; intermediate flow rate gave the best attachment of bacteria over time. Simply by lowering flow-speed of medium,our system yielded higher biofilm formation which was evident when there was dramatic production of acid in the latter stages of the chemostat; although more work had to be done to establish an optimum rate for media flow.

Chemostat

Readings were not taken over weekends, and therefore excluded in Figures 4,5,6,7,8.

During the running of the chemostat, the production of acetic acid initially decreased then increased over time. This meant we had not achieved the “real chemostat stage” in which there is a constant inflow of substrate and outflow of harvest and the concentration of the products, substrate, oxygen and biomass should stay almost constant.

This could have been attributed to the fact that we did not have a lot of time set aside for running the chemostat. Our Acetobacterculture was still not performing very well when we switched it to the Chemostat.

Acetic acid concentration

Concentration of acid was getting poorer in the batch process, very likely due to erratic temperature conditions and the lack of sufficient oxygen, and thus restricted the ability and activity of the Acetobacter.

We measured nearly 5.9% m/v of acetic acid at peak, before the eventual demise of our chemostat. Effect of ethanol concentration on acid formation could be explained. Acetic acid bacteria were able to grow and oxidize ethanol at 9% without a lag phase. When the concentration was increased to 10%,, the bacteria overcame this inhibitory effect after an initial lag phase, (Saeki et al., 1997b). Acetobacter in our column had acquired some form of tolerance to ethanol after we increased the ethanol concentration to from 5% to 10% batch to chemostat. Saeki et al., 1997b also showed that increase in ethanol concentration decrease the probability for the acetic acid to survive. Data obtained from the experiment supported this phenomenon, see Figure 4, chemostat.

GC Analysis

Assuming that 10% of ethanol was in the medium, theoretically, we should expect almost 10% of acetate production as accordingly to the following equation:

CH3CH2OH + O2 -> CH3COOH + H2O

The maximum concentration of the acetic acid in chemostat mode was approximately 5.9% m/v. The amount of acetic acid produced could have been higher if we had managed to chill the product to prevent loss of volume to the atmosphere.

Another possibility for lower acid concentration than expected was perhaps due to accumulation of the intermediate product; acetaldehyde, before further oxidation into acetic acid. Which probably meant that feed rate of ethanol into the reactor should be lowered down, supported by the fact that we had significant presence of ethanol in our harvest vessel. Conversion rate of ethanol to acetic acid was not established due to downtime of GC equipment.

pH and Temperature

Commercially, temperature of vinegar production is controlled strictly at 30oC, acid production in out experiment probably suffered because of that. Actual kinetics and bacteria behaviour would be better studied under conditions of a fixed and constant temperature of 30oC throughout the whole experiment

We wondered about the actual effect of pH on the bacteria, since its optimum pH for survival is at about 5.4-6.3 (Krieg, et al.1989). Whilst it was also obvious that lower pH implied a stronger concentration of acetic acid present. This parameter could be scrutinized more closely.

Oxygen supply

The whole trickle-bed setup was ran without the aid of a pump to supply oxygen to the bacteria. Instead we simply relied on the bacteria to perform its metabolism via natural process of using oxygen from the surroundings in the laboratory. Higher amounts of oxygen supplied to bacteria would have given us a better chance of more biofilm formation and also help the bacteria for its proliferation purposes.

Conclusion

Though there were problems aplenty encountered during the whole course of the experiment, our simple trickle-bed reactor still managed to produce roughly 5.9% compared to commercially available table vinegar which is typically 5%. More desirable results would surely follow with better governance of temperature, pH and oxygen supply. With the help of plenty literature and first-hand experience after our experiment, we learnt much about how to produce good levels of acetic acid in context to all the parameters discussed in our report

References

A. Kraigsley, P. De Ronney ., S. E. Finkel.Hydrodynamic influences on biofilm formation and growth.