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Chemostat project: Acetic acid fermentation Joshi, Vivek Ranaweera, Ishan (30655523) Sain, Minhaz. Ong, Vin. INTRODUCTION Acetic acid (CH3COOH) is a weak carboxylic acid with a pungent odour that exists as a liquid in room temperature (Myers R.,2007).The word acetic acid comes from the word acetum which in Latin means “sour” and relates to the fact that acetic acid is responsible for the bitter tastes of fermented juices. Acetic acid is produced naturally and synthetically in large quantities for industrial purposes. It is formed when ubiquitous bacteria of the genera acetobacter and clostridium convert alcohols and sugars to acetic acid (Myers R., 2007). Acetobacter , especially Acetobacter aceti , are more efficient acetic acid bacteria and produce much higher concentrations of acetic acid compared to clostridium. The acetobacter is a gram negative and aerobic bacterium (Boone et al., 2005). Vinegar is a dilute aqueous solution for acetic acid (Maga et al, 1995).The literal meaning of vinegar is “sour wine”. Vinegars are produced from ciders, grapes (wines), sucrose, glucose, or malt by successive alcoholic and acetous fermentations (Maga et al, 1995). In the first step, sugars in the plant material are converted into alcohol and carbon dioxide through the action of the enzymes produced by yeasts. The next step involves the conversion of alcohol into vinegar by acetobacter. This bacteria provides the enzymes to convert the alcohol first to acetaldehyde and then to acetic acid(Maga et al, 1995) .The three distinct approaches used in vinegar fermentation are the open vat method, the trickle method and the bubble method .In all the three methods the air supplies the oxygen needed for the conversion of alcohol to vinegar. Vinegar is used as an acidifier, flavor enhancer, flavoring agent, pH control agent, pickling agent, solvent, and used very often for its anti microbial properties. Vinegars are extensively used in food as it is of low cost and also has an antimicrobial action on foods (Maga et al, 1995). . Acetic acid is more effective in limiting yeast and bacterial growth than mold growth. It works by lowering the pH below the optimum levels for growth (Maga et al, 1995). Acetobacter can tolerate acetic acid since they are often associated with it in fermented products such as pickles and vinegar. It is also an effective antifungal agent at pH 3.5 against bread molds (Boone et al., 2005). Basic Principle of Chemostats A chemostat is an experimental chamber in which the dynamics of small, usually asexually reproducing organisms are studied under controlled laboratory conditions. most of the times, chemostats are maintained in a steady state conditions (Haefner.,2005) .A steady state chemostat consists of a growth chamber into which a constant concentration of nutrients are pumped at a constant rate. Organisms are placed into the chamber and allowed to take up nutrients and grow. The growth medium and the microorganisms are removed from the chamber at a constant rate in order to maintain a constant volume(Haefner.,2005) .The purpose of this arrangement is to permit the microorganism to grow in constant abiotic conditions (Haefner.,2005). These systems are used in research labs for physiological studies, in industries as a method to produce large quantities of chemical by-products useful in research and medicine, and in sewage treatment plants (Haefner, 2005). A batch wise culture system where the culture medium and inoculums are added into a culture vessel at the beginning of the fermentation process and then incubated at a suitable temperature and gaseous environment for a period of time. This is called a batch culture (Lee., 2006) .The batch culture system goes through a lag phase, accelerating growth phase, exponential growth phase decelerating growth phase, stationary phase and sometimes the decline phase depends on the end product (Lee., 2006).The substrate concentration in the culture medium and growth parameters changes throughout the growth phases and therefore the product formation is confined to a certain period of cultivation (Dworkin et al,. 2006). The batch culture is used widely in industrial processes such as the brewery industry for its ease of operation and less stringent sterilization (Grigarova et al., 1990). Batch cultures chemostats ,offer the possibility of maintaining growth conditions and thus selecting pressure constant for prolonged periods of time(Grigarova et al.,1990).This may encompass conditions of programmed change resulting in, for example pH,temperature or aerobic or anaerobic transitions, thus making the chemostat a precise tool for selection (Grigarova et al.,1990). Nowadays, in most chemostat, a continuous culture system is used to provide a culture growing permanently in an exponential fashion at a constant sub-maximum growth rate (Dworkin et al,2006) .Continuous culture approaches to a chemostat, the continuously changing conditions characteristic to a batch culture are eliminated and relatively large populations of cells with constant physiological state can be maintained in the presence of low concentrations of a limiting nutrient (Dworkin et al,2006) . In a chemostat ,there is a continuous culture system which consists of a limiting nutrient and growth rate is determined by the rate at which the fresh medium is fed into the culture vessel and an equal volume of effluent is collected (McCall et al, 2000). Industrial production of acetic acid The acetic acid fermentation is a highly aerobic process essentially a biotransformation by acetic acid bacteria, which involves the incomplete oxidation of ethanol to acetic acid (J.Waites, 2001). The ethanol may be derived from many different sources including wine, cider, beer or fermented fruit juice, or it may be made synthetically from natural gas and petroleum derivatives (J.Waites, 2001). Industrial acetic acid bacteria which are members of Acetobacter can be obtained by bacterial species such as A. aceti, A. rancens, A. xylinum and A. europheus (J.Waites, 2001).Since the conversion of acetic acid is an oxidation process , there need to be a sufficient amount of oxygen to facilitate the oxidation. The following equations illustrate this need for oxygen. CH3CH2OH + ½ O2 CH3CHO + 1/2O2 CH3CHO + H20 CH2COOH In the first equation, ethanol oxidises to acetaldehyde with a NAD or NADP specific alcohol dehydrogenase. In the next step acetaldehyde dehydrogenase oxidises to acetic acid. Figure 1A – Oxidation of Ethanol by acetobacter In figure 1A oxidation of ethanol to acetic acid proceeds via acetaldehyde is shown in detail. This is a very membrane associated process with membrane bound alcohol dehydrogenase and aldehyde dehydrogenase uses pyrrolo-quinoline quinone (PQQ) which is a redox cofactor in bacteria to donate electrons to a ubiquinone embedded in the membrane phospholipids which produces ubiquinol which is then oxidized by a terminal oxidase, an activity associated with cytochromes a1, b and d in different Acetobacter species. Thus the oxidation of ethanol results in the net translocation of protons across the cell's plasma membrane (Adams, 1999). Effect of temperature There are many problems associated with the production of acetic acid from ethanol. One such problem is the need for strict temperature control. The temperature usually has to be 30°C for the production of vinegar in both batch culture or in a chemostat (W, Tesfaye,M. L Morales,M .C García-Parrilla and A. M Troncoso;, 2002). . A temperature increase by 2–3°C causes a serious failure in both fermentation rate and fermentation efficiency.. Another problem faced in ethanol production is the evoporation of volatile compounds including the substrate ehtanol, intermediate acetaldehyde as well as the final product of acetic acid. This results in a reduced yield.(I. Caro, L. Pérez and D. Cantero, 1992). The Aim of this experiment is to run an Acetobnactor . sp obtained from Anchor foods using a chemostat process to produce acetic acid (vinegar) from ethanol. This experiment will analytically derive acetic acid and ethanol concentration by titrations and measurement of pH. 2.0 Materials and Methods 2.1 Materials 2.1.1 Bacteria The bacteria used in this study consisted of mixed Acetobacter strains that were obtained from Anchor Foods Pty Ltd in O’Conner, Perth, Western Australia. 2.1.2 Medium The medium used was also supplied from the same branch of Anchor Foods Pty Ltd in Perth, Western Australia. Due to the unavailability of the information of the medium’s actual recipe, an assumption was made that the contents of the medium provided by Anchor Foods Pty Ltd were similar as that of the previous reports, which also gathered their resources from Anchor Foods Pty Ltd. Composition Amount Dextrose 24 kg Diammonium phosphate 12 kg Malt beer 400 L Raw vinegar 3000 L Ethanol 2800 L Water 24000 L 2.2 Reactor Design Three types of reactors were used in this project; a batch culture reactor, a chemostat reactor, and a column trickle reactor. 2.2.1 Batch Culture Reactor Diagram 2.1 Schematic representation of the batch culture reactor set-up. The batch reactor was set-up as illustrated in Diagram 2.1.The air filter vessel contained water to moisten the air molecules to reduce evaporation as it bubbles through the sparge from the air pump prior to entering the reactor vessel. The aquarium heater was set to maintain a constant temperature of 32°C in the water-filled aquarium. The 1L reactor vessel contained 0.5L of Acetobacter culture and 0.5L of media. The reactor vessel was aerated at 100L/hour with a stirring rate of 5. The aquarium was covered with aluminium foil to create a suitable dark environment for the bacteria culture to grow. The hot plate was not used as it is unreliable at maintaining constant temperatures. 2.2.2 Chemostat Reactor The chemostat reactor was set-up, as illustrated in Diagram 2.2, in week 2; 7 days after the batch culture reactor was run. The chemostat is a reactor with continuous feeding of growth medium to the Enterobacter to optimize acetic acid production. The 5L feed vessel contained the medium and is pumped into the 1L reactor vessel via a bidirectional pump with a timer. The bidirectional pump with timer was used to provide equal flow rates of feed into the reactor and removal of acetic acid from the reactor to the harvest vessel. The reactor vessel was aerated at 100L/hour with a stirring rate of 5. A constant temperature of 32°C in the aquarium was maintained. Initially, the 5L harvest vessel did not have an air vent, causing a pressure build-up in the bottle which led to tube leaching. An air vent was then added to the corresponding vessel to prevent future pressure build-ups. 2.2.3 Column Trickle Reactor Diagram 2.3 Schematic representation of the column trickle reactor set-up. The column trickle reactor was set-up, as illustrated in Diagram 2.3, and started at the same time as when the batch culture reactor was running. The reactor consists of 2 measuring cylinders of 2 different sizes. The larger measuring cylinder was used to hold the second measuring cylinder, its packing materials, and the bacteria culture and medium. The second measuring cylinder contains small plastic pieces which acted as adherence materials for the bacteria to grow on. The second measuring cylinder, with a small hole at the bottom which connects to a silicone tube, was fitted into the larger measuring cylinder. The silicone tube was connected to the inlet of a one-way pump, and the outlet was connected back to the top of the second measuring cylinder where it would trickle and pass through the column with the plastic pieces. The trickle reactor was set-up and ran parallel and concurrently with the batch culture and acted as a back-up plan should anything go wrong with the initial batch culture or chemostat reactors. This proved useful when the chemostat reactor was run for the first time without an air vent in the harvest vessel, causing a massive leaching of essential materials. A second chemostat was set-up using the bacteria culture cultivated from the trickle reactor. 2.3 Sampling Techniques 2.3.1 pH An electronic pH probe was used to measure the pH of the batch culture reactor. 2.3.2 Titration 0.5M sodium hydroxide solution was prepared (20g NaOH solid pellets in 1L dH2O) and titrated against 5mL samples taken from the batch culture reactor. Bromothymol blue was used as an indicator. 2.4 Results Table 2.4.1 pH and titre measurements of the batch culture taken at different days. Date Day pH Titrated acid (M) 24/9 1 2.64 - 25/9 2 2.74 0.0312 28/9 5 2.60 0.0925 2/10 9 2.72 0.061 Table 2.4.1 shows the pH and titration results taken during from the batch culture reactor during the first week of the project. There was a significant increase in the acid contents from day 2 to day 5 (Table 2.4.1). On day 9 of the experiment, 2 days after the first chemostat commenced, the pH measured was 2.72 and the titrated acid was calculated at 0.061M. A human error occurred while using the heaters, which resulted in the experiment being forced to be discontinued. Hence no further results were obtained from the chemostat. Discussion Due to numerous problems faced during this experiment, the aim of producing acetic acid from ethanol via a chemostat process was not obtained. However, when the batch culture was used at first before switching to chemostat, 1.91g/L was obtained on the first day while 5.56g/L of acetic acid was obtained on the last day before the process was converted from a batch culture to a chemostat process. The amount of acetic acid that was obtained from the batch culture is comparable to that obtained from Anchor Foods Cider Vinegar which was 4.08g/L. However the significance of our results is greatly reduced due to the fact that the data obtained from our experiment was not at steady state hence no accurate comparisons can be made. Acetobacter sp which is a gram negative bacteria used in this experiment has an absolute requirement for oxygen and can only survive at temperatures between 30-35°C (Boone et al.,2005). To maintain the temperature, an aquarium heater was used instead of the normal bench heaters as this was done to minimize operator error. According to Tesfaye et al, (2002), there are many problems associated with the production of acetic acid from ethanol. One such problem is the need for strict temperature control as the temperature usually has to be 30°C for the production of vinegar in both batch culture or in a chemostat. A temperature increase by 2–3°C causes a serious failure in both fermentation rate and fermentation efficiency. Another problem faced in ethanol production is the evoporation of volatile compounds including the substrate ehtanol, intermediate acetaldehyde as well as the final product of acetic acid hence resulting in a reduced yield (I. Caro et al,. 1992) and to overcome the problem of the strict temperature limitation of 30°C, scientists have found thermotolerant acetic acid bacteria which are useful for vinegar fermentation at higher temperatures at around 38–40°C (A. Saeki et al,. 1997). Similarly to overcome the evoporation of volatile substances such as ethanol, acetaldehyde and acetic acid a special fermentation system with gas recirculation has been devoloped. The application of this procedure makes possible to operate in a closed system, hence preventing losses in fermentation yields due to evaporation that occur in open systems and reduce the loss of volatile compounds (J.M. Gómez et al,. 1994). Analysis of results by previous workers. In previous experiments done by (Horiuchi, et al., 1999), the acetic acid productivity using a packed bed bioreactor is much higher despite taking 180days to allow this process to undergo completion when compared to the acetic acid production by Fregapane et al, (1998) who had done this experiment using a semi- continuous bubble column with novel dynamic sparger which was completed in 37 hours. In the experiment by (Horiuchi, et al., 1999) the productivity was 3.9/g/L/h whereas the productivity of acetic acid by (Fregapane, et al., 1998) was 1.8g/L/h. Productivity was higher using the packed bed bioreactor because of the high surface area which contained ions such as magnesium, potassium, calcium, phosphorus and iron (Horiuchi, et al., 1999) which enabled the acetobacter to adsorb on to the surface and multiply steadily. According to (Moo-Young, 1985), the design and the matrix used for a bioreactor allow more productivity of acetic acid. In our experiment we had used plastic shavings as the matrix used to immobilise the microbes where as in the experiment done by (Horiuchi, et al., 1999), waste mushroom medium was used as of their bacterial affinity and high specific area and low production cost. Problems and Insights Firstly, our group had problems adjusting the temperature of the water bath as two of our aquarium heaters were not working properly as our temperatures varied between 25-40°C. This huge variation of temperature was not ideal as it could have killed of our bacteria. The second problem we faced was that the feed vessel was empty the first day we changed our batch culture to a chemostat. This was because of two reasons, firstly was that a very high flow rate was used hence consuming all the feed. Secondly, this problem was also due to the fact that wrong instructions were given on how to use the pump timer in the context of when the pump should be on and when it should be off. From previous years’ report the pump was supposed to be ON for 12 seconds (T1) and OFF for 48 seconds (T2) however on our instruction manual, pump on was T2 and pump off was T1. The third problem that our group faced was the leakage of both the water bath and culture which occurred due to the peristatic pumps wearing the tubes out mainly from over-pressing and general wear of the tubing. During the experiment, our harvest feed had leaked and burst as this was due to pressure build up in tubes. This could have been avoidable if we had inserted a syringe as this would have released the pressure that was accumulating. The most disastrous problem that our group faced was that our aquarium tank containing water, our culture had cracked and thus leaking everywhere onto the table. This was a catastrophe as we could not recover any of our culture and hence had to stop our chemostat project as there was insufficient time to start it all over again. Should you choose this project? Patience and commitment This is a self-paced project/experiment so don’t be tempted to try to finish early and run-off from the lab. Be patient and make sure everything is working perfectly (or close to perfect) and in order before you leave, especially for the weekend when the labs aren’t open. And make sure effort is taken by every group member to come into the lab to help out, even during study breaks Help It may take awhile before everyone in the group really knows what’s going on. Help each other out to make it easier to understand, and update and talk to your supervisor (Ralf) about it. Ralf will give you valuable feedback on whether your project is heading the right direction. If assistance is required to get specific materials or equipments, or even just to figure out how to get an equipment working, ask for the help! Ralf and the lab staff would be more than willing to provide support. Some Additional things to consider, - Read up on papers that other students have written and draw up a simple diagram to show to Ralf Make sure everyone is present when the experiment is being set up so everyone in the group understands what is actually going on. Make sure you know your full inventory of pieces before starting up as this will allow getting required equipment earlier rather than freeloading around later. If you are doing a trickle bed reactor, look for different substrate to act as immobilisation matrix as compressibility is a large factor Label all tubes (example feed in, feed out, harvest in and harvest out) Try using an aquarium pump first which can later be backed up by an air inlet as in our experiment the air valve was shut off before we brought our bacteria culture. This is critical as the bacteria have absolute requirement of oxygen to survive. - Contact Max (Anchor Foods) the moment you choose this experiment as he provides this bacteria culture and he may not be available all the time. - Determine OUR and D.O to determine steady states as this will assist with the understanding the concepts of a chemostat Measure the pH as pH decreases acetic acid concentration increases Check tubing daily for problems Lastly communicate and delegate task between group members. Use the notebook to write down day to day basis what you have encountered, questions that need answers as well as your results. This is also a way in which the group knows who did what on which day. Well this is a damage control report as expected. However we agreed that you would analyse some data either from previous groupg or the literature, specifically on the use of chemostats for vinegar production. What you wrote is clear and it covers something on vinegar and on chemostat but nothing on vinegar production by chemostats, or the problems anticipated (e.g. toxicity, lack of growth at the low pH values, …) 5.5/10 References M, Adams, . R. (1999). VINEGAR . Encyclopedia of Food Microbiology , 2258-2263. D,Boone ., Castenholz R., Brenner D. and Garrity G.,2005, Bergey's manual of systematic bacteriology, Volume 2, Part 3,Springer publishing L, Caro, Pérez and D. Cantero. (1992). Modelling of ethanol evaporative losses during batch alcohol fermentation. Chemical Engineering Journal , 42, B15–B22. M, Dworkin and Falkow S., 2006, The Prokaryotes: Symbiotic associations, biotechnology, applied microbiology, Springer publishing. J.M. Gómez, L.E. Romero, I. Caro and D. Cantero. (1994). Application of a gas recirculation system to industrial acetic fermentation processes. Biothecnology Techniques , 8, 711–716. R,Grigorova .and Norris J.,1990, Techniques in microbial ecology, Academic Press publishing J,Haefner .,2005, Modeling biological systems: principles and applications, Springer publishing. Y,Lee .,2006, Microbial biotechnology: principles and applications, World Scientific Publishing. J,Maga . And Tu A.,1995,Food additive toxicology ,CRC press publishing D, McCall .,Stock D. and Achey P.,2000,Introduction to Microbiology,Wiley-Blackwell Publishing. R,Myers .,2007,The 100 most important chemical compounds : a reference guide., greenwood publishing. A. Saeki, G. Theeragool, K. Matsushita, H. Toyama, N. Lotong and O. Adachi. (1997). Development of thermotolerant acetic acid bacteria useful for vinegar fermentation at higher temperatures. Bioscience. Biotechnology and Biochemistry , 61, 138–145. J.Waites, M. (2001). Industrial microbiology:an introduction. Oxford: Blackwell Science Ltd. W, Tesfaye,M. L Morales,M .C García-Parrilla and A. M Troncoso;. (2002). Wine vinegar: technology, authenticity and quality evaluation. Trends in Food Science & Technology , 13 (1), 12-21. Sivalingam J., Driessen S. and Allan R. Acetic acid fermentation (semi-continuous) [Report]. Perth : [s.n.], 1999. Fregapane G., Fernandez H.R. and Nieto J. and Salvador,M.D. Wine Vinegar Production Using a Noncommercial 100-litre Bubble Column Reactor Equiped with a Novel Type Dynamic Sparger [Journal]. - Madrid : Department of Food Technology, 1998. - 14 : Vol. 63. Horiuchi J., Tabata K. and Kanno T. and Kobayashi, M. Continuous Acetic Acid Production by a Packed Bed Bioreactor Employing Charcoal Pellets Derived from Waste Mushroom Medium [Journal]. - Japan : Journal of Bioscience and Bioengineering, 1999. - 2 : Vol. 89. Moo-Young M. Comprehensive Biotechnology- The Principles,Applocation and Regulation of Biotechnology in Industry, Agriculture and Medicine [Journal]. - New York : Pergamon Press, 1985. - Vol. 3. Appendix Summary of entries in log book 18.09.09 Planning and task distribution among group members. A dummy batch culture was set up using water and left to run through the weekend to ensure equipments were working and not faulty. 21.09.09 Air pumps in the lab stopped working. Used electric operated aquarium air pumps for air flow. 23.09.09 Acetobacter culture and media were collected from Anchor Foods Pty Ltd. Batch culture started. 24.09.09 Trickle reactor started. Plastic pieces were used in the column. 25.09.09 0.5 NaOH solution was prepared. Titration against 5mL sample from batch culture using Bromothymol blue as indicator. 28.09.09 Titration against 5mL sample from batch culture using Bromothymol blue as indicator. 30.09.09 Chemostat reactor started. 01.10.09 Pressure build-up in harvest vessel of chemostat caused severe leaching. Assessment of damage and mode of action to recover (if possible). 02.10.09 Started second chemostat using back-up bacteria culture from trickle reactor. 05.10.09 Human error occurrence with heaters. Aquarium broke. Materials not salvageable. Assessment of damage and mode of action to recover (if possible). Concluded that experiment was unable to continue. Experiment discontinued. Set-up was dismantled.