Chemostat Report
Group 1
Rodney Ling – 32140372, Kristen Wolter – 31250128, King Zheng (Sam) Lim – 31544501 Rodney Ling – 32140372, Kristen Wolter – 31250128, King Zheng (Sam) Lim – 31544501
10/1/2013
Abstract
Biocementation process has utilized the ability of microbes to promote precipitation of CaCO3. This in turns will be used on repairing lime stone and strengthening concrete. Moreover, such ability has been employed for applications such as preventing soil avalanching and compacting soil from reclaimed land. One of such bacterium(of what such? What the word such referring to?) is S.pasteurii. The aim of this experiment was to investigate the viability of S.pasteurii growing in a non-sterile Chemostat by providing harsh environment at pH 10 to prevent other types of microbial growth. Good In addition, various parameters such as varying hydraulic retention time (HRT) of the chemostat, urea concentration and yeast concentration in feed media have been carried out to improve the purity of culture and to improve urease production. This explained what you have done. Now, I hope you explain what you have found. In result, it was found that by increasing the HRT, biomass productivity increased; however it did not increase the specific urease activity of culture. Furthermore, results also showed that by performing yeast extract increment in the media unclear (adding YE?), the purity of the culture decreased quite significantly, along with a sign of decreasing specific urease activity. Nonetheless, such alteration caused a rise in biomass concentration. Besides, increasing urea concentration in the media not only did not show an increase in purity of the culture, but also showed a lower growth and specific urease activity of S.pasteurii in the culture vessel. Lastly, the highest urease activity product obtained from the Chemostat has shown success in biocementation of silicon sand. Reasonable abstract. Could refer to pH or a causal relationship between conditions and effects on urease activity.
Keywords: Sporosarcina pasteurii, urease, biocementation
Introduction
In almost every environment on Earth there are microbes which participate in mineralization processes such as calcium carbonate precipitation (Achal et al, 2009). In both laboratory and natural settings, microbes have shown to induce the precipitation of calcium carbonate and are thus regarded as important factors in the formation of carbonate sediments and deposits (Achal et al, 2009). Bioremediation processes have utilised this ability of microbes to create a microbial plugging process, known as biocementation, to promote the precipitation of calcium carbonate in the form of calcite (Achal et al, 2009). This is then used for various applications such as the consolidation of sand columns, the repair of limestone monuments and the repair of cracks in concrete (Achal et al, 2009). Furthermore, using biocementation to enhance the strength and stiffness of soil, soil avalanching can be prevented, soil on reclaimed land sites can be compacted, and the effects of liquefaction can be alleviated (Ivanov et al, 2008).
One such microbe which has been shown to contribute to calcium carbonate precipitation is Sporosarcina pasteurii (previously known as Bacillus pasteurii), an endospore-forming, alkaliphilic, soil bacterium, which grows at an optimum pH of 9 (Achal et al, 2009). S.pasteurii produces a nickel containing enzyme called urease which catalyses the hydrolysis of urea to ammonia and carbon dioxide:
NH2-CO-NH2 + H2O → 2NH4++CO32-
Nice and clear to here.
This results in an increase in pH in the immediate environment, which when in the presence of calcium and carbonate ions causes them to precipitate together as calcium carbonate (Stocks-Fischer et al, 1999). Thus, by integration of S.pasteuriiwith urea and calcium chloride, biocementation can occur. It has also been found that the presence of high concentrations of ammonia does not inhibit growth of S.pasteurii, nor does it prevent urease production as it does in other microorganisms which produce urease such as Pseudomonas aerogenes (Morsdorfet al, 1989). This combined with the high pH optimum of S.pasteurii, allows for selective growth conditions even in a non-sterile environment. Source? (Cheng paper?)
From the equation above, the production of ammonium ion can result in ammonia gas formation. As a consequence, it can cause some degree of pH drop and affect the culture purity. Such reaction can be explained by using the Le’ Chatelier’s principle (Appendix G), with support of the spontaneity of the reaction using Gibbs free energy. However, it has also been found that the presence of high concentrations of ammonia does not inhibit growth of S.pasteurii, nor does it prevent urease production as it does in other microorganisms which produce urease such as Pseudomonas aerogenes (Morsdorf et al, 1989). This combined with the high pH optimum of S.pasteurii, allows for selective growth conditions even in a non-sterile environment.
In this project, a chemostat was run for two weeks under non-sterile conditions containing a culture of S.pasteurii. A constant temperature of 280 Celsius and a pH of approximately 10 were used for the chemostat. The aim of the experiment was to test the feasibility of S.pasteurii production of urease under continuous conditions, in a non-sterile chemostat, employing harsh conditions to minimize contamination and to optimize conditions to enhance the production of urease. Nicely defined aim. This elevates the introducition to a more quality level. Various methods were employed to enhance the production of the enzyme including, altering the concentration of urea and yeast extract in the feed, changing the hydraulic retention time of the chemostat and controlling the pH of the environment to limit contamination. Following the two weeks of inoculation, a harvest sample with the highest specific urease activity was used to attempt biocementation to determine if production of urease in a non-sterile condition is feasible. I would better finish the intro with the aim rather than pre-empting results.
Methods
Chemostat Set Up: A 600ml bioreactor was placed in a 28oC water bath with an overhead drill stirrer, an airflow tube and feed and harvest tubes connected into it.The airflow tube was connected to an airflow meter which was connected to an air source. Both the feed and harvest tubes were connected to separate pumps, which had corresponding tubes connected to a feed bottle and a harvest bottle. The pumps were also connected through a labjack data card to a computer so that the chemostat system could be controlled via the computer. A diagrammatic representation of the chemostat set up can be seen in Figure 1. Note: as the pH pump was not working correctly from batch 1 result thus, it was disassembled from the set up for batch 2 culture. Could explain more how the pH was maintained.
Figure 1: Diagrammatic representation of chemostat set up. good
Chemostat Innoculation and Running: 600ml of S.pasteurii culture was inoculated into the bioreactor at the beginning of the two weeks. The chemostat was continuously fed and harvested over the two weeks with retention times altered according to needs of the experiment. The harvest pump was set at the 600ml mark to enable a consistent volume of 600ml in the chemostat. The stirring speed was set at a constant 400rpm, and airflow rates were varied according to experiment needs. Temperature of the chemostat was kept at a constant 28oC, and pH was manually controlled to maintain an approximate pH of 10.
Urease Activity: The urease activity of the culture was determined using a conductivity assay. This was achieved by adding10ml of 3M urea, 8ml of deionized water and 2ml of culture to a 50ml centrifugation tube. A conductivity probe was then placed into the tube and was used to gently mix the solution. Conductivity changes were then measured over 10 minutes at room temperature. Following this the centrifugation tube was placed overnight in a fume hood to de-fume. The conductivity probe was calibrated prior to each test following instructions provided by the manufacturer. Could briefly explain the principle
Chemostat Feed: 20g of yeast, 10.21g of urea, 20g of sodium acetate and 2ml of nickel chloride (50mM stock solution) were combined and deionized water added to make up to 900mls. The pH was then adjusted to 10 using 10M sodium hydroxide before deionized water was added to make up the total volume of 1L. Following this, any further pH adjustments were made to maintain a pH of 10. Alterations in feed media were made by changing the amount of yeast or urea added.
Dissolved oxygen, Optical Density and pH: Dissolved oxygen in the chemostat was measured using a DO probe. The probe was calibrated before every use following manufacturer instructions and measurements made in ppm. Optical density measurements were made using a spectrophotometer with a wavelength of 600nm, and using feed solution as the blank. To obtain a reading under 1, various dilutions were made and the resultant reading multiplied by the dilution factor to determine true optical density. The pH was also measured using a pH probe. The probe was calibrated prior to each use following manufacturer instructions and the pH adjusted up to 10 using 10M sodium hydroxide.
Biocementation: Based on the production of carbonate ions from urease activity, biocementation was performed by adding 10ml of harvest culture with a urease activity of 31 mM/min into a 50ml syringe filled with silicon sand. After the excess harvest culture had been flashed out via rubber tube, 10ml of 1M calcium chloride and 1M of urea was added. Subsequently, 20ml of 1M calcium chloride and 1M of urea was added every 7 hours. By providing calcium ions and urea to S.pasteurii, formation of carbonate ions can be formed by urease activity and precipitation of calcium carbonate to the sand would occur.
Figure 2. Sand Column set up.
good methods section
Results and Discussions
Starting up of chemostat in a non-sterile condition (Batch 1)
During the first week of chemostat project, various experimental parameters were determined prior to start up, in order to achieve a successful running chemostat. Parameters such as the retention time and feed media selection were considered and were varied accordingly.
For 24 hours of retention time operation, an initial optical density (OD) of the culture in the bioreactor was read at 0.585 absorbance (abs). Based on Liang’s findings for the conversion of cell turbidity to culture biomass concentration (Cheng & Cord-Ruwisch, 2013), the biomass concentration was found to be 0.2574 g/L. show calculation, this value sounds too low to me. In the meantime, an initial urease activity test was carried out and its activity was found to be 4.889 mM/min. Thus, initial specific urease activity was determined to be 8.3325 mM/min/OD (Refer to Appendix B, Batch 1, Day 0).
An issue with the harvest pump resulted in a slight delay, with time taken to trouble shoot required, and the discovery that the harvest pump was unserviceable. Consequently, this fault caused the culture media in the bioreactor to be diluted, thus giving a low optical density reading. Therefore, the logical decision was made to increase the retention time to 48 hours in order to increase the biomass. Could go in appendix. The reader is interested in how you test your idea of producing more of the enzyme (hypothesis / scientific method)
Optical density was determined to be 0.828 abs after the change in hydraulic retention time. This relates to a higher biomass concentration which was found to be at 0.364g/L. Subsequently, its specific urease activity was found to be 21.8867 mM/min/OD (Refer to Appendix B, Batch 1, Day 1). Such alteration on the HRT had significantly increased the biomass concentration and its specific urease activity, which suggested that the culture growth and urease production had increased to its steady state.
High NaOH consumption due to pH meter inaccuracy
An unexpected event, involving an error related to the pH Pump, resulted in an additional 50ml of 10mM NaOH being added overnight. A re-calibration was then necessary in order to bring the system back to the required state. At the same time, due to the jumpiness of the raw data measured by the pH probe, some improvement on filtering the unwanted raw data was done by implementing an error range filtration in the Chemostat program, within Labview. After debugging the program, the measured pH of the culture was seen to drastically reduce to 6.58. Due to the fact the apparent pH value was below the optimum (pH10), 5M of NaOH was added continuously by running the NaOH pump for approximately 15 minutes. After a further measurement revealed the pH had overshot to 14, it was again adjusted accordingly with the addition of 150ml of 5M HCl. This action was effective in raising the pH to the acceptable level of 10.24. Fair enough, the exposure to pH 14 could explain loss of the culture , which would be much nicer shown as time course figure than a table.
The chemostat was left for 48 hrs to sequence a batch culture, with the airflow rate set at 100 L/h, and stirrer at 200 rpm. Later when checked, the bioreactor was found overflowing with a frothing culture that was sticky and pale-white in colour. This gooey-textured growth, found swarming around the reactor as well as the water bath, was not S.pasteurii according to its morphology. The reactor was therefore confirmed to be contaminated and the chemostat was re-inoculated with new culture.