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Overall comments: Not good yet. Should have more: Structure, Clarity in writing Details in explanations Evidence of understanding Urease activity by Sporasarcina pasteurii By Group 3 & 4 1 Abstract (not done yet) Make sure you cover in the abstract the background, goal, what was done and most of all what was found. It should be understandable without having read the background to the project 2 Introduction Limestone or calcite is a natural formation found in rocks and in many instances it acts as a form of natural cement which holds together rock and sand in a composite structure. As a natural formation of erosion and sedimentation by water flow it has formed many natural landscapes. The dykes for instance in Holland which keeps it from being flooded due to the country being below sea level is dependent on limestone which is beginning to erode. Interest has been expressed in utilizing bacteria as a countermeasure (Cord-Ruwisch 2008). Sporosarcina pasteurii is a useful alkaphilic bacteria which enzymatically digests urea to produce ammonia via the action of urease a nickel containing enzyme (Karplus et. al. 1997). The result of this reaction is two fold: 1) it can raise the pH making its environment more basic by the influence of ammonia and 2) it can produce carbonate. This can be summed up by the following equation: (NH2)2CO + H2O = CO3-2 + 2NH4+ Urea + Water = carbonate + 2 moles ammonia Figure 1: Equation of the breakdown of urea by urease enzyme into carbonate and ammonia (Whiffin et. al. 2004). The increase in pH also has a synergistic effect as the precipitation of Ca2+ and CO3-2 and its calcification can be enhanced by a more basic pH. Principally the carbonate produced can react with calcium provided or in the soil to form hardened limestone. In order to function effectively urease requires the presence of nickel ions to act as a cofactor. Each molecule of urease requires 2 nickel ions to work effectively. Our aim was to culture the bacteria in a bioreactor under septic conditions to simulate the conditions the bacteria is intended to be used in. To achieve successful calcification a specific activity of 2mM urea min-1 was set as the goal under septic chemostat conditions. Previous experiments showed that S. pasteurii grew optimally at pH 9.25 based on its ideal pKa value illustrated by this graph add source. Figure 2: Chemical speciation of ammonia (empty dots) and ammonium (black dots) in relation to pH. The pKa value was found to be pH 9.25 which is the point of half the dissociation constant for the NH3/NH4+ equilibrium. This is the state where the concentration of ammonia and ammonium created from the activity of urease within the cell are at equilibrium causing an external pH of 9.25 which is the measured pH. This is illustrated by this diagram: Figure 3: Urea hydrolysis and ATP generation intracellularly in S. pasteurii. (Jahns et. al. 1999) It is clear from this that the growth optimum and ATP-production optimum are the same which require an equal amount of NH3 and NH4+ to be present (Whiffen et. al. 2004). Our aim was to set the conditions of the reactor such that the cultured S. Pasteurii would grow nominally while decominating the reactor. We then intended to study the urease activity by using conductivity tests, biomass by optical density and dry cell weight and determining the specific activity from these results. 3 Materials & Method 3.1 Trial 1 3.1.1 Bacteria strain and cultivation An aliquot of 50 mL of S. pasteurii (from Chen Liang, Murdoch University, Western Australia) was cultured in 500 mL rich medium with 20 g/L yeast extract, 170 mM ammonium sulphate and 0.1 mM NiCl. pH was adjusted to 9 using NaOH. Bacterium was grown as batch culture for 24 hours at 30°C in a shaking water bath. 3.1.2 Chemostat setup The entire batch culture of 500 mL was transferred to the chemostat reactor. The batch culture medium was used as the feed medium. A 2L glass reactor was put into the water bath at the temperature of 30°C. A stirrer set at 250 rpm. One Chemaster TM peristaltic pump set to turn on and off with a timer was used to pump the feed medium into the reactor. Another pump and timer was used to pump product out of the reactor into a collection bottle. Both timers were set and synchronised to switch on for 2 seconds and switched off for 3 minutes. A sparger set at 250 qnL/h was used to aerate the system. 3.1.3 Sampling procedure 3.1.3.1 Dissolved oxygen Oxygen probe was used to measure the oxygen concentration and temperature in the reactor. 3.1.3.2 pH The outflow was measured using Hanna instruments HI 8424 pH meter. 3.1.3.3 Conductivity A 1:11 dilution was done by adding 4 mL of sample into 40 mL of 1.5 M urease solution. Conductivity was measured by using Hanna Instruments HI8733. Readings of conductivity (mS) was taken at intervals of 20 seconds for 30 minutes. The urease activity (mM urea hydrolysed min-1) was calculated for each sample, using the conversion factor of 11.11 mS min-1 = 1 mM urea hydrolysed min-1 (Salwa’s thesis). 3.1.3.4 Optical Density The sample with urease solution was taken after conductivity test, for the absorbance measured at 600nm in a spectrophotometer. 3.1.3.5 Dry Cell Mass 1.5 mL of outflow product was centrifuged. Supernatant was discarded and pellet was left to dry in a 37°C oven overnight. The dry cell mass was weighed the next day. 3.2 Trial 2 3.2.1 Bacteria strain and cultivation An aliquot of 50 mL of a new batch of S. pasteurii (from Chen Liang, Murdoch University, Western Australia) was cultured in 500 mL rich medium with 20 g/L yeast extract, 100 mM ammonium sulphate, 0.1 mM NiCl and 100 mM urea. pH was adjusted to 9 using NaOH. Bacterium was grown as batch culture for 24 hours at 30°C in a shaking water bath. 3.2.2 Chemostat setup The entire batch culture of 250 mL was transferred to the chemostat reactor. The batch culture medium was used as the feed medium. The pH was raised to 9.25 by adding NaOH. A 2L glass reactor was put into the water bath at the temperature of 30°C. A stirrer set at 300 rpm and the stirring rate was increased to 500 rpm on day 7. One Chemaster TM peristaltic pump set to turn on and off with a timer was used to pump the feed medium into the reactor. Another pump and timer was used to pump product out of the reactor into a collection bottle. Both timers were set and synchronised to switch on for 2 seconds and switched off for 3 minutes. A sparger set at 250 qnL/h was used to aerate the system. 3.2.3 Sampling procedure 3.2.3.1 Dissolved oxygen Oxygen probe was used to measure the oxygen concentration and temperature in the reactor. 3.2.3.2 pH The outflow was measured using Hanna instruments HI 8424 pH meter. 3.2.3.3 Conductivity A 1:15 dilution was done by adding 4 mL of sample into 56 mL of 1.5 M urease solution. Conductivity was measured by using Hanna Instruments HI8733. Readings of conductivity (mS) was taken at intervals of 20 seconds for 30 minutes. The urease activity (mM urea hydrolysed min-1) was calculated for each sample, using the conversion factor of 11.11 mS min-1 = 1 mM urea hydrolysed min-1 (Salwa’s thesis). 3.2.3.4 Optical Density The sample with urease solution was taken after conductivity test, for the absorbance measured at 600nm in a spectrophotometer. 3.2.3.5 Dry Cell Mass 1.5 mL of outflow product was centrifuged. Supernatant was discarded and pellet was left to dry in a 37°C oven overnight. The dry cell mass was weighed the next day. 4 Results 4.1 Trial 1 Optical Density 0.8 OD (at 600nm) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 3 4 5 6 7 8 9 Experiment Day Fig.1. Changes in the OD The OD of the S. pasteurii culture was used to measure the concentration of bacterial cells. The concentration of bacterial cells peaked at Day 4 before falling at Day 5 and remained more or less constant for the rest of the days. Specific Urease Activity (mM urea min1/OD) Specific Urease Activity 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 3 5 7 9 Experiment Day Fig.2. Specific Urease activity (mM urea hydrolysed/ min/ OD) The specific urease activity (SUA) is an index of the efficiency of urease production by S. pasteurii. The SUA peaked at Day 2 before falling at Day 3. It increased again at Day 4 before decreasing for the rest of the days. Productivity 0.006000 Productivity (g/L/h) 0.005000 0.004000 0.003000 0.002000 0.001000 0.000000 3 4 5 6 7 8 9 Experiment Day Fig.3. Productivity of urease over the days of experiment The productivity of urea was increasing steadily at a slow rate over days 3 to 5. There was a steep increase from day 5 onwards. Oxygen Uptake Rate (OUR) OUR was calculated from the dissolved oxygen. The OUR obtained from the experiment was decreasing, however, the trend of OUR cannot be determined as there are too few data points. The kLa obtained was 0.0626 h-1. find out what’s the significance of kla pH 8.4 8.3 pH of Outflow 8.2 8.1 8 7.9 7.8 7.7 7.6 3 4 5 6 Experiment Day Fig.4. Change of pH over days of experiment 7 8 9 The drop of pH at Day 4 resulted in poor productivity. The increasing pH from day 4 onwards resulted in an increased productivity as the bacteria thrive at a pH of 9.25 (Whiffin, 2004). The change of the feed pH from 9 to 9.5 resulted in the increase in pH. Trial 2 Optical Density 7 6 OD (at 600 nm) 5 4 3 2 1 0 0 2 4 6 8 10 12 Experiment day Fig.1. Changes in the OD OD increased in Day 5 because the feed pumps were increased hence more feed was given to S. pasteurii. As a result, higher growth was obtained. After sampling on day 7, the stirring rate was changed from 300 rpm to 500 rpm, resulted in a rise in OD. After which, OD dropped slightly. Specific Urease Activity Specific Urease Activity (mM urea min-1/OD) 4.2 1.40000 1.20000 1.00000 0.80000 0.60000 0.40000 0.20000 0.00000 0 2 4 6 Experiment Day 8 10 12 The specific urease activity was obtained using urease activity obtained from the conductivity test and divided by the OD. What is the units and relevance?? 5-7 high OD was achieved; this meant tht there was high bacteria concentration. Day 4…drastic drop from day 1-4 because not enough feed pumped into the reactor for bacteria growth resulting in lesser urease production. Day 6…outflow was higher than inflow resulting in washout of bacteria. Hence S. pasteurii in the outflow therefore when the conductivity test was conducted, there was not enough urea in the solution for the bacteria to degrade. Too much bacteria cells.unlikely to have contaminitiaon as ph is high.. Productivity 3.5 Productivity (g/L/h) 3 2.5 2 1.5 1 0.5 0 0 2 4 6 8 10 12 Experiment Day Dilution x biomass = productivity Noted that dilution rate was increasing from Day 4 to Day 7 however, dry cell mass collected decreased during that period. Day 4…drastic drop from day 1-4 because not enough feed pumped into the reactor for bacteria growth resulting in lesser urease production. Day 6…outflow was higher than inflow resulting in washout of bacteria.. Oxygen Uptake Rate (OUR) pH 9.6 pH of outflow 9.4 9.2 9 8.8 8.6 8.4 8.2 0 2 4 6 8 10 12 Experiment Day 5 Discussion Our studies showed that during experimentation the activity decreased in reactor conditions due to contamination consequently we attempted to maintain high pH conditions and a high dilution rate. To achieve this pH we provided extremely high ammonia and urea concentrations in the feed medium of 100mM ammonium sulphate and 500mM dissolved urea. We also attempted to maintain pH directly by using 10 M concentrated NaOH solution as the pH would acidify when exposed to oxygen. We needed to continually aerate the culture by direct bubbling and stirring to provide oxygen for the bacteria. (still under construction) 6 Recommendations 1) Starting early is important. The reactor can thankfully be restarted within 24 hours as new culture can be grow in less than 24 hours and inoculated into a clean bioreactor. 2) To maintain high pH and reactor sterility with only the chosen bacterial strain use feed medium containing 500 mM urea. 3) Ensure that amount of feed media is able to last the weekend. Reduction of reactor volume and starvation periods can hurt the bacteria. 4) Make sure when using the pumps that the same type of tubes with the same diameters are used. Some pumps have two sets of tubes with different diameters causing vastly different inflow and outflow rates lowering or raising the reactor volume rather than keeping them constant. Also try to have sufficient reactor volume to distance the bubbling from the outflow tube, push the tube as far down as possible this helps. Be warned this can result in complete reactor drainage if the inflow fails. Add a small syringe tube to try and limit the build up bubbles in the outflow tube, this causes a reduction in outflow raising the reactor volume. 5) Try to set up a periodic dripper using a small syringe with a needle to continually add anti-foam agent. This is needed for extended periods when the chemostat is not observed like over the weekend as outflow removes the antifoam agent. Without it foaming occurs and reactor volume is quickly lost. 7 Conclusion 8 References Ralf Cord-Ruwisch. Personal Communication and Advice 2008. T. Jahns. Ammonium/urea-dependant generation of a proton electrochemical potential and synthesis of ATP in Bacillus Pasteurii. Journal of Biotechnology 178 pg. 403 -409. 1999. P.A. Karplus, M.A. Paerson. 70 years of crystalline urease: What have we learned. Acc. Chem. Res. Issue 30 pg. 330 – 337. Victoria S. Whiffin. Microbial CaCO3 precipitation for the production of biocement. Thesis paper for the School of Biological Sciences & Biotechnology of Murdoch University 2004. 9 Appendices Trial 1 Oxygen Uptake Rate (OUR) Average temperature, 28.75°C cS = 468 ÷ (28.75 +31.6) = 7.75 mg/L kLa = 0.0461 h-1 0.1 y = 0.0461x - 0.2277 OUR (mg/L/h) 0.08 0.06 0.04 0.02 0 -0.02 0 2 4 -0.04 cS-cL (mg/L) 6 8 Trial 2 OUR Average temperature = 29.6 cS = 468 / (29.6 + 31.6) = 7.65 mg/L kLa = 0.0176 h-1 0.1 OUR (mg/L/h) 0.05 y = 0.0176x - 0.1142 0 0 1 2 3 4 -0.05 -0.1 -0.15 cS-cL (mg/L) 5 6 7 8