A NON-STERILE CHEMOSTAT CONTAINING UREASE-PRODUCING BACILLUS PASTEURIIBy Craig Blake (31963253), Priyanka Mariyappa (32165861) and Kristina Reklev (32096833).

AbstractA culture of Bacillus pasteurii with a known urease production rate of 11.11 units of urease produced/L/min was placed in a non-sterile (open) chemostat. The pH, oxygen flow, stirrer rate, dilution rate and feed concentration was adjusted over a period of 406.5 hours in order to investigate how specific changes in these parameters affected the biomass concentration and the urease production rate of the bacteria. An ideal system would contain a low amount of bacteria which yielded a high amount of urease. Trial and error showed that B. pasteurii was capable of growth at a relatively high pH, but preferred a lower food concentration and a lower dilution rate than what was expected - as this allows the production of biomass while simultaneously limiting contaminants. It was concluded that these unexpected results were due to B. pasteurii being subjected to substrate inhibition and/or could not tolerate with the high concentration of ammonia gas from the increased rate of metabolism.

IntroductionBacillus pasteurii (currently named as Sporasarcina pasteurii) from the Planococcaceae family is a Gram-positive, motile, spore-forming bacterium of spherical shape that is predominantly found in soil, however it has also been isolated from water and sewage systems (Yoon et al. 2001). \The optimum conditions for culturing B. pasteurii in vitro includes alkaline media with a pH ranging from 7-10, and temperatures around 25° C (Boriello et al. 2005).

Urease is an enzyme produced by B. pasteurii which catalyses the hydrolysis of urea to form ammonium and carbon dioxide as shown in the reaction below (Krajewska 2009).

(NH2)2CO + 3H2O CO2 +2NH4OH

Urease produced by B. pasteurii is industrially important due to its capacity to precipitate calcite and solidify sand if both calcium and urea is provided (Siddique and Chahal 2011). This ability has been applied to solve issues regarding soil liquefaction, amend cracked stone and cement, as well as for petrifying wood (Bang et al. 2001, Cheng and Cord-Ruwisch 2013, Jonkers and Schlangen 2007, Ramachandran et al. 2001).

A chemostat is a bioreactor in which the amount of feed flowing into the system is the same as the effluent flowing out maintaining a constant volume in the reactor (Novick and Szilard 1950). The bioreactor is inoculated with the desired microbe and given all the necessary components for growth and metabolism in the feed, oxygen is supplied

to the reactor via aeration and distributed throughout the reactor by a stirrer (Novick and Szilard 1950). The purpose of a chemostat is to create an environment that is optimised for the specific microbe present to produce the maximum amount of yield of the desired product, which is achieved by optimising parameters such as the dilution rate, pH, oxygen supply and feed concentration.

The aims of this experiment was to run a chemostat culture containing B. pasteurii and produce urease at the highest possible productivity through the application of scientific method. This involved changing the conditions under which the chemostat was run, including changes made to the pH, oxygen supply, stirrer rate, feed concentration and dilution rate.

Materials and MethodsA bacterial culture of Bacillus pasteurii with a known urease activity of 11.11 mmol urease/L/min was provided. The bacteria was placed in a 600 mL container, where the biomass was recorded to be 0.71 g/L. The container was kept in a water bath at 29° C, and 50 L/h of air was constantly supplied while the culture was being stirred at 194 rpm by an automatic stirrer. Note that the oxygen supply and stirring speed was raised to 75 L/h and 260 rpm, respectively, after 155.5 hours of running the chemostat.Feed (brand: Difco yeast extract) was provided to the chemostat culture. For the first 87.5 hours of the experiment, the feed provided was at a normal concentration, before the concentration was doubled for the subsequent 294.5 hours. For the final 24.5 hours, the feed added was at a normal concentration. The feed was added at various flow rates, where for the first 7.15 hours the flow rate of feed was 0.033 L/h, before it was reduced to 0.02 L/h after 24.5 hours, and again increased to 0.029 L/h after 36.5 hours. The flow rate was then kept steady until 333.7 hours had passed, at which stage it was increased to 0.04 L/h, before it was finally reduced and kept at 0.035 L/h after 382 hours.A pH, oxygen, biomass and urease activity reading was taken from the chemostat culture twice a day over the period of 406.5 hours of which the experiment was run. The pH reading was done using a pH probe, where the pH was adjusted by automatically by adding 10 M of sodium hydroxide when the pH dropped below the desired level. The pH was maintained at 10 for the first 216 hours, before being raised to 10.2 for the next 49.9 hours, and for the remaining 140.6 hours it was fluctuating between 10.3, 10.2, 10.3, 9.6, 10.3 and 10.2.

The biomass reading was done using a spectrophotometer. The absorbance was set at 600 nm and 3 mL of the feed was used as a blank for the culture to be measured against. The optical density was converted using the conversion method described by Cheng and Cord-Ruwisch to give the biomass in g/L (Cheng and Cord-Ruwisch 2013). Note that if the absorbance measurement was greater than 1, the sample was diluted with feed to give an accurate absorbance reading.

The amount of dissolved oxygen present in the chemostat was measured using an oxygen probe. Oxygen readings were mostly used as a guide to ensure the culture was not experiencing limiting oxygen conditions as the readings given by the oxygen probe were generally imprecise. The oxygen levels were recorded when the reading were at a relatively stable level.

The urease activity of the bacterial culture was examined by mixing 8 mL of urea, 10 mL of deionised water and 2 mL of culture, stirring the solution gently, and recording the amount of ions produced every minute over 10 minutes with a voltmeter. The final reading taken after 10 minutes was subtracted from the initial reading taken at 1 minute (in order to allow the solution to stabilise), and the units of enzymes produced was calculated from this data.

Figure 1 below shows an overview of the chemostat used to performed this experiment. As seen in the figure, both feeding, harvesting, addition of sodium hydroxide, stirrer and airflow could be adjusted either manually or automatically by the computer.

Figure 1: The above diagram shows all of the components of the chemostat used in this experiment. (The diagram has been sourced from “Chemostat Projects – Student Notes”)

Results600 mL of a bacterial culture containing B. pasteurii was used to inoculate a chemostat culture. The pH of the chemostat was set to 10, and the culture was known to have an activity of 11.11 units of urease produced per liter minute. The amount of biomass within the chemostat was measured to be 0.71g/L, and the dilution rate was set to 0.055 /h. The pH, oxygen, biomass and urease data was recorded over a period of 406.5 hours.

Figure 2 shows the change in the dilution rate over the 406.5 hours of which the experiment was conducted. The dilution rate was kept relatively stable throughout the experiment, starting off at 0.055 /h, before decreasing the dilution rate to 0.033 /h. A dilution rate of 0.048 /h was used for the majority of the experiment, but after 333 hours, the dilution rate was increased to 0.066 /h. The dilution rate was then reduced to 0.058 /h after 382 hours.

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Time (h) against Dilution Rate (/h)

Time (h)

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Figure 2: The above graph is showing the changes in dilution rates over the 406.5 hours of which the experiment was conducted. The dilution rate was at its lowest in the initial stages of the experiment, but kept relatively stable throughout the experiment, before being slightly raised towards the end of the experiment.   

The pH of the chemostat culture was recorded over the 406.5 hours of which the experiment, and the data is shown in Figure 3. As seen in the figure below, the pH was kept at 10 for 216 hours, before it was increased steadily over the next 118 hours to 10.3. At this stage, irregularities occurred with the computer responsible for automatic adjustment of the pH. Note that the pH never wavered outside the range of which B. pasteurii is capable of growing.

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The change in pH over time (/h)

Time (h)

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Figure 3: The above figure shows the changes in pH over the 406.5 hours of which the experiment was conducted. The pH was kept stable at 10 until 216 hours had passed, before it was increased to 10.3 over the subsequent 118 hours. After this stage, the pH fluctuated between 9.6 and 10.3.

The oxygen concentration (ppm) in the chemostat was recorded over the 406.5 hours of which the experiment was conducted, and the data is described in Figure 4. As seen in the figure, the amount of dissolved oxygen fluctuated greatly throughout the experiment, mainly due to use of imprecise oxygen probes. The oxygen reading was, however, used as a guide to ensure that the dissolved oxygen was never below a critical level. The lowest dissolved oxygen level recorded was 0.4 ppm at 382 hours, at which stage the biomass was remained viable - acting as a confirmation that at no point where the oxygen level too low for normal cellular function.

The oxygen transfer rate was calculated by reading the amount of dissolved oxygen for a period of 105 seconds during 5 second intervals when the stirring speed was set at 260 rpm and the airflow rate was set to 75 L/h. These results were plotted onto a graph where the gradient equals the oxygen transfer rate (OTR), which was determined to be 135 mg/L/h. The kLa was then calculated by taking the OTR (135 mg/L/h) and divide it by the steady state dissolved oxygen reading of the culture (4.12 mg/L), giving a kLa value of 32.78 /h. The kLa is an important constant to know as it quantifies and describes the performance of a bioreactor and its ability to provide oxygen to microbes.

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Dissolved oxygen (ppm) vs Time (h)

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Figure 4: The above figure shows the change in oxygen concentrations (ppm) over time. The oxygen concentration varied greatly over the 406.5 hours of the experiment, mostly due to use of imprecise oxygen probes.

The biomass concentration (g/L) and urease production rate of B. pasteurii (units of urease/mL/h) was recorded over the 406.5 of which the experiment was conducted, and is shown in Figure 5. The biomass stayed relatively constant until the experiment had been running for approximately 90 hours, before it steadily increased until 150 hours had passed - at which stage the feed concentration was doubled. This caused the biomass to decrease, before it picked up again at around the 340 hour mark, before it again decreased and stayed relatively low. The rate of urease production rate decreased over the first 67 hours, before it significantly increased for the next 93 hours. The urease production rate then decreased over the next 200 hours – reaching a low of 4.44 units of urease/mL/hour. For the last 56.5 hours, the urease activity rapidly increased to reach a maximum of 14.43 units of urease produced per mL per hour.

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Biomass (g/L) and units of urease production rate (L/min) vs. time (h)

Units urease/L/min Biomass (g/L)

Time (h)

Figure 5: The above figure shows the amount of biomass (g/L) and the urease production rate (mL/h) over the 406.5 hours of which the experiment was conducted. As seen in the figure, the biomass was somewhat stable, with peaks in biomass occurring around 150 and 340 hours of conducting the experiment. The urease production rate varied greatly over throughout the experiment, where it peaked around 150 and 400 hours of running the experiment. The shaded area indicates the time over which double feed concentration was used.

The urease (units of urease) produced by the biomass (g) over time (h) was calculated and depicted in a graph (see Figure 6a). As seen in Figure 6a, the specific urease production rate started off high when the system was least likely to be affected by contaminants. The specific urease productivity then declined for the first 50 hours, before it rapidly increased to be more than 1 units of urease/biomass/h after around 100 hours. The specific urease productivity declined and stayed low for the following 250 hours where the feed concentration was doubled, before it peaked at the end of the experiment - where it was approximately 1.1 units of urease/biomass/hour.

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Figure 6a: The above graph shows the specific urease productivity of B. pasteurii over the length of the experiment, this demonstrates the units of enzyme produced per gram of bacteria per hour. The shaded area indicates the time over which double feed concentration was used.

Separate data obtained from another group performing the same experiment is shown in Figure 6b. As seen in the graph, the urease activity produced per biomass per hour overall increased for the first 90 hours, before it rapidly declined and stayed low for the remainder of the experiment.

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Specific Urease Productivity

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Figure 6b: The above graph shows the specific urease productivity of B. pasteurii produced by Group 1 over the length of the experiment. The yellow area indicates where the culture was run under batch conditions.

Figure 7 is a visual representation of the effect on biomass concentration and urease production rate considering changes in the pH and dissolved oxygen. As already depicted in Figure 6a, the specific urease production rate was at its maximum around the 100 and 400 hour mark. In the figure below, we can see that the pH was around 10 for both of these times, and the dissolved oxygen was around 7 ppm. Furthermore - according to Figure 7 indicates a peak of urease productivity around  150 hours, but the biomass concentration was also very high at this stage, meaning the level of contaminants was high. Figure 6a also shows that the specific urease production rate was at its minimum around the 200 and 350 hour mark. As seen below in Figure 7, at 200 hours, the pH was increased from 10 to 10.2, and the reduction in urease activity is likely due to conditions being too harsh for B. pasteurii. At 350 hours, the dilution rate was increased, which likely resulted in the chemostat experienced a washout and explains the reduction in urease production rate. Also note that between these times, the urease production rate declined, which was most likely caused by the doubling of the feed concentration.

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Compiled graph of the pH, urease produc-tion rate, biomass and dissolved oxygen vs.

time (h)

Units urease/L/min Biomass (g/L)pH Dissolved Oxygen (ppm)

Time (h)

Figure 7: The above figure shows a comparison between the pH, concentration of dissolved oxygen (ppm), units of urease produced per mL per hour, and amount of biomass (g/L) present in the chemostat over the 406.5 hours of which the experiment was conducted. The shaded area indicates the time over which double feed concentration was used.

DiscussionThe system was given 155 hours to stabilise, at which point the first hypothesis of increasing urease production rate by doubling the feed concentration was implemented. Theoretically, doubling the feed concentration should result in a doubling of the biomass, and thus a doubling of the urease production rate (Novak et al. 1997). As seen in Figure 5, at 150 hours the biomass concentration was high, but the urease production rate was low - meaning the system was heavily contaminated. Increasing the food concentration would increase the concentration of urea present in the system - which is readily converted to ammonia gas by B. pasteurii, making the conditions harsher for contaminants (Cheng and Cord-Ruwisch 2013).

In an effort to further reduce contaminating bacteria in the non-sterile system, the second hypothesis of increasing the urease production rate by increasing the pH to 10.2 was implemented at 240 hours (refer to Figure 3). Increasing the pH affects the system in two ways, firstly it favours the presence of gaseous ammonia over ammonium in the solution (Cheng and Cord-Ruwisch 2013). Secondly, a higher pH will contribute to the elimination of contaminants that cannot tolerate such alkaline conditions (Brito et al. 2013). As expected, the increased pH caused the biomass to decrease, and a brief increase in the specific urease production rate was apparent - indicating the amount of contaminants had indeed become reduced, before it dipped and remained low - implying that the conditions might be too alkaline for B. pasteurii.

After increasing the pH and reducing the amounts of contamination, and a third hypothesis of increasing the urease production rate by increasing the dilution rate was implemented at 330 hours. This hypothesis was based upon the theory that an increase in dilution rate would

reduce the escape of gaseous ammonia, it would eliminate contaminants with slow generation times, and it would also increase the substrate availability for B. Pasteurii - allowing for higher biomass production and thus elevated urease production rate. However, it was found that the higher dilution rate produced the opposite effect of what was expected, as both biomass and urease production rate decreased drastically (see Figure 5). These results indicate that the reactor was experiencing washout - which occurs when the dilution rate is too high, and the biomass is washed out faster than it is able to replicate. Note that in our system, the maximum dilution rate for B. pasteurii was calculated to be 0.0833 /h, and that the dilution rate was kept well below this level in order to prevent washout.

An increase in dilution rate will also cause the retention time of ammonia gas (a product of urease activity) to reduce. Gaseous ammonia is a toxic compound to many bacterial species - but B. pasteurii is capable of tolerating relatively high levels of the substance (Leejeerajumnean et al. 2000).  The expected results were therefore a decreased amount of contaminants and an increased amount of B. pasteurii and urease production rate. However - as already mentioned, the increased dilution rate caused the biomass and the specific urease production rate to decrease, which can be interpreted as the concentration of gaseous ammonia was above the level of tolerance of B. pasteurii.  

A new hypothesis of increasing the urease production rate was implemented at 382 hours. This hypothesis involved changing the feed concentration back to normal. The increased feed concentration which had been used from the 150 hour mark and up until this point yielded an overall decrease in urease production rate. This was most likely caused by substrate inhibition of B. pasteurii, and/or a build-up of ammonia gas (a toxic product of urease - effects already discussed), making the conditions too harsh for the bacterium (Sokic-Lazic and Minteer 2008, Cheng and Cord-Ruwisch 2013). Substrate inhibition occurs when the substrate is saturating and the sugars are continuously taken up by the culture. Since the first step of glycolysis where the sugar is converted into acetate is much faster than the TCA cycle and the electron transport chain (ETC), acetate will accumulate. Acetate inhibits the TCA cycle and the ETC, causing the essential products of these cycles (ATP and NAD+) to become limiting, meaning that bacterial growth and enzyme production will be affected (Sokic-Lazic and Minteer 2008). As the feed concentration was decreased, a significant increase in the urease production rate (see Figure 5) and specific urease production rate (see Figure 6a) became apparent.

Figure 6b illustrates the urease production rate produced by the biomass in group one’s chemostat. Group one’s chemostat maintained constant conditions throughout their experiment, but periods of batch conditions were utilised to increase the total biomass and thus the productivity of the reactor. Initially this method resulted in much higher urease production rates than those achieved by our experiment. However, as the experiment continued, the high specific urease productivity dropped significantly. This was likely due to the system being non-sterile with conditions that favoured contaminants once they inevitably appeared.

As a conclusion, further investigation into changes in feed concentrations without subsequent increase in urea concentration should be undertaken in order to determine whether or not substrate inhibition or high ammonia concentrations is responsible for the low urease production rate experienced at high feed concentrations. Although the overall urease production rate only increased marginally over the entirety of the experiment, we have concluded from our findings that substrate inhibition, the presence of too much toxic ammonia gas and too alkaline conditions are detrimental for B. pasteurii’s ability to produce urease. Additional testing of different conditions is required to achieve maximum urease production rates by B. pasteurii in an open system such as this.

Nice clear, concise report that actually tests a few hypotheses and interpretes the results skilfully. The results section is perhaps the weakest part as it is quite descriptive and showing disconnected time courses rather than potential causal relationships. 8.5/10

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