Continuous Production of Urease by Bacillus Pasteuriiunder Non-Sterile Conditions in A

Continuous production of urease by Bacillus pasteuriiunder non-sterile conditions in a chemostat

Abstract

The enzyme urease has a great deal of commercial potential, especially in its ability to produce ‘bio-cement’. As such, optimizing its production is an important factor in achieving viable production levels. In this experiment we attempted to find the optimum conditions under which Bacillus pasteurii could produce urease by adjusting flow rates, feed components and pH levels. Our results, however, proved inconclusive as we were unable to achieve good levels of enzyme production until the last day of the experiment and thus couldn’t determine how sustainable the conditions were.(The last statement is not useful. Instead you should list whatever effects you have measured and what is explainable and wht is not. I would discourage students to use as the key outcome of a project to write it was inconclusive)

Abstract should contain what was attempted, what was found not just what was concluded, in particular if the conclusion was considered to be “inconclusive”.

Introduction

In general all bacteria require the presence of some inorganic chemical components for growth, such as, potassium, phosphorus, and sulphur. Moreover, most bacteria also requires an energy yielding carbon source, such as glucose or lactate for example. In addition to these elements different kinds of bacteria require more complex compounds, for instance, amino acids, which they are not capable to synthesizing. (Novick, et. al. 1950)Not incorrect, but not sure how it helps introducing this particular topic.

Basically, the growth rate of a bacterial strain may be within wide limits of the chemicals concentration, organic or inorganic growth factors, but since at zero concentration, the growth rate is also zero, in other words, there are necessities at low concentrations of the growth factor. I could not find any sense I this sentence.

On the other hand, it should be possible to maintain a bacterial population over an indefinite period of time growing at a normal growth rate simply by controlling growth factors at a sufficient level to cultivate a bacterial strain, as is done, for example, in a Chemostat. (Novick, et. al. 1950))(The literature you use is very outdated)

Urease

(There is a sudden jump from chemostat to urease which could be lessened by making a transition between the two topics) Urease (EC 3.5.1.5), is a nickel dependent metalloenzyme which catalyzes the hydrolysis of urea to yield ammonia and carbonate and the latter compound spontaneously hydrolyzes to form carbonic acid and another molecule of ammonia (Andrews, et. al. 1984). The reaction occurs as follows:

(NH2) 2CO + H2O → CO2 + 2NH3

Functionally, ureases belong to the super family of amidohydrolases and phosphotriestreases (Holm & Sander, 1997). Urease activity tends to increase the pH of the environment because it produces ammonia as a product and ammonia is a basic molecule. Ureases are found in numerous bacteria, fungi, plants and some invertebrates, as well as in soils, as a soil enzyme (Krajewska, et.al. 2012). The urease activity can be used in the biocalcification process of soils (Mahantyet. al. 2013), and some clinical studies related to the virulence of pathogenic bacteria (Collins et, al. 1993).The jump from chemostat to urease is sudden and there is not link that helps the reader understanding what the introductory background is

Bacillus pasteurii

Bacillus pasteurii, also called as Sporosarcinapasteurii is a bacterium that carries out cementation via the precipitation of calcite and solidifies sand with urea and calcium. This bacteria is a highly urease active alkaliphile that can thrive in high ammonia environment. (Lee et al. 2000)

The focus of this project is to investigate the production of urease via alkalinicBacilluspasteurii in a non-sterile condition, and maximize the productivity of this process for stable urease production.(As background info, the paper provided to you by Cheng et al.) should have been used. It is essential background that B. pasteurii can be grown in an open chemostat)

Materials and Methods

The Chemostat was set up by the help of the Lab technicians.

The following diagram illustrates this:

Figure 1: Set up for the chemostat Bioreactor

Nice drawing. You could indicate the author.

Culture conditions

- Temperature: 30o

- pH 10

- Mixing rate: Approx. 400rpm

- Airflow rate: Approx 50 L/H

The set-up of the Bioreactor

On day 1, the Bioreactor was set up as shown in Figure 1. The volume of the reactor was made up to 600ml. This was made by using 552ml of Inoculum and 48ml of the feed as the volume of the inoculum provided was not exactly 600ml. After this, 2 drops of anti-form was added to bioreactor.

Preparation of the enrichments for the Micro-organisms

On day 1 to Day 13, the feed of the Urease positive bacteria was prepared as follows:

- 20g/L of yeast extract was used

- 10.21g/L of 0.17M of Urea was used

- 20g/L Sodium acetate was used

- 2ml of 50mM stock/L of 0.1mM of NiCl2was used.

And the contents were shaken. The pH of the feed was adjusted to 10 by using NaOH.

On day thirteen the yeast extract concentration was increased to 40g/L and the urea concentration was increased to 20.4g/L..(This would be an experimental test and goes into results section.)

pH Controls

The pH was constantly being monitored by a pH meter which was calibrated(you mean to say that the setpoint was pH 10, not the calibration) at a pH of 10, if the pH dropped below 10 the sodium hydroxide pump would commence and the pH would be brought back to 10.NaOH pump was kept at a constant rate of 1sec and with the set point of 60 sec .Statement could be clearer. The pH probe was calibrated with the computer when the bioreactor first commenced and also during the project to make sure that the pH probe was giving the correct readings Good. In general use full stops at the end of all sentences.However, on the day 13, pH was measured at 11.7 due to error in calibration or the computer system. Then the whole system was analysed again and it was found that the Ph probe was not working so the PH probe mistakenly underestimated the exact PH and so the pump would falsely pump NaOH into the culture so as to restore the pH to what it believed to be 10 again. After this, the problem was solved state exactly how the problem was rectified, most likely the probe was not “broken”.and the pH was back to normal again.

Operation of the system

The chemostat consisted of a glass bioreactor with an operating volume of 864mL for the first seven days that the chemostat was running and then reduced to 670mL for the remaining seven days. This culture was continuously fed with fresh medium for the 2 weeks at a hydraulic retention time (HRT) of 32 hours initially and then at 16.5 hours from day 8 onwards. Nice and clear.The decanting pump was set at a faster rate than the influent pump to ensure constant volume. The decant tube was set at a length of 5cm whereas the influent was triple that in measurement. So, as of day eight, we instead used the harvest pump tube to pump feed into reactor and the feed tube for the harvest bottle. The stirring speed was set at 400 rpm and the airflow was a constant 50 L/h throughout the experiment. The pH of the reactor was controlled to a set point of 10 by computer program that pumped NaOH, which was changed to 10.2 on the seventh day and lowered to 10.1 on the thirteenth. The temperature of the culture was kept constant at 30oC. The culture was then tested regularly for the urease activity and optical density and hence enzyme activity and biomass concentration were recorded.

Normally the daily changes whether on purpose or not belong to the results section as they are not methods.

Monitoring methods

Biomass measurement (OD 600 nm):

On a daily basis, the biomass concentration was recorded with the feed being used as the blank. If the biomass’s absorbance measured at a wavelength of greater than or equal to 2 then the stock was diluted to get the absorbance value down to a level at which the spectrophotometer could measure it acurately.

This was calculated for converting the absorbance reading into biomass concentration is as follows:

C ( Biomass concentration. g/L) = 0.44 x OD (600 nm)

(Liang.Cheng& Ralf Cord- Ruwisch.2013)

Urease activity:

Urease is a constitutively expressed enzyme that hydrolyzes urea to NH4+ and CO32 The urease activity was determined by measuring the rate of change of conductivity over time due to the conversion of non ionic substrate Urea to ionic products NH4+ and CO32 - (Liang Cheng and Ralf Cord-Ruwisch, 2014). Thus, 2ml of the culture was added to 10 ml of 3M urea and 8 ml of deionised water and the conductivity was recorded over a period of 10 minutes in 1 minute intervals at room temperature (25oC). The mS per minute obtained from this was converted into units of enzyme activity per litre by multiplying the value obtained by 11100. Sounds good this part. Could say what the definition is of one unit in terms of urease converted per time.

Dissolved oxygen

Oxygen concentration was measured by using the Dissolved Oxygen probe. Oxygen concentration needs to be monitored as it is important to ensure that the oxygen concentration does not become a limiting factor. To ensure the accuracy of the readings, the dissolved oxygen probe needs to be calibrated before use.. During the measurement, feed/harvest bung in the chemostat was removed and the probe was carefully introduced through the aperture.

Results

Initial conditions

The initial culture used to inoculate the chemostat had a pH of 10, an optical density of 1.463 and an activity of 4051.5 units of enzyme per litre. 600mL of this culture was added to the chemostat vessel and made up to 864mL with feed. The pumps were set to turn on for one minute out of every 20, giving a flow rate of 27mL/h. From Thursday the 4th (day 1) through to Monday the 8th (day 4) the pH was not regulated and was measured at about 9.5. Good start for results section. What the reader wants to know now is the purpose, aim of your experiments and the hypothesis you are going to test.

Results of Initial running conditions

Between the beginning of the experiment (day 0) and day 4 the conditions were maintained as described above for days 0, 1 and 4, with the pumps being turned off for days 2 and 3. Over this time an increase in biomass productivity was observed (figure 1 and 3). This was determined by measuring the optical density and multiplying the value obtained by 0.44 to give the g of biomass/L (Cheng, L., Cord-Ruwisch, 2013).There is an inconsistency between your explanation leading to g/L and the units used in the figure (g/L/h). It would be good to list your dilution rate such that the calculation can be verified.(Your calculation does not seem to take into account the dilution rate. So is it really the biomass level in g/L that you record?)

In addition we observed a decrease in the enzyme activity and production (Figure 2 and 3). Enzyme activity was determined by measuring the rate of change in the conductivity of a urea solution when exposed to the culture (see materials and methods above) and multiplying the rate by to give activity in Units of enzyme/LNo need to repeat, it was explained in the method (Cheng, L., Cord-Ruwisch, 2013).

Figure 1: Biomass productivity over the first four days showing a sharp increase in biomass by day 4.Always write the units next to the axes.

Figure 2: Enzyme productivity over the first four days, showing a marked decrease between day 0 and day 1, with a smaller decrease by day 4.

Figure 3: Biomass concentration and enzyme activity, showing the decrease in enzyme activity in relation to the amount that the biomass increased.

This increase in biomass coupled with a drop in enzyme activity implied to us that the culture had become contaminated. Correct conclusion. However to the reader it is a bit surprising as you have not lined out in the intro, aims, methods or results that this is an open chemostat subject to contamination when the pH and ammonia don’t generate “extreme’ conditions.

It is evident that this was occurring as of the first day as the enzyme activity dropped markedly while the biomass concentration remained relatively steady, indicating a higher proportion of non-urease producing bacteria in the culture. The sharp decrease in enzyme activity over the first day is likely due to the dilution of the original culture with the feed mediaDilution would result in both OD and enzyme activity to decrease. So I am not sure whether this explanation is backed up by data.but the slight increase in biomass over this time, despite the dilution, implies that bacteria were growing. The increase in biomass and decrease in enzyme activity over the next three days implies that non-urea producing bacteria continued to be dominant and the number of active Bacillus pasteurii continued to decrease. Our next goal was to correct this by removing the contaminant organisms.Yes, you are leading to the objective for the next period. That is good. It should be done by formulating a hypothesis and testing it. It is not so much removing contaminants but favouring the growth of Bacillus relative to that of contaminants.

Removing Contaminants

On day four the NaOH pump was turned on and the chemostat was set to be regulated at 10pH. Where are the pH recordings ver time and what was the pH in the previous period?This was maintained until day 7, when the pH pump was recalibrated and the setting changed again. The purpose of the increased pH was to create an environment in which Bacillus pasteurii could survive but which would inhibit or prevent the growth of other micro-organisms. Should have explained in the intro what the specific extromphilic nature is of Bacillus p. Between day four and day 7 there were problems with the computer program such that the feed and harvest pumps were not running for significant periods of time. Other conditions such as feed rate, airflow rate, feed concentration and reactor volume were maintained during this time. Despite the increase in pH we continued to experience an increase in biomass concentration and a decrease in enzyme activity during this period (figure 4).

Figure 4: Biomass concentration and enzyme activity between days 4 and 7 of the experiment, showing further increase in biomass and decrease in enzyme activity despite the increased pH.Vague.where is the precise pH monitored?

Increasing Enzyme Production Rate

Despite our lack of success in removing contamination it was decided that our focus should shift to increasing the enzyme production rate. In order to do this we increased the flow rate to 40.5mL/h and decreased the reactor volume to 670mL from day 7. This meant that our dilution rate changed from 0.03125h-1 to 0.060448h-1, almost doubling. Reads OK, but you should explain here why an increase in dilution rate is expected to improve the situation, without that it would seem it is trial and error rather than using the scientific method.Also, avoid excessive number of digits which imply precision that was not realistic.This had the effect of initially increasing our theoretical enzyme production rate (figure 5) although attempts to actually harvest this were frustrated by further problems(specifically what? feel free to admit that operator errors were made too) with the computers, leading to the feed and harvest pumps again not functioning for much of the time. In addition, the pH setting was increased to 10.2 and the pH probe recalibrated. We believe that at this point the pH probe was mis-calibrated and the actual pH achieved was closer to 11.7. All other conditions were maintained as stated above and these were maintained until day thirteen with the exception that the reactor was turned off on days 9 and 10.(That is not too bad as you can now discuss the expected and found effect of pH11.7 on this culture. Will it kill the culture or the enzyme activity, will it kill more contaminants than alkalophilic strain?)

Figure 5: Enzyme productivity between days 7 and 13, showing the increase in production between days seven and eight and the subsequent drop in enzyme productivity.

Following this both the enzyme activity and biomass concentrations dropped drastically (figure 6). The initial assumption would have been washout due to the increased harvest rate It is not initial assumption, it is the most likely explanation if the biomass concentration decreases. Correctly spotted but then the issue is confused by referring to pump problems.but the failure of the computer program and subsequent lack of harvest discounted this possibility. On day 13 the pH metre was recalibrated and the actual pH (which had been measured at 10.1) was shown to be 11.7. (Still? Is this an accidental repeat writing, or has the pH not been able to be corrected since the last observation of 11.7?) Assuming that this error had occurred when the pH metre was recalibrated on day 7 this would explain the drop in both biomass and enzyme activity as such a high pH would kill off both contaminant organisms and Bacillus pasteurii.Well done, this is nice evidence based explanation what is likely to have happened.

Figure 6: Biomass concentration and enzyme activity between days 7 and 13 showing the decrease in both. We believe that this was due to a high pH induced by a pH metre mis-calibration. The slight increase in enzyme activity on day 13 may imply that Bacillus pasteurii performs better at these extreme pH levels than other microorganisms and had begun to recover.

Recovery of Culture

When the pH problem was discovered on day 13 it was immediately reset to a pH of 10.1 in an effort to allow the culture to recover. To aid this the feed media was also changed, increasing the yeast extract to 40g/L and the amount of urea increased to 20.4g/L. These changes were intended to increase the growth rate of Bacillus pasteurii and decrease the stress on it so that it could recover to useful levels. This was achieved as the levels of both biomass and enzyme activity increased (figure 7). The enzyme activity in particular increased more than fivefold, while the biomass increased by about a third, possibly indicating that there is still contamination in the culture. (No, it indicates regrowth of the target strain only, as would be evidenced by plotting the specific enzyme activity).Well done, you improved your report quality from measurements on the last two days.