Production of urease active bacteria by non-sterile...

Production of urease active bacteria by non-sterile chemostat

Group Members:

Devashi Devi Nemdharry (31624575) Priyadarshinee Boojhawon (31668482) Hu Shi (32082998)

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Abstract

This experiment describes the production of the enzyme Urease in a chemostat. This was achieved by culturing the bacteria Bacillus Pasteurii in a non- sterile chemostat. Urease is a nickel containing enzyme that catalyzes the hydrolysis of urea to carbon dioxide and ammonia (Liang Cheng & Ralf Cord Ruwisch, 2013)Unusual way of citing by using first name. Microbial Ureases are of high benefit to the chemical and Biotechnology Industry; among which one of crucial process that can make use of this enzyme is the precipitation of Calcium Carbonate in the presence of Calcium and Urea (Liang Cheng & Ralf Cord Ruwisch, 2013). In this experiment, we have used a pH of 10 and about 0.2M urea in a yeast extract- based medium to grow ureolytic bacteria under aerobic chemostat operation at a hydraulic retention times of about 10h after two days. Good.Urease production was optimized under the above mentioned conditions and 1ml of 10uMm Nickel solution was added to the growth media (feed) resulting in an increase in urease activity by the further addition of 20g/L of yeast extract.

However, after day 2, we encountered problems with the bioreactor and noticed that there was no enzyme activity or little. Too detailed for abstract. The root cause for this problem was due to several factors namely: we had only one pump where both the feed tubing and harvest tubing were connected. This therefore means that we could not increase the capacity/ speed of the harvest pump individually as doing so will result in the same change in the feed pump. The other reason was due to the presence of contaminants. Therefore, to favour the growth of the desired bacteria, we made the conditions harsher so that the other competitors cannot survive and only the S. Pasteurii strain can grow. The experiment was a success after overcoming several problems during the experiment. Second half too detailed for abstract. Rather focus on results obtained such as what was your productivity and how does it relate to literature.

Introduction

Sporosarcina pasteurii once known as Bacillus Pasteurii is a common bacterium in soil and is very much studied for it great ability to produce high concentration of Urease (Laprotox, 2011). This enzyme is an alkaliphiles no the bacterium is and grows in conditions that can be very toxic to other bacteria and also the presence of free ammonia makes the condition harsher for other competitors to survive. Good. Could say first where the ammonia comes fromThe Urease enzyme hydrolyses substrate Urea to give ammonia and carbamate according to the following equation:

H2 N-CO-NH2 + H2O NH3 +H2N-CO-OH

The carbamate formed is then decomposed to form another molecule of ammonia and carbonic acid as per the following equation: give source for that

H2N-CO-OH + H2O NH3 + H2CO3

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The 2 ammonia molecule and carbonic acid then equilibrate in water with their deprotonated and protonated forms thereby resulting in an increase in pH. This is illustrated as per the equation below (Al Sawat use correct spelling , 2008):

H2CO3 H+ + HCO3

2NH3 + 2H2O 2NH4+ + 2OH-

(Al Sawat, 2008)

The Urease enzyme which releases carbonate therefore causes Calcium ( Ca2+) to be precipitated as CaCO3; also known as biocement. According to studies, the alkaline pH in which the enzyme works is the prime reason for the calcite precipitation (Al Sawat, 2008). However, the formation of CaCO3 is still a mystery and it could not be understood at both genetic and molecular level (Al Sawat, 2008).

Biocalcification also known as Microbiologically induced Calcite precipitation (MICP) has many novel applications such as to improve properties of bricks by soil bacteria (Sarda et al, 2009), improvement of the durability of cementations materials and Biocementation (Liang Cheng & Ralf Cord Ruwisch, 2013)

Aim of Experiment:

The main aim of this experiment was to produce urease active bacteria by non-sterile chemostat culture and to test whether urease would produce biocement. Good to see the aim well defined. The urease production can be increased by using yeast extract (non- sterile media) and urea which is also a good gfrowth mediuma for other micro-organisms that can act as contaminants to the S. Pasteurii Species. This seems to be a claim without giving sources. It would be better to conclude from your experiments rather than postulate what you think is supposed to h appen. To boost the production of Urease, the substrate concentration in the media was increased; the pH was increased to 10. After, the biomass production was determined by the measurement of the optical density and the production of the enzyme was determined by measuring the urease activity. We were expected to obtain an increase in biomass of the S. Pasteurii bacteria and in urease activity in order to make sure that the experiment was a success. If ever an increased in biomass but decrease in urease activity was obtained, this could mean the presence of contaminants in the bioreactor. After introducing the aim at the end of the introduction

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Materials and Methods

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

The following diagram illustrate this:

Figure 1: Set up for the chemostat BioreactorNice sketch. A bit unusual that it is a photo from paper

Culture conditions

- Temperature: 28- 30o

- pH 10

- Mixing rate: Approx. 400rpm

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- Airflow rate: Approx 100 L/H which was then changed to 150 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 3, 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 NiCl2 was used.

And then distilled water was added to make a total volume of 1L and contents was shaken. The pH of the feed was adjusted to 10 by using NaOH.

On day 4 onwards, the ingredients of the feed was doubled as follows (explanation provided later in the report under the discussion part)

- 40g/L of yeast extract was used

- 20.42g/L of 0.17M of urea was used

- 40g/L Sodium acetate was used

- 4ml of 50mM stock of 0.1mM of NiCl2 was used.

And then distilled water added to make contents up to a volume of 1L. The pH of the feed was then adjusted to 10 by using NaOH.

The same was repeated for the preparation of feed during the 2 weeks.

The Bioreactor was incubated in a water bath at 28oC for the 2 weeks with a stirring speed of 400 rpm. The pH was constantly being monitored by a pH meter which was calibrated at a pH of 10. However, on the second day, pH increased to 10.4. Then the whole system was analysed again and it was found that the PH probe was not working well as so the PH probe mistakenly read the exact PH underlevelled and the pump could falsely pump NaOH into the cuture so as to restore the pH to 10 again. After this, the problem was solved and the pH was back to normal again.

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Operation of the system

The chemostat consisted of a glass bioreactor with an operating volume of 600mL. This culture was continuously fed with fresh medium for the 2 weeks. The Hydraulic retention time (HRT) of 24h for first three days and was changed to 10h from day 4 onwards was used by using a pump at a speed of 1.88L/h. Explain to the reader your purpose in changing the HRTWe could not use a separate pump to decant the harvest as the one we were provided with was a faulty one. So instead, we used the harvest pump to pump feed into reactor and to pump decanting fluid into the harvest bottle. The stirring speed was set at 400 rpm and the airflow for day 1 was 100 L/h and then changed to 150 L/h and remained so for the other days. The pH of the reactor was controlled to a set point of 10.1. The temperature of the culture was kept constant at 28oC. The culture was then tested regularly for the urease activity and hence biomass concentration was recorded.

Monitoring methods

Biomass Concentration:

On a daily basis, the biomass concentration was recorded. The feed was used as the blank. Better to blank against water and subtract the OD of feed.This was calculated as follows:

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

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

Urease activity:

The urease activity was determined by measuring the rate of change of Conductivity over time. Conductivity can be measured with the help of a conductivity probe and there exist a flow of current due to the conversion of non ionic substrate Urea to ionic products NH4

+ and CO3

2 - (Liang Cheng and Ralf Cord-Ruwisch, 2013). Thus, 2ml of the culture was added to 10ml of 3M urea and 8ml of deionised water and the conductivity was recorded over a period of 10 minutes at 25oC.

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Results

Biomass Measurement (OD600)

Here you need to explain your strategy. What specifically was changed and for what reason. Biomass monitoring was carried out by measuring the Optical density of the culture using a spectrophotometer (600 nm). The correlation of biomass concentration and optical density was established as per the following equation: (this is still methods text)

Biomass Concentration (g/L) = 0.44 x OD (600 nm)

Table 1 Shows the Biomass concentration over the days we ran the experiment.

Time ( Day ) Optical Density (600nm) Biomass Concentration, g/L1 1.780 1.30532 0.827 0.60653 0.760 0.55734 1.276 0.93575 1.880 1.37876 2.110 1.54737 2.203 1.61558 1.739 1.2753

Tables and figures should be always first introduced by the text.

0 1 2 3 4 5 6 7 8 90

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8Biomass Concentration against Time

Biomass Concen-tration agaisnt T...

Time (Days)

Biom

ass c

once

ntra

tion

(g/L

)

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Figure 2: Graph showing the Biomass concentration (g/L) over time

From Figure 2, from day 1 to 3 there was a decrease in concentration of biomass. Yes here you introduced the table by the text. This was because of the increase in pH to an extreme 10.4, as discussed in the discussion section. From day 3 to day 8, biomass concentration was increased because of the drop in pH to 10.0.

Productivity of the urease enzyme (Engl.)

Table 3 shows the productivity of the Urease Enzyme over the course of the experiment.

Time courses are much more visual than tables. They also allow to view possible trends and causal relationships between parameters such as HRT and urease concentration.

Dilution rate= Flow rate / Volume

Volume of reactor: 600ml = 0.6L

For Day 1-Day 3, flow rate= 0.025L/h

Therefore dilution rate= 0.025/0.6= 0.0417 h-1

For day 4 to day 8, the flow rate was 0.06L/hShow example calculations in the appendix

We had to increase flow rate so as to make the conditions more favourable for the B.Pasteurii Strain due to other micro-organisms being present in the bioreactor. Explain the principle behind that. Without such explanation the report does not demonstrate the understanding that your group actually had.

Therefore dilution rate for Day 4 to Day8 = 0.06 / 0.6 = 0.1 h-1

Table 3: Table showing Productivity of Urease over time avoid excessive use of decimal places of numbers. You multiplied biomass concentration by dilution rate. This gives you the

biomass productivity but provides no information about the enzyme levels or the urease productivity.

DAY DILUTION RATE (h-1) BIOMASS CONCENTRATION (g/L) PRODUCTIVITY (g/L/h)1 0.0417 1.3053 0.05442 0.0417 0.6065 0.02533 0.0417 0.5573 0.02324 0.1 0.9357 0.09365 0.1 1.3787 0.13796 0.1 1.5473 0.15477 0.1 1.6155 0.16168 0.1 1.2753 0.1275

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0 1 2 3 4 5 6 7 8 90

0.020.040.060.08

0.10.120.140.160.18

Productivity against Time

Productitivy against Time

Time (days)

Prod

uctiv

ity (g

/L/h

)

Figure 3: Graph showing the productivity of Urease over time ok good to see the productivity plotted. Still, if plotted together with other

parameters (e.g. HRT, pH, etc, OD) it could easier demonstrate relationships between the parameters

From Figure 3, it can be noted that the productivity of the urease enzyme decreased from Day 1 to day 3. This is because the increase in pH as the PH probe not calibrated well on day 1 resulted in more OH- being added by the computer programme and hence kills our desired bacterial. . The effect of pH is best shown on the concentration of the enzyme and the concentration of biomass and hence as the specific enzyme concentration (enz/biomasss) not so much on productivity, as productivity also depends on the HRT

The increase was due to the NaOH pump being faulty. After fixing (could explain the problem and how it was fixed) the NaOH pump, the pH was checked again and a pH of 10.1 was obtained. It was left at this pH so that other micro-organisms cannot survive in this condition but our desired ones. Need to explain the reasoning for this. The next day, DAY 3, we measured the OD of the culture and it was still low, (0.760) what does that mean? Is it good or bad news? and no obvious sign of increase of conductivity measurement which means the Urease activity had vanisheddied. . This data point (zero activity can’t be seen from your figures and tables) We discarded all the culture and replaced it with Group 2 culture (200ml) with 400 ml feed and left of overnight for a batch culture. Hence, the next day, the bacteria reproduced and they were again hydrolysing Urea to Ammonia and Carbon Dioxide which explains the rise in productivity after day 3.

Urease Activity in Culture

The urease activity of the culture was determined by measuring the rate of change of conductivity. According to Whiffin, 1mS cm-1 min-1 corresponds to 11.11 umol min-1ml-1 of urease activity. Conductivity can be measured with the help of a conductivity probe and there exist a flow of current due to the conversion of non ionic substrate Urea to ionic

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products NH4+ and CO3

2-. Thus, 2ml of the culture was added to 10ml of 3M urea and 8ml of deionised water and the conductivity was recorded over a period of 10 minutes at 25oC.

The following table shows the conductivity over the 8 days that we ran the experiment This table and figure 5 is raw data and should go in appendix. We just want to know the enzyme

activity on each day.

Time Conductivity Conductivity Conductivity Conductivity Conductivity Conductivity Conductivity Conductivity Conductivity

min ms ms ms ms ms ms ms ms ms

Day0 Day1 Day 2 Day3 Day 4 Day 5 Day6 Day7 Day8

1 9.28 6.12 4.26 5.46 7.93 9.42 9.3 8.55 8.34

2 9.65 6.12 4.29 5.46 8.37 9.11 9.51 8.74 8.56

3 9.98 6.12 4.36 5.46 8.57 9.16 9.67 8.9 8.77

4 10.24 6.12 4.48 5.46 8.78 9.51 9.89 9.14 8.96

5 10.55 6.12 4.52 5.46 8.95 9.71 10.16 9.37 9.19

6 10.84 6.12 4.62 5.46 9.08 9.76 10.4 9.66 9.41

7 11.16 6.12 4.67 5.46 9.21 9.88 10.65 9.88 9.59

8 11.39 6.12 4.75 5.46 9.35 10.04 10.92 10.19 9.78

9 11.64 6.12 4.9 5.46 9.47 10.29 11.19 10.5 9.99

10 11.9 6.12 4.95 5.46 9.58 10.44 11.45 10.82 10.4

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0 2 4 6 8 10 120

2

4

6

8

10

12

14

Conductivity vs Time

Day0 Linear (Day0)Day 2 Linear (Day 2)Day 4 Linear (Day 4)Day 5 Linear (Day 5)Day1 Linear (Day1)Day6 Linear (Day6)Day3 Linear (Day3)Day7 Linear (Day7)Day8 Linear (Day8)Linear (Day8)

Time(min)

Cond

uctiv

ity(m

s)

Figure 5: Graph showing the rate of change of Conductivity

Table 5: table shows the rate of change of conductivity over time.

Finally!! You show the most important results the enzyme activity expressed as change in conductivity. Why do you not plot this most relevant set of results as a function of time next to your

biomass concentrations? Units are missing in this table.

Day EquationRate of change of conductivity

0 y=0.2885x+9.076 0.28851 y=6.12 02 y=0.0794x+4.1433 0.7943 y=5.46 04 y=0.1689x+8 0.16895 y=0.1394x+8.9653 0.13946 y=0.2417x+8.9847 0.24177 y=0.2528x+8.1847 0.25288 y=0.2164x+8.1087 0.2164

From Table 5, it can be seem that, the rate of change of conductivity of day 6 and day 7 was the highest among these days of experiment except the original raw culture, which suggested that these two days, urease activity was the highest. Yes The reason being for the day 6 and day 7, the feed had been doubled the concentration (this should be verifiable by the reader from a plot where feed

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concentration and urease activity are plotted for each day as well as applied at a retention time of 10h, faster stirring rate and a suitable PH of 10.1 played a part also.

Discussion

Throughout the experiment, the production of urease was optimised and the productivity of the enzyme increased. Data of the productivity (production rate of the enzyme are actually missing this would be something like: (mS/min)/h This was achieved by running the chemostat under the following conditions, Flowrate: 100-150 L/h too high, Urea concentration in media: 0.2M, Stirrer speed: 400 rpm but has been changed over time, pH was 10 at first but has also been changed to 10.1 over time in order to obtain a more suitable environment for our desired bacterial to grow. Beter to point to causal relationships rather than listing all conditions including those that were actually not changed and hence did not contribute to improving urease production.

Urease activitydegradation: ???The activity of the urease could be measured and obtained in non-aseptic sterile conditions using a conductivity probe in the presence of urea. The basis for this is that the hydrolysis of urea by urease results in the production of ions such as NH4+ and HCO3-. The rising levels of NH4+ and HCO3- contributes a charge that can be measured using a conductivity probe (Grunwald.P, 1984). Well explained, but does not belong to discussion.The amount of ions produced from the urea hydrolysis increased very slowly at first but after doubling concentration of the ingredients and fixing the right pH, the amount of ions produced increased proportionally over time and when plotted on a graph should display a linear rise. The linear rise corresponds to enzyme activity and was measured in mS/min. goodThere were many factors that were thought to affect urease production and therefore, the productivity of the enzyme in the chemostat.

Some of the factors that were thought to affect productivity are listed and discussed below:

Effects of pH change on the chemostat culture: For the first few days the pH was kept constant did you not say above the pH was uncontrolled? Missing in this report is the plot of pH value for each day – 10. However, normally, the Biomass concentration should have increased over the days but there was a sudden drop in Biomass concentration from Day 2 to Day 3. This is because the pH of the culture increased from 10 to 10.4. The reason being our PH probe was not well calibrated, so that the reading our PH probe showed a lower value as it was supposed to be and as the consequence the pH probe would detect a lower value of the exact value in our bioreactor so that the computer programme would direct NaOH into the culture until pH is adjusted back to 10 again. But in our scenario, the NaOH pump was not working properly and hence this explains the possibility of the drastic change in pH. For the last two days of our experiment, the pH was maintained at 10.1.This pH was discovered to have little or no effect on the specific enzyme activity where is this shown? but to kill other micro-organisms unclear to me. The biomass was also increasing which

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suggests that the suitable pH could be more favourable for growth as well as for the production of urease. These results were corroborated in other literature (Whiffin, 2004). In what way? You did not use the literature provided on chemostat production of urease

Effects of increasing stirring rate: At first the stirring rate was kept constant at 414 rpm but at day 7 and 8 the stirring rate was increased up to 600 rpm. The effects of oxygen limitation were also considered to see if a lack of oxygen was the reason for the lack of production of enzyme activity. It was documented before, that a lack of oxygen could reduce the enzyme production in bacteria (Mukherjee et al, 1999). As dissolved oxygen concentrations in our experiment remained consistently low (Day 0 – Day 8), where are the data supporting this claim? it was postulated that the lack of oxygen could be one of the main reasons for the decrease in specific enzyme activity. Otherwise correct reasoning

Based on this theory, the stirrer speed was increased from 414 rpm to 600 rpm to bring up the dissolved oxygen concentration levels in the bioreactor. Would be nice to see stirrer speed, dissolved oxygen and enzyme concentration in one plot to allow the reader to see the measurements that support your statements.

The increase in stirring rate brought about an increase in oxygen concentrations and biomass. Evidence? The increase in oxygen had an effect on the specific activity of the enzyme Fig 5. Evidence? Fig 5 does not show specific activity (activity per g of biomass e.g. (mS/min)/g/L.From the results obtained, we concluded that the oxygen was not limited since the growth of the microbes was still continuing and the activities were favourable. This could be indicative that perhaps the conditions were favourable toward biomass production and urease production.

Effects of increasing flowrate: In order to check and see if the conditions in the chemostat were indeed more favourable for bacterial growth but not urease, we decided to change the flowrate. At day 7 and day 8 the flowrate was increased from 100 to 150 (units? Better to show dilution rate). If the environment was not conducive for enzyme production, an increased in flowrate should merely increase the biomass,no, this is not what chemostat theory would predict (the biomass concentrationwould stay similar (slightly lower) while the biomass productivity would be increased. while keeping the enzyme activity minimal. The Initial flowrate was determined to be 100 and was changed to 150. A resultant increase and continued rise in enzyme activity. The increase in dilution rate also correspondingly increased the enzyme productivity approximately 10 fold from 0.04 to 0.10 mMurea/min/h (Day 3 – Day 8) suggesting that pH and oxygen deficiency were not the only cause of the low specific activity.

One of the reasons for the initial drop in activity could be due to the lack of sufficient urea,true, but can you explain to the reader why? hence the ingredients were doubled as from day 2. Supporting texts have shown that urease may not be constitutively expressed (produced all the time regardless of substrate concentration) as once thought (Morsdorf &

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Kaltwasser, 1989)i, but in fact, subject to strict regulation (Whiffin, 2004)ii especially in the absence of urea.

Effects of increasing urea concentration: Concluding that the amount of urea present was directly correlated to the urease activity, basal media urea concentrations along with the other ingredients were doubled as from day 4. This resulted in an increase in the urease production.

+ Good Labwork done: 3.5/4

+Some reasonable explanation showing understanding

-Key data and relationships not plotted (pH, urease concentration, specific urease activity

-structure could be improved by showing important plots and moving raw data to appendix

-Use of Scientific Method not demonstrated

-Lots of small inadequecies, hence many comments and corrections

Report quality as scientific report (evidence based conclusions using the Scientific Method):

Total mark: 6/10

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i

Referenceii

Grunwald, P., (1984). Experimental section: Imparting some biochemical fundamentals in the course of basic education of chemistry students with the system urease/urea as an example. Biochemical Education. Vol (12), pp 170-173.

Mukherjee, J., et al., (1999). Studies on nutritional and oxygen requirements for production of L-asparaginase by Enterobacter aerogenes. Applied Microbiology and Biotechnology. Vol (53), pp 180-184.

Morsdorf, G., Kaltwasser H., (1989). Ammonium assimilation in Proteus vulgaris, Bacillus pasteurii and Sporosarcina urea. Archives of Microbiology. Vol (152), pp 125-131.

Whiffin, V. S., (2004). Microbial CaCO3 precipitation for the production of biocement. Thesis for doctorate in philosophy Murdoch University Research Repository.

What about the paper that was given to you on chemostat cultivation of Bacillus pasteurii?

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