BIO301: Industrial Bioprocessing and Bioremediation
Title of Report:
Cost effective continuous urease production
by Bacillus pasteurii under non-sterile
conditions by a chemostat
Group 4:
Shin Lau
James Kossen
Haikle Lee Ning
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Abstract
The production of urease by Bacillus pasteurii in a chemostat was investigated
over the duration of 8 days. The specific urease activity was on a decreasing
trend from day 2 to day 4 . The maximum specific urease activity was on day 3
(0.045 ms/OD/min). As too many unforeseen circumstances had arisen during
the duration of the experiment, we were unable to carry out a successful
experiment to produce urease via Bacillus in a feasible manner. All groups had
problems. Here you can state what wanted to achieve and what you did achieve
to quantify the shortfall.
1 Introduction
Urease belongs to the family of amidohydrolases and phosphotriestreases. The
nickel containing metalloenzymes catalyzes the hydrolysis of urea into carbon dioxide
and ammonia: (NH2)2CO + H2O → CO2 + 2NH3. It can be found in numerous
bacteria, plants, fungi, algae, as well as in soil enzyme. Urease-catalyzed hydrolysis of
urea is important and has great potential in medical and agricultural applications. It is
also a catalyst in the precipitation of calcium carbonate. The product of urease
activity, especially ammonia increases the pH of the reaction environment and is
toxic to most bacteria.
Bacillus pasteurii, also known as Sporosarcina pasteurii is a bacterium that is
capable of carrying out cementation via the precipitation of calcite and solidifies
sand with urea and calcium. The bacterium is a highly urease active alkaliphiles that
can thrive in high ammonia environment. It can grow at ammonia concentrations
greater than 500 mmole L-1. (Leejeerajumnean et al. 2000)
The catalytic property of microbial urease in calcium carbonate is gradually
becoming an important subject in biocementation. Biocementation is a natural
method in soil strengthening which requires the urease role in nitrogen metabolism.
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However, the production of sterile urease in large scale can be costly as any
contamination of yeast culture will lead to process failure. (Ivanov & Chu, 2008)
The aim of this project is to investigate whether the production of urease via
alkalinic Bacillus pasteurii in a non-sterile condition is feasible. Aim is correct. Could
explain on what the aim is based (selective enrichment with high pH etc.).
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2 Materials and Methods
The chemostat culture was supplied with an airflow rate of 100L/h, 400rpm of
mixing and maintained at pH 10 to avoid the growth of other microorganisms. For
the cultivation of Bacillus pasteurii, the culture was added with 1L of pH 10 the pH of
the feed was not 10 feed containing 20g of yeast extract, 10.21g of 0.17M urea, and
20g of sodium acetate and 2mL of 50mM stock/L.
For 8 days, the culture was pumped with feed and harvest collected every half
hour. Observations and control of the chemostat was done through Labview software.
NaOH was added if the software detects a decrease in pH levels.
Could explain the basis of pH control.
Biomass was monitored daily by the measurement of optical density via
spectrophotometer at 600nm with 3mL of culture sample and 3mL of water as blank.
Measurement of urease productivity was conducted to compare against the optical
density in order to detect the presence of contaminants, eg: other biomass.
Urease productivity was measured in a mixture containing 10mL of 3M urea,
8mL of deionised water and 2mL of culture. Recording of the conductivity was
conducted every minute for 10 minutes with the conductivity probe placed in the
solution. The probe was calibrated at the start of the experiment and with the stirrer
turned on.
Specific activities were observed throughout 8 days and calculated via the plot of
gradient of conductivity over optical density reading for the day. good
2.1 Determination of biomass concentration
The biomass concentration is determined by measuring the optical density which is
the absorbance value of the cultural suspension at 600nm. The correlation of optical
density (OD600) and biomass concentration was previously established by the
regression equation Y = 0.44X; where Y is the biomass concentration measured in g/L
and X is the OD600
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3 Results
In all reports the results section should start with text that explains the purpose and outcome of
the figure first and then refers to the figure.
3.1
pH
pH over Time
10.1
10
9.9
pH
9.8
9.7
9.6
9.5
9.4
9.3
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Time
Figure 3.1
The optimal pH 10 is not maintained from day 3 onwards, this could lead to the growth of
microorganisms other than B. pasteurii.
Biomass (Optical Density)
Biomass concentration
4
Biomass concentration (g/L)
3.2
3.5
3
2.5
2
1.5
1
0.5
0
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Figure 3.2
Biomass concentration calculated using equation Biomass concentration = 0.44*OD
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3.3
Conductivity
Conductivity (ms)
Conductivity over Time
0.28
0.26
0.24
0.22
0.2
0.18
0.16
0.14
0.12
0.1
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Time
Figure 3.3
Conductivity level decreases on day 1 onwards and gradually increases on day 4 onwards.
3.4
Specific Urease Activity
Specific Urease Activity (ms/OD/min)
Specific Urease Activity
0.05
0.045
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Figure 3.4
The specific urease activity is an indication of the efficiency of urease production by
B. pasteurii. Specific urease activity decreases from Day 3 and gradually increases.
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The results section could not be understood as it had no
text but merely a collection of figures.
Discussion
Many factors were manipulated to determine the optimum growing condition for B.
pasteurii. Factors include:
PH
As B. pasteurii requires high pH for growth (it is a alkalophile), pH was maintained at
pH 10. Such high pH can also inhibit the growth of other microbes (contamination) by
disrupting plasma membrane, denaturing proteins and decreasing availability of
nutrients (Prescott et al, 2005).
NaOH was automatically added when pH lower than 9 was registered.
Literature
FEED CYCLE
The system was initially running on a 12-hour cycle on Day 0, 1, 2 but was changed to
10-hour cycle on Day 3, 4, 5 and 8-hour cycle on Day 6 and Day 7.
FEED CONCENTRATION
After data were collected on Day 2, the chemostat system had been altered to run on
double of the yeast feed.
Many issues that could give inaccurate results were encountered. Issues encountered
were as following:
i)
ii)
iii)
iv)
There were several occasions when the yeast feed had run out
completely before it was replaced.
The pH probe was found faulty during Day 4. The pH reactor was reading
33.8, which the lab team speculated, could have been faulty.
The duration of experiment included 2 days of weekends when the lab
was locked up and no observation could be made.
Miscommunication between group members.
RESULTS EXPLAINATIONS
Figure 3.2 Biomass concentration is a measurement of the concentration of biomass
created. Biomass concentration initially increased from Day 0 to Day 2 and started
decreasing from there to Day 5 then started increasing again.
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The specific urease activity is an indication of the efficiency of urease production by B.
pasteurii. The specific activity of B. pasteurii peaked at Day 3, which could be caused by
the change in feed concentration and feed cycle, although it is not clear which one of the
factors was responsible for the change.
Lowest pH on Day 4 corresponded with lowest urease activity on Day 4. A thesis by
Whiffin in 2004 suggested that low specific urease activity was not due to depletion
of urea nor due to the high concentrations of the main reaction product, ammonium.
Although pH levels were shown to have a regulatory effect on urease but it was
evident that another co-regulating mechanism existed.
An explanation was suggested that the sudden decrease of urease activity on Day 3
and 4 (Figure 3.4) was due to the drop in pH (Figure 3.1). Reason of this is because B.
pasteurii is a spore-forming organism that forms spores under stress condition to
survive (Prescott et al, 2005). These spores do not need nutrients to grow, and so do
not produce urease at all. The drop in pH could have caused the culture to
experience stress. Potentially reasonable attempt to explain, but missing out on the
whole point of using pH control in the presence of high ammonia levels.
On Day 6, a strange odor was observed which could indicate the growth of other
microbes, hence a contamination. Although on Figure 3.4, it showed that specific
urease activity for Day 6 was on an increasing trend, showing little/no sign of
contamination.
RECOMMENDATION
i)
ii)
iii)
iv)
Manipulate one factor at a time for easier interpretation of data.
Devise a way to ensure anti-foam is added when needed. This is
especially needed for the weekends that nothing can be done to the
chemostat.
Double check pH probes before setting up the chemostat.
Record results at the same time of the day to ensure consistency, ie.
v)
records all data either in the morning or afternoon.
Derive relationship between conductivity and specific urease activity.
CONCLUSION
It can be concluded that urease was successfully produced through an open
chemostat culture. This statement contradicts your summary.The project is
incomplete, as the final step of biocementation is not carried out. Further
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experiment completing the biocementation step is recommended.
-
Scientific method (as requested) was not used (as
+ Understanding of the project in the class seemed OK.
+ Reasonable operation of the chemostat
- Data processing as in bioprosim simulation (as requested) not carried out
-No evidence of understanding the fundamentals of how contamination was
controlled in this report
The report is more observational than with purpose.
5.5/10
References
Ivanov, V., & Chu, J. (2008). Applications of microorganisms to geotechnical
engineering for bioclogging and biocementation of soil in situ. Reviews in
Environmental Science and Bio/Technology, 7(2), 139-153.
Prescott, L.M., Harley, J.P. & Klein, D.A. (2005). Microbiology, 6th Edition. 122. Boston:
McGraw Hill.
Whiffin, Victoria S. (2004) Microbial CaCO3 precipitation for the production of
biocement. PhD thesis, Murdoch University.
Appendix
Specific Urea Activity
Date
Day
Conductivity
Activity (ms/min)
OD
(ms/OD/min)
17-Sept
0
0.133
0.1319
5
0.01
18-Sept
1
0.266
0.2507
5.86
0.02251
19-Sept
2
0.252
0.2087
7.65
0.03278
20-Sept
3
0.172
0.1364
4.61
0.04527
20-Sept
4
0.159
0.1588
3.1
0.01369
23-Sept
5
0.178
0.1642
1.21
0.01312
24-Sept
6
0.189
0.174
6.65
0.01978
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7
0.244
0.247
7.6
0.02616
Day 0 Conductivity over time
Conductivity (ms)
9
8
7
6
5
4
3
2
1
0
y = 0.1319x + 6.5058
R² = 0.9914
0
5
10
time (min)
Day 1 Conductivity over time
Conductivity (ms)
12
y = 0.2507x + 6.9092
R² = 0.9992
10
8
6
4
2
0
0
5
10
time (min)
Day 2 Conductivity over time
Conductivity (ms)
25-Sept
y = 0.2087x + 4.7606
R² = 0.9496
8
7
6
5
4
3
2
1
0
0
5
10
time (min)
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Conductivity (ms)
Day 3 Conductivity over time
y = 0.1364x + 4.7612
R² = 0.918
7
6
5
4
3
2
1
0
0
5
10
time (min)
Day 4 Conductivity over time
Conductivity (ms)
9
8
7
6
5
4
3
2
1
0
y = 0.1588x + 6.486
R² = 0.9916
0
5
10
time (min)
Conductivity (ms)
Day 5 Conductivity over time
y = 0.1642x + 5.9233
R² = 0.9854
8
7
6
5
4
3
2
1
0
0
5
10
time (min)
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Conductivity (ms)
Day 6 Conductivity over time
y = 0.174x + 5.417
R² = 0.9878
8
7
6
5
4
3
2
1
0
0
5
10
time (min)
Conductivity (ms)
Day 7 Conductivity over Time
9
8
7
6
5
4
3
2
1
0
y = 0.247x + 5.2945
R² = 0.9986
0
5
10
time (min)
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