Cost effective continuous urease production by Bacillus
pasteurii under non-sterile conditions by a chemostat.
Amanda Edwards
Ednara da Costa Sampaio Alvino
Nicole Elliot
Shin Lau
Abstract.
Many technological applications utilise microbes, such as bacterium to covert
a substrate to a particular desired product. Two systems that can be set up to
exploit such bacterial processes are a batch culture and a chemostat. In this
experiment a non-sterile continuous chemostat containing a culture of B.
pasteurii was set up and run for the duration of 11 days, with the aid of
computer software. This was done in order to produce the product urease and
attempt to optimize such production. The chemostat was also run to observe
what issues emerged and to overcome such issues. It was found that various
technical issues with pumps (controlled by the computer software) arose.
Likewise various chemostat parameters, such as pH and dissolved oxygen
and dilution rate had to be adjusted throughout the experiment. Such issues
had detrimental impacts on productivity and unease production, however once
these were overcome the productivity and urease production increased. (This
abstract does not give any of your findings (e.g. urease could be produced
under non sterile conditions provided the pH…))
Introduction:
A batch culture is a contained environment with a finite running time, whereas
a chemostat is a continuous culture with inflow and outflow. The
disadvantages that affect batch culture such as nutrient depletion and toxic
product inhibition can be overcome by using an open system with
continuously growing culture. A chemostat can be either a sterile or nonsterile system and is characterised by a production vessel housing a microbial
culture, with an inlet through which fresh media is pumped, and an outlet
through which excess cells and product leave; through this design steady
chemical conditions can be maintained. The growth rate of the culture is
controlled by the dilution rate, which can be calculated by dividing the flow
rate by the volume of the culture vessel and altered for optimal growth. The
chemostat’s main advantage over batch culture is that through constant
dilution with fresh media the growth rate as well as biomass, substrate and
product concentrations can be kept constant throughout the process,
achieving high productivity (Najafpour, 2007). Maintaining optimal conditions
for the particular culture strain, such as pH is extremely important in most
non-sterile chemostat cultures as any deviation from this may result in
contamination by other microbes, which can greatly affect the desired
outcome, such as the production of a specific enzyme.
The pH of the culture vessel must be maintained at a level appropriate to the
species of microbe grown. The structural and functional integrity of a
microbe’s internal proteins rely heavily on it’s cytoplasmic pH. If the microbe
cannot maintain it’s internal pH then such proteins cannot function and growth
and replication can be compromised. Consequently other microbes may
colonise the culture vessel and compete with the desired microbe.
The microorganism used for this experiment was the alkalophilic bacterium
Bacillus pasteurii, which requires a pH of 10 (Padan et al., 2005). B. pasteurii
is an a soil bacterium that grows in alkali conditions containing high
ammonium salt concentrations. It has been shown that ammonium is
converted to free ammonia inside the cell for amino acid transport, namely
glutamine. It has been assumed that through the direct activation of a sodiumtranslocating ATPase, an electrochemical sodium potential is produced, which
the cell requires for the transport of substrate. This stimulation is caused by
ammonium, which requires an alkaline pH to function. (Jahns, 1995). When
ammonium salts are substituted for urea, B. pasteurii produces urease, an
enzyme that hydrolyses urea to ammonia and carbamate (Bang, 2000).
Urea is a substance ubiquitous throughout nature, and large amounts are
excreted into the environment in the waste of mammals, birds, reptiles and
most terrestrial insects through many biological actions and its release is
involved in the biodegradation of nitrogenous compounds such as arginine.
The hydrolysis of urea is catalysed by the enzyme urease, which is
synthesised by many eukaryotic and prokaryotic microorganisms. Microbial
ureases are a significant substance both medically and ecologically. Bacterial
ureases are associated with the pathogenesis of many bacteria species and
have been implicated in clinical conditions such as urinary catheter
encrustation, peptic ulceration, ammonia encephalopathy and the formation of
infection stones. Conversely microbial ureases have a significant role in the
rumen and gastrointestinal tract of sheep, cattle and other ruminants. Animal
derived urea is a major source of nitrogen for commensal bacteria colonising
the rumen and is recycled there where its enzymatic activity releases
ammonia. The hydrolysis of urea also occurs in humans, mice and other
monogastrics however its significance is such animals is far less important
than that of ruminants (Mobley and Hausinger, 1989)
In this experiment a non-sterile chemostat was run for the duration of two
weeks containing a culture of B. pasteurii. The bacteria were fed on a media
comprised of yeast extract, urea, sodium acetate and nickel chloride. The
chemostat was run at 28°C and a pH of 10; this was maintained throughout
the experiment using a sodium hydroxide pump which tested the pH every 20
seconds and adjusted it accordingly. The culture was continuously aerated
and stirred using a an electronic stirrer to ensure the culture was well
oxygenated. The feed, harvest and sodium hydroxide pumps were all
controlled via a computer program designed for running a chemostat.
The main aim of this experiment was to run a non-sterile chemostat under
continuous conditions through reproducing a suitable environment for the
bacterium B. pasteurii in order to maximise its urease production over the twoweek period. The urease activity of B. pasteurii was tested and recorded daily,
along with the optical density and dissolved oxygxx en concentration. A
second, equally important aim of this experiment was to become familiar with
the setting up and operating of a continuous, non-sterile chemostat while
overcoming the various issues that arose, such as contamination, adjusting
flow rates to maximise productivity and dissolved oxygen concentrations.
(Introduction is very well written and it provides in depth background to what is
needed to understand your project. Towards the end of the intro where it is
supposed to lead to the objective and the purpose of this experiment there is
less focus such that in fact the objective is not clearly enough specified. The
fact that it is an open culture in which harsh conditions are intended to be
used to favour the desired organism B. pasteurii over contaminants, is not
coming out clear.)
Materials and method:
Chemostat Set Up:
A 600ml bioreactor was placed in a 280C water bath, the water level to be
kept at the same level as that in the bioreactor and water to be kept at
constant temperature. Attached to a stand and clamp, a drill stirrer was placed
into the bioreactor from above. An airflow tube was inserted into the
bioreactor; the tube was connected to an airflow source. Both feed and
harvest bottles were connected to separate pumps via tubes; the tubes then
ran from the pumps to the bioreactor. In the bioreactor, the harvest tube was
shorter than the feed tube to ensure that the bioreactor was never fully
drained into the harvest bottle. The feed and harvest pumps were also
connected to the labjack data card, which was then connected to the
computer, which controlled the Chemostat and recorded data with the
LabView software. A pH probe was inserted into the bioreactor, which was
connected to a pH pump responsible for pumping NaOH into the system
automatically when LabView recognised a change in pH. (Diagrammatic setup of entire chemostat pictured below, figure 1.1).
Chemostat Inoculation and Running:
At the beginning of the experiment, 600ml of Bacillus pasteuri was inoculated
into the bioreactor. The culture was provided with feed for the duration of the
experiment with varied retention times; harvest was also pumped out of the
system. The harvest tube was placed at the 600ml mark of the culture vessel
to ensure that 600ml of culture was always present. The harvest pump was
set at a higher flow rate than that of the feed pump to ensure that the culture
did not overflow into the water bath. The temperature of the water bath was
constantly kept at 280C and the pH kept at close to 10. The stirring rate was
kept at 400rpm.
Chemostat Feed:
Lab technicians prepared feed media daily, which had a pH of 10. 1L of feed
was made up using 20g of yeast, 10.21g of urea, 20g of sodium acetate and
2ml of 50mM stock solution of NiCl2. Deionised water was then added to
make up the 1L volume. The feed was stored in the fridge and then in a tray
filled with ice while being used for the Chemostat to avoid contamination
(pictured below, figure 1.2).
Urease Activity:
A conductivity assay was used to determine the cultures’ urease activity. This
involved adding 10ml of 3M urea and 8ml of deionised water to a 50ml
centrifugation tube. The conductivity meter was then turned on and set to mS
to 2 decimal places. The conductivity probe was calibrated to 12.88mS before
being rinsed with deionised water. With 10 mins on the stopwatch, 2ml of
culture was added to the centrifugation tube, immediately followed by the
conductivity probe. The probe was used to gently mix the solution, ensuring
that all of the holes on the probe were fully submerged and filled with solution.
The initial conductivity reading was recorded and the stopwatch started. The
conductivity reading was recorded every minute over 10 minutes at room
temperature. The overall change in conductivity was measured over the 10minute period. The conductivity of the culture indicated the urease activity
within the culture and, more specifically, how fast the Bacillus pasteuri
converted H2N-CO-NH2 into NH4+. It was expected that the activity would
increase over the 10-minute period. (How did this essay allow you to derive
the units showing in figure 2.1?)
Optical Density (O.D.):
Approximately 2-3ml of feed was transferred into a cuvette and 2-3ml of
culture was transferred into another cuvette. Once the spectrophotometer was
switched on, the wavelength was set to 600nm. Using the feed sample as a
blank, the spectrophotometer was ‘Auto Zeroed’. Replacing the feed sample
with the culture sample in the carrier the optical density was taken and the
absorbance displayed on the screen was recorded. If the O.D. was above 2,
the culture sample was diluted with feed media and the process was repeated
until an absorbance of less than 2 was recorded. The true optical density
could be calculated by multiplying the absorbance reading by the dilution
factor.
Dissolved Oxygen:
The dissolved oxygen probe was turned on and left to initialise on the desk for
5-10 mins. The probe was then held in the air to be calibrated, after which the
reading should have read close to 100%. The range was then changed to
mg/L (ppm) before being placed in the culture, ensuring that it was fully
submerged. The reading from the screen was then recorded.
pH:
The pH was measured using a pH probe. The probe was calibrated and left
submerged in the culture for the duration of the Chemostat experiment. When
the LabView software detected a deviation from 10, the pH was adjusted by
pumping in 10M NaOH.
Figure 1.1: Relative diagram of entire chemostat set up (sourced from
“Chemostat Projects- Student Notes”).
Figure 1.2: Feed media used for chemostat is placed in tray full of ice.
Urease Activity
mmol/min/ml
Results
Results always start with text that introduces the figures not with unintroduced figures.
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Days of Experiment
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Figure 2.1: Urease activity over the time of experiment. The dilution rate was
0.035 h-1 from day 1 to day 8, and 0.075 h-1 from day 8 to day 11.
pH
Urease Activity:
During the experiment time the urease activity went up and down as can be
seen in figure 2.1. On day 3 and day 8, which were after the weekends (when
the feed and harvest pumps were turned off), it was observed that the urease
activity dropped. Day 3 and 8 had the lowest recorded urease activity
throughout the experiment. After both decreases on day 3 and 8 the activity
increased again steadily. In the case of the day 8, in order to increase the
urease activity, it was decided to double the dilution rate (from 0.035 to 0.075
h-1). As can be seen on the graph 1, the urease activity increased after day 8.
10.2
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Days of Experiment
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Figure 2.2: pH over the days of Experiment.
Figure 2.2 shows the pH for the duration of the experiment. As can be seen
the pH started lower than required, around 9. On day 5 the pH increased to
the appropriate level of 10 and remained here for the duration of the
experiment.
Optical Density:
Another measurement taken was the optical density (OD 600), which was
used to measure the concentration of bacterial cells. From the value obtained
of OD the biomass was calculate by the following equation:
C (biomass concentration, g/L) = 0.44 x OD (600mn)
Biomass Productivity (g/L/h)
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Days of Experiment
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Figure 2.3: Biomass productivity over the time of experiment. The dilution rate
was 0.035 h-1 from day 1 to day 8, and 0.075 h-1 from day 8 to day 11. The
biomass was calculated from relation of biomass and dilution rate.
Figure 2.3 shows the biomass productivity. As can been seen biomass
productivity changed slightly between day 1 and day 8; however after
doubling the dilution rate (from day 8) the biomass productivity doubled as
was expected.
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D.O (mg/L)
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Days of Experiment
D.O (mg/L)
Figure 2.4: Dissolved Oxygen.
Dissolved Oxygen (D.O)
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Figure 2.4 shows the dissolved oxygen concentration for the duration of the
experiment. During the experiment the measurement of oxygen was not as
expected. However it was found the oxygen probe not work properly and
almost every time the oxygen concentration was measured, it was not
reaching a stable value. Once a new oxygen meter was acquired more stable
and suitable oxygen concentrations were recorded.
(Your results section has compiled all findings in figures and then displayed
the figures without demonstrating intent. You don’t say what the purpose was
of particular periods of the chemostat operation, what parameters were
varied and what the effect was of changing certain parameters.)
Discussion:
As an ureolytic organism, B. pasteurii has the ability to produce urease
enzymes when urea is given as an energy source (Sarda et al., 2009). Urease
is produced in order to hydrolyse urea for ATP production for microbial
growth. The increase in both urease and biomass productivity shown in
figures 2.1 and 2.3 confirmed that urease was readily being produced and as
a result, more biomass was also produced. (Correct. You were able to
continuously run the chemostat. What about the key objective of the
experiment to improve the urease production rate (productivity)?)
It is important to note that B. pasteurii is a spore-forming organism, which
sporulates under stressful conditions for survival (Prescott et al., 2005). As
recorded on Day 3, which was followed after a weekend of non-feeding, it is
possible that the microorganisms had sporulated under such stressful
condition. In its spore form, urease is not produced. This theory could explain
the massive drop in urease activity but not in the biomass level shown if
figures 2.1 and 2.3, respectively. (Could be correct but it is speculative as you
have not evidence to back up this claim).
The dilution rate was set to 0.035 h-1 on Day 1 to 8, then increased to 0.075
h-1 on Day 9 onwards by altering the pumps on/off times. By increasing the
dilution rate, it is only natural that productivity increased according to the
equation - “ Productivity = Dilution Rate * Biomass Level”. The results shown
in figures 2.1 and 2.3 confirmed the increase in productivity after the change
in dilution rate after Day 9. (This could be a key part of your report (trying to
increase productivity). However in this particular case you don’t want to
produce bacteria (OD) as that may well be contaminants that overgrow your
producing strain, but you want to produce the enzyme.)
B. pasteurii is an alkalophile and its optimum pH is around 10 (Wiley &
Stokes, 1962). Controlling the pH to around 10 with the help of LabView, f
high pH-tolerant bacterial strains can grow. This restricts the growth of
unwanted strains, which is especially important in open (non-sterile) system.
(So did you change the pH ? Did you feel there was contaminants growing as
well for example by dividing the enzyme activity by biomass to obtain specific
activity?)
Several problems were encountered during the eleven-day span of this
experiment. The biggest problem was the technical issue with the computer
software, where the pump would be stopped for many hours, sometimes all
night before it was fixed. This irregularity of feeding and harvesting surely had
impacts on the biomass and usearse production of the B. pasteurii culture.
Other problems include the malfunction of the oxygen probe and the wrong
set up for pH pump. As seen in figure 2.2, pH reached 8.95 on Day 1, due to
no sodium hydroxide being pumped into the system. (What effect did you
expect this to have and did it have the expected effect?) The pump was found
to have a tube diameter too small for the pump. This meant that although it
was registering a lower pH thean required and was pumping, no sodium
hydroxide was reaching the culture vessel. These issues contributed to the
fluctuation in results.
Overall, the continuous open (non-sterile) chemostat proved to be effective in
growing a selective strain, if grown under with the suitable condition. It is
useful and economical in industrial processes that do not require sterility such
as biogas digestion and wine making (Cheng & Cord-Ruwisch, 2013). Under
the optimum condition, high productivity can be achieved with low cost.
For future replication of this experiment, a longer period of experiment is
highly recommended. Factors such as concentration of urea in feed,
optimizing temperature and pH should be altered in order to optimize the
experiment.
(Good and clear writing. Understanding seems also good. There was actually
no real test run or hypothesis tested in your project. Admittedly equipment
malfunction can cause problems but effects caused by malfunction can still be
attempted to be explained using the background understanding. For example
did you think you have contaminants present (no specific enzyme activity data
shown (enzyme activity/ biomass present). 7/10
References:
Bang SS., Galinat JK and Ramakrishnan V. (2000) Calcite precipitation
induced by polyurethane-immobilised Bacillus pasteurii, Enzyme and
Microbial Technology 28, 404-409
Cheng, L., & Cord-Ruwisch, R. (2013). Selective enrichment and production
of highly urease active bacteria by non-sterile (open) chemostat culture.
Journal Of Industrial Microbiology \& Biotechnology, 40(10), 1095--1104.
Jahns T. (1995). Ammonium/Urea-Dependant Generation of a Proton
Electrochemical Potential and Synthesis of APT in Bacillus pasteurii, Journal
of Bacteriology 178, 403-409
Mobley HLT and Hausinger RP. (1989). Microbial Ureases: Significance,
Regulation and Molecular Characterisation, Microbiological Reviews 53, 85108
Najafpour, G.D. (2007). ‘Biochemical Engeneering and Biotechnology’.
Elsevier. Amsterdam, pp 84-86
Padan E., Bibi E., Ito M and Krulwich A. (2005). Alkaline pH homeostasis in
bacteria: New insights, Biochemica et Biophysica Acta 1717, 67-68
Prescott, M. P., Harley, J. P., Klein, D. A., (1999).
Edition, McGraw Hill, USA
Microbiology Fourth
Sarda, D., Choonia, H., Sarode, D., & Lele, S. (2009). Biocalcification by
Bacillus pasteurii urease: a novel application. Journal Of Industrial
Microbiology & Biotechnology, 36(8), 1111--1115.
Wiley, W., & Stokes, J. (1962). Requirement of an alkaline pH and ammonia
for substrate oxidation by Bacillus pasteurii. Journal Of Bacteriology, 84(4),
730--734.