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Urease activity by Sporasarcina pasteurii
By Group 3 & 4
1
Abstract
(not done yet)
Make sure you cover in the abstract the background, goal, what was done and most of
all what was found. It should be understandable without having read the background
to the project
2
Introduction
Limestone or calcite is a natural formation found in rocks and in many
instances it acts as a form of natural cement which holds together rock and
sand in a composite structure. As a natural formation of erosion and
sedimentation by water flow it has formed many natural landscapes. The
dykes for instance in Holland which keeps it from being flooded due to the
country being below sea level is dependent on limestone which is beginning to
erode. Interest has been expressed in utilizing bacteria as a countermeasure
(Cord-Ruwisch 2008).
Sporosarcina pasteurii is a useful alkaphilic bacteria which enzymatically
digests urea to produce ammonia via the action of urease a nickel containing
enzyme (Karplus et. al. 1997). The result of this reaction is two fold: 1) it can
raise the pH making its environment more basic by the influence of ammonia
and 2) it can produce carbonate. This can be summed up by the following
equation:
(NH2)2CO + H2O = CO3-2 + 2NH4+
Urea + Water = carbonate + 2 moles ammonia
Figure 1: Equation of the breakdown of urea by urease enzyme into carbonate
and ammonia (Whiffin et. al. 2004).
The increase in pH also has a synergistic effect as the precipitation of Ca2+ and
CO3-2 and its calcification can be enhanced by a more basic pH. Principally the
carbonate produced can react with calcium provided or in the soil to form
hardened limestone.
In order to function effectively urease requires the presence of nickel ions to
act as a cofactor. Each molecule of urease requires 2 nickel ions to work
effectively.
Our aim was to culture the bacteria in a bioreactor under septic conditions to
simulate the conditions the bacteria is intended to be used in. To achieve
successful calcification a specific activity of 2mM urea min-1 was set as the
goal under septic chemostat conditions. Previous experiments showed that S.
pasteurii grew optimally at pH 9.25 based on its ideal pKa value illustrated by
this graph add source.
Figure 2: Chemical speciation of ammonia (empty dots) and ammonium (black dots)
in relation to pH.
The pKa value was found to be pH 9.25 which is the point of half the dissociation
constant for the NH3/NH4+ equilibrium. This is the state where the concentration of
ammonia and ammonium created from the activity of urease within the cell are at
equilibrium causing an external pH of 9.25 which is the measured pH. This is
illustrated by this diagram:
Figure 3: Urea hydrolysis and ATP
generation intracellularly in S.
pasteurii. (Jahns et. al. 1999)
It is clear from this that the growth optimum and ATP-production optimum are the
same which require an equal amount of NH3 and NH4+ to be present (Whiffen et. al.
2004).
Our aim was to set the conditions of the reactor such that the cultured S. Pasteurii
would grow nominally while decominating the reactor. We then intended to study the
urease activity by using conductivity tests, biomass by optical density and dry cell
weight and determining the specific activity from these results.
3
Materials & Method
3.1
Trial 1
3.1.1 Bacteria strain and cultivation
An aliquot of 50 mL of S. pasteurii (from Chen Liang, Murdoch University,
Western Australia) was cultured in 500 mL rich medium with 20 g/L yeast
extract, 170 mM ammonium sulphate and 0.1 mM NiCl. pH was adjusted to 9
using NaOH. Bacterium was grown as batch culture for 24 hours at 30°C in a
shaking water bath.
3.1.2 Chemostat setup
The entire batch culture of 500 mL was transferred to the chemostat reactor.
The batch culture medium was used as the feed medium. A 2L glass reactor
was put into the water bath at the temperature of 30°C. A stirrer set at 250
rpm. One Chemaster TM peristaltic pump set to turn on and off with a timer
was used to pump the feed medium into the reactor. Another pump and timer
was used to pump product out of the reactor into a collection bottle. Both
timers were set and synchronised to switch on for 2 seconds and switched off
for 3 minutes. A sparger set at 250 qnL/h was used to aerate the system.
3.1.3 Sampling procedure
3.1.3.1 Dissolved oxygen
Oxygen probe was used to measure the oxygen concentration and temperature
in the reactor.
3.1.3.2 pH
The outflow was measured using Hanna instruments HI 8424 pH meter.
3.1.3.3 Conductivity
A 1:11 dilution was done by adding 4 mL of sample into 40 mL of 1.5 M
urease solution. Conductivity was measured by using Hanna Instruments
HI8733. Readings of conductivity (mS) was taken at intervals of 20 seconds
for 30 minutes. The urease activity (mM urea hydrolysed min-1) was
calculated for each sample, using the conversion factor of 11.11 mS min-1 = 1
mM urea hydrolysed min-1 (Salwa’s thesis).
3.1.3.4 Optical Density
The sample with urease solution was taken after conductivity test, for the
absorbance measured at 600nm in a spectrophotometer.
3.1.3.5 Dry Cell Mass
1.5 mL of outflow product was centrifuged. Supernatant was discarded and
pellet was left to dry in a 37°C oven overnight. The dry cell mass was weighed
the next day.
3.2
Trial 2
3.2.1 Bacteria strain and cultivation
An aliquot of 50 mL of a new batch of S. pasteurii (from Chen Liang,
Murdoch University, Western Australia) was cultured in 500 mL rich medium
with 20 g/L yeast extract, 100 mM ammonium sulphate, 0.1 mM NiCl and 100
mM urea. pH was adjusted to 9 using NaOH. Bacterium was grown as batch
culture for 24 hours at 30°C in a shaking water bath.
3.2.2 Chemostat setup
The entire batch culture of 250 mL was transferred to the chemostat reactor.
The batch culture medium was used as the feed medium. The pH was raised to
9.25 by adding NaOH. A 2L glass reactor was put into the water bath at the
temperature of 30°C. A stirrer set at 300 rpm and the stirring rate was
increased to 500 rpm on day 7. One Chemaster TM peristaltic pump set to turn
on and off with a timer was used to pump the feed medium into the reactor.
Another pump and timer was used to pump product out of the reactor into a
collection bottle. Both timers were set and synchronised to switch on for 2
seconds and switched off for 3 minutes. A sparger set at 250 qnL/h was used to
aerate the system.
3.2.3 Sampling procedure
3.2.3.1 Dissolved oxygen
Oxygen probe was used to measure the oxygen concentration and temperature
in the reactor.
3.2.3.2 pH
The outflow was measured using Hanna instruments HI 8424 pH meter.
3.2.3.3 Conductivity
A 1:15 dilution was done by adding 4 mL of sample into 56 mL of 1.5 M
urease solution. Conductivity was measured by using Hanna Instruments
HI8733. Readings of conductivity (mS) was taken at intervals of 20 seconds
for 30 minutes. The urease activity (mM urea hydrolysed min-1) was
calculated for each sample, using the conversion factor of 11.11 mS min-1 = 1
mM urea hydrolysed min-1 (Salwa’s thesis).
3.2.3.4 Optical Density
The sample with urease solution was taken after conductivity test, for the
absorbance measured at 600nm in a spectrophotometer.
3.2.3.5 Dry Cell Mass
1.5 mL of outflow product was centrifuged. Supernatant was discarded and
pellet was left to dry in a 37°C oven overnight. The dry cell mass was weighed
the next day.
4 Results
4.1
Trial 1
Optical Density
0.8
OD (at 600nm)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
3
4
5
6
7
8
9
Experiment Day
Fig.1. Changes in the OD
The OD of the S. pasteurii culture was used to measure the concentration of
bacterial cells. The concentration of bacterial cells peaked at Day 4 before
falling at Day 5 and remained more or less constant for the rest of the days.
Specific Urease Activity (mM urea min1/OD)
Specific Urease Activity
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
3
5
7
9
Experiment Day
Fig.2. Specific Urease activity (mM urea hydrolysed/ min/ OD)
The specific urease activity (SUA) is an index of the efficiency of urease
production by S. pasteurii. The SUA peaked at Day 2 before falling at Day 3.
It increased again at Day 4 before decreasing for the rest of the days.
Productivity
0.006000
Productivity (g/L/h)
0.005000
0.004000
0.003000
0.002000
0.001000
0.000000
3
4
5
6
7
8
9
Experiment Day
Fig.3. Productivity of urease over the days of experiment
The productivity of urea was increasing steadily at a slow rate over days 3 to
5. There was a steep increase from day 5 onwards.
Oxygen Uptake Rate (OUR)
OUR was calculated from the dissolved oxygen. The OUR obtained from the
experiment was decreasing, however, the trend of OUR cannot be determined
as there are too few data points.
The kLa obtained was 0.0626 h-1. find out what’s the significance of kla
pH
8.4
8.3
pH of Outflow
8.2
8.1
8
7.9
7.8
7.7
7.6
3
4
5
6
Experiment Day
Fig.4. Change of pH over days of experiment
7
8
9
The drop of pH at Day 4 resulted in poor productivity. The increasing pH from
day 4 onwards resulted in an increased productivity as the bacteria thrive at a
pH of 9.25 (Whiffin, 2004). The change of the feed pH from 9 to 9.5 resulted
in the increase in pH.
Trial 2
Optical Density
7
6
OD (at 600 nm)
5
4
3
2
1
0
0
2
4
6
8
10
12
Experiment day
Fig.1. Changes in the OD
OD increased in Day 5 because the feed pumps were increased hence more
feed was given to S. pasteurii. As a result, higher growth was obtained. After
sampling on day 7, the stirring rate was changed from 300 rpm to 500 rpm,
resulted in a rise in OD. After which, OD dropped slightly.
Specific Urease Activity
Specific Urease Activity (mM urea
min-1/OD)
4.2
1.40000
1.20000
1.00000
0.80000
0.60000
0.40000
0.20000
0.00000
0
2
4
6
Experiment Day
8
10
12
The specific urease activity was obtained using urease activity obtained from the
conductivity test and divided by the OD. What is the units and relevance??
5-7 high OD was achieved; this meant tht there was high bacteria concentration.
Day 4…drastic drop from day 1-4 because not enough feed pumped into the reactor
for bacteria growth resulting in lesser urease production.
Day 6…outflow was higher than inflow resulting in washout of bacteria. Hence S.
pasteurii in the outflow therefore when the conductivity test was conducted, there was
not enough urea in the solution for the bacteria to degrade.
Too much bacteria cells.unlikely to have contaminitiaon as ph is high..
Productivity
3.5
Productivity (g/L/h)
3
2.5
2
1.5
1
0.5
0
0
2
4
6
8
10
12
Experiment Day
Dilution x biomass = productivity
Noted that dilution rate was increasing from Day 4 to Day 7 however, dry cell mass
collected decreased during that period.
Day 4…drastic drop from day 1-4 because not enough feed pumped into the reactor
for bacteria growth resulting in lesser urease production.
Day 6…outflow was higher than inflow resulting in washout of bacteria..
Oxygen Uptake Rate (OUR)
pH
9.6
pH of outflow
9.4
9.2
9
8.8
8.6
8.4
8.2
0
2
4
6
8
10
12
Experiment Day
5
Discussion
Our studies showed that during experimentation the activity decreased in
reactor conditions due to contamination consequently we attempted to
maintain high pH conditions and a high dilution rate.
To achieve this pH we provided extremely high ammonia and urea
concentrations in the feed medium of 100mM ammonium sulphate and
500mM dissolved urea. We also attempted to maintain pH directly by using 10
M concentrated NaOH solution as the pH would acidify when exposed to
oxygen. We needed to continually aerate the culture by direct bubbling and
stirring to provide oxygen for the bacteria.
(still under construction)
6
Recommendations
1) Starting early is important. The reactor can thankfully be restarted within 24
hours as new culture can be grow in less than 24 hours and inoculated into a
clean bioreactor.
2) To maintain high pH and reactor sterility with only the chosen bacterial strain
use feed medium containing 500 mM urea.
3) Ensure that amount of feed media is able to last the weekend. Reduction of
reactor volume and starvation periods can hurt the bacteria.
4) Make sure when using the pumps that the same type of tubes with the same
diameters are used. Some pumps have two sets of tubes with different
diameters causing vastly different inflow and outflow rates lowering or raising
the reactor volume rather than keeping them constant. Also try to have
sufficient reactor volume to distance the bubbling from the outflow tube, push
the tube as far down as possible this helps. Be warned this can result in
complete reactor drainage if the inflow fails. Add a small syringe tube to try
and limit the build up bubbles in the outflow tube, this causes a reduction in
outflow raising the reactor volume.
5) Try to set up a periodic dripper using a small syringe with a needle to
continually add anti-foam agent. This is needed for extended periods when the
chemostat is not observed like over the weekend as outflow removes the antifoam agent. Without it foaming occurs and reactor volume is quickly lost.
7
Conclusion
8
References
Ralf Cord-Ruwisch. Personal Communication and Advice 2008.
T. Jahns. Ammonium/urea-dependant generation of a proton electrochemical
potential and synthesis of ATP in Bacillus Pasteurii. Journal of Biotechnology
178 pg. 403 -409. 1999.
P.A. Karplus, M.A. Paerson. 70 years of crystalline urease: What have we
learned. Acc. Chem. Res. Issue 30 pg. 330 – 337.
Victoria S. Whiffin. Microbial CaCO3 precipitation for the production of
biocement. Thesis paper for the School of Biological Sciences &
Biotechnology of Murdoch University 2004.
9
Appendices
Trial 1
Oxygen Uptake Rate (OUR)
Average temperature, 28.75°C
cS = 468 ÷ (28.75 +31.6)
= 7.75 mg/L
kLa = 0.0461 h-1
0.1
y = 0.0461x - 0.2277
OUR (mg/L/h)
0.08
0.06
0.04
0.02
0
-0.02
0
2
4
-0.04
cS-cL (mg/L)
6
8
Trial 2
OUR
Average temperature = 29.6
cS
= 468 / (29.6 + 31.6)
= 7.65 mg/L
kLa = 0.0176 h-1
0.1
OUR (mg/L/h)
0.05
y = 0.0176x - 0.1142
0
0
1
2
3
4
-0.05
-0.1
-0.15
cS-cL (mg/L)
5
6
7
8