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Should have more:
Structure,
Clarity in writing
Details in explanations
Evidence of understanding
rease activity !y Sporasarcina pasteurii
By Group 3 & 4
" #!stract
(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
$ %ntroduction

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. s a natural formation of erosion and
sedimentation by water flow it has formed many natural landscapes. !he
dykes for instance in "olland 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 e#pressed in utili$ing bacteria as a countermeasure
(%ord&'uwisch ())*).
Sporosarcina pasteurii is a useful alkaphilic bacteria which en$ymatically
digests urea to produce ammonia via the action of urease a nickel containing
en$yme (+arplus et. al. ,--.). !he result of this reaction is two fold/ ,) it can
raise the p" making its environment more basic by the influence of ammonia
and () it can produce carbonate. !his can be summed up by the following
e0uation/
(1"
(
)
(
%2 3 "
(
2 4 %2
5
&(
3 (1"
6
3
7rea 3 8ater 4 carbonate 3 ( moles ammonia
&igure ": 90uation of the breakdown of urea by urease en$yme into carbonate
and ammonia (8hiffin et. al. ())6).
!he increase in p" also has a synergistic effect as the precipitation of %a
(3
and
%2
5
&(
and its calcification can be enhanced by a more basic p". :rincipally the
carbonate produced can react with calcium provided or in the soil to form
hardened limestone.
In order to function effectively urease re0uires the presence of nickel ions to
act as a cofactor. 9ach molecule of urease re0uires ( nickel ions to work
effectively.
2ur aim was to culture the bacteria in a bioreactor under septic conditions to
simulate the conditions the bacteria is intended to be used in. !o achieve
successful calcification a specific activity of (mM urea min
&,

was set as the
goal under septic chemostat conditions. :revious e#periments showed that S.
pPasteurii grew optimally at p" -.(; based on its ideal p+a value illustrated
by this graph add source.
&igure $: %hemical speciation of ammonia (empty dots) and ammonium (black dots)
in relation to p".
!he p+a value was found to be p" -.(; which is the point of half the dissociation
constant for the 1"
5
<1"
6
3
e0uilibrium. !his is the state where the concentration of
ammonia and ammonium created from the activity of urease within the cell are at
e0uilibrium causing an e#ternal p" of -.(; which is the measured p". !his is
illustrated by this diagram/
&igure ': 7rea hydrolysis and !:
generation intracellularly in S.
pasteurii. (=ahns et. al. ,---)
It is clear from this that the growth optimum and !:&production optimum are the
same which re0uire an e0ual amount of 1"
5
and 1"
6
3
to be present (8hiffen et. al.
())6).
2ur aim was to set the conditions of the reactor such that the cultured S. Pasteurii
would grow nominally while decominating the reactor. 8e 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.
' (aterials ) (ethod
5., !rial ,
5.,., >acteria strain and cultivation
n ali0uot of ;) mL of S. pasteurii (from %hen Liang, Murdoch 7niversity,
8estern ustralia) was cultured in ;)) mL rich medium with () g<L yeast
e#tract, ,.) mM ammonium sulphate and )., mM 1i%l. p" was adjusted to -
using 1a2". >acterium was grown as batch culture for (6 hours at 5)?% in a
shaking water bath.
5.,.( %hemostat setup
!he entire batch culture of ;)) mL was transferred to the chemostat reactor.
!he batch culture medium was used as the feed medium. (L glass reactor
was put into the water bath at the temperature of 5)?%. stirrer set at (;)
rpm. 2ne %hemaster
!M
peristaltic pump set to turn on and off with a timer was
used to pump the feed medium into the reactor. nother pump and timer was
used to pump product out of the reactor into a collection bottle. >oth timers
were set and synchronised to switch on for ( seconds and switched off for 5
minutes. sparger set at (;) 0
n
L<h was used to aerate the system.
5.,.5 @ampling procedure
5.,.5., Aissolved o#ygen
2#ygen probe was used to measure the o#ygen concentration and temperature
in the reactor.
5.,.5.( p"
!he outflow was measured using "anna instruments "I *6(6 p" meter.
5.,.5.5 %onductivity
,/,, dilution was done by adding 6 mL of sample into 6) mL of ,.; M
urease solution. %onductivity was measured by using "anna Instruments
"I*.55. 'eadings of conductivity (m@) was taken at intervals of () seconds
for 5) minutes. !he urease activity (mM urea hydrolysed min&,) was
calculated for each sample, using the conversion factor of ,,.,, m@ min&, 4 ,
mM urea hydrolysed min&, (@alwaBs thesis).
5.,.5.6 2ptical Aensity
!he sample with urease solution was taken after conductivity test, for the
absorbance measured at C))nm in a spectrophotometer.
5.,.5.; Ary %ell Mass
,.; mL of outflow product was centrifuged. @upernatant was discarded and
pellet was left to dry in a 5.?% oven overnight. !he dry cell mass was weighed
the ne#t day.
5.( !rial (
5.(., >acteria strain and cultivation
n ali0uot of ;) mL of a new batch of S. pasteurii (from %hen Liang,
Murdoch 7niversity, 8estern ustralia) was cultured in ;)) mL rich medium
with () g<L yeast e#tract, ,)) mM ammonium sulphate, )., mM 1i%l and ,))
mM urea. p" was adjusted to - using 1a2". >acterium was grown as batch
culture for (6 hours at 5)?% in a shaking water bath.
5.(.( %hemostat setup
!he entire batch culture of (;) mL was transferred to the chemostat reactor.
!he batch culture medium was used as the feed medium. !he p" was raised to
-.(; by adding 1a2". (L glass reactor was put into the water bath at the
temperature of 5)?%. stirrer set at 5)) rpm and the stirring rate was
increased to ;)) rpm on day .. 2ne %hemaster
!M
peristaltic pump set to turn
on and off with a timer was used to pump the feed medium into the reactor.
nother pump and timer was used to pump product out of the reactor into a
collection bottle. >oth timers were set and synchronised to switch on for (
seconds and switched off for 5 minutes. sparger set at (;) 0
n
L<h was used to
aerate the system.
5.(.5 @ampling procedure
5.(.5., Aissolved o#ygen
2#ygen probe was used to measure the o#ygen concentration and temperature
in the reactor.
5.(.5.( p"
!he outflow was measured using "anna instruments "I *6(6 p" meter.
5.(.5.5 %onductivity
,/,; dilution was done by adding 6 mL of sample into ;C mL of ,.; M
urease solution. %onductivity was measured by using "anna Instruments
"I*.55. 'eadings of conductivity (m@) was taken at intervals of () seconds
for 5) minutes. !he urease activity (mM urea hydrolysed min&,) was
calculated for each sample, using the conversion factor of ,,.,, m@ min&, 4 ,
mM urea hydrolysed min&, (@alwaBs thesis).
5.(.5.6 2ptical Aensity
!he sample with urease solution was taken after conductivity test, for the
absorbance measured at C))nm in a spectrophotometer.
5.(.5.; Ary %ell Mass
,.; mL of outflow product was centrifuged. @upernatant was discarded and
pellet was left to dry in a 5.?% oven overnight. !he dry cell mass was weighed
the ne#t day.
* +esults
6., !rial ,
Optical Density
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
3 4 5 6 7 8 9
Experiment Day
O
D

(
a
t

6
0
0
n
m
)
Dig.,. %hanges in the 2A
!he 2A of the @. pasteurii culture was used to measure the concentration of
bacterial cells. !he concentration of bacterial cells peaked at Aay 6 before
falling at Aay ; and remained more or less constant for the rest of the days.
Specific Urease Activity
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 3 5 7 9
Experiment Day
S
p
e
c
i
f
i
c

U
r
e
a
s
e

A
c
t
i
v
i
t
y


(
m
M

u
r
e
a

m
i
n
-
1
/
O
D
)
Dig.(. @pecific 7rease activity (mM urea hydrolysed< min< 2A)
!he specific urease activity (@7) is an inde# of the efficiency of urease
production by @. pasteurii. !he @7 peaked at Aay ( before falling at Aay 5.
It increased again at Aay 6 before decreasing for the rest of the days.
Productivity
0.000000
0.001000
0.002000
0.003000
0.004000
0.005000
0.006000
3 4 5 6 7 8 9
Experiment Day
P
r

!
u
c
t
i
v
i
t
y

(
"
/
#
/
$
)
Dig.5. :roductivity of urease over the days of e#periment
!he productivity of urea was increasing steadily at a slow rate over days 5 to
;. !here was a steep increase from day ; onwards.
Oxygen Uptake Rate (OUR)
27' was calculated from the dissolved o#ygen. !he 27' obtained from the
e#periment was decreasing, however, the trend of 27' cannot be determined
as there are too few data points.
!he kLa obtained was 0.0626 h
-1
. find out whats the significance of kla
pH
7.6
7.7
7.8
7.9
8
8.1
8.2
8.3
8.4
3 4 5 6 7 8 9
Experiment Day
p
%

f

O
u
t
f
&

'
Dig.6. %hange of p" over days of e#periment
!he drop of p" at Aay 6 resulted in poor productivity. !he increasing p" from
day 6 onwards resulted in an increased productivity as the bacteria thrive at a
p" of -.(; (8hiffin, ())6). !he change of the feed p" from - to -.; resulted
in the increase in p".
6.( !rial (
Optical Density
0
1
2
3
4
5
6
7
0 2 4 6 8 10 12
Experiment !ay
O
D

(
a
t

6
0
0

n
m
)
Dig.,. %hanges in the 2A
2A increased in Aay ; because the feed pumps were increased hence more
feed was given to @. pasteurii. s a result, higher growth was obtained. fter
sampling on day ., the stirring rate was changed from 5)) rpm to ;)) rpm,
resulted in a rise in 2A. fter which, 2A dropped slightly.
Specific Urease Activity
0.00000
0.20000
0.40000
0.60000
0.80000
1.00000
1.20000
1.40000
0 2 4 6 8 10 12
Experiment Day
S
p
e
c
i
f
i
c

U
r
e
a
s
e

A
c
t
i
v
i
t
y

(
m
M

u
r
e
a

m
i
n
-
1
/
O
D
)
!he specific urease activity was obtained using urease activity obtained from the
conductivity test and divided by the 2A. 8hat is the units and relevanceEE
;&. high 2A was achievedF this meant tht there was high bacteria concentration.
Aay 6Gdrastic drop from day ,&6 because not enough feed pumped into the reactor
for bacteria growth resulting in lesser urease production.
Aay CGoutflow was higher than inflow resulting in washout of bacteria. "ence @.
pasteurii in the outflow therefore when the conductivity test was conducted, there was
not enough urea in the solution for the bacteria to degrade.
!oo much bacteria cells.unlikely to have contaminitiaon as ph is high..
Productivity
0
0.5
1
1.5
2
2.5
3
3.5
0 2 4 6 8 10 12
Experiment Day
P
r

!
u
c
t
i
v
i
t
y

(
"
/
#
/
$
)
Ailution # biomass 4 productivity
1oted that dilution rate was increasing from Aay 6 to Aay . however, dry cell mass
collected decreased during that period.
Aay 6Gdrastic drop from day ,&6 because not enough feed pumped into the reactor
for bacteria growth resulting in lesser urease production.
Aay CGoutflow was higher than inflow resulting in washout of bacteria..
Oxygen Uptake Rate (OUR)
pH
8.2
8.4
8.6
8.8
9
9.2
9.4
9.6
0 2 4 6 8 10 12
Experiment Day
p
%

u
t
f
&

'
, Discussion
2ur studies showed that during e#perimentation the activity decreased in
reactor conditions due to contamination conse0uently we attempted to
maintain high p" conditions and a high dilution rate.
!o achieve this p" we provided e#tremely high ammonia and urea
concentrations in the feed medium of ,))mM ammonium sulphate and
;))mM dissolved urea. 8e also attempted to maintain p" directly by using ,)
M concentrated 1a2" solution as the p" would acidify when e#posed to
o#ygen. 8e needed to continually aerate the culture by direct bubbling and
stirring to provide o#ygen for the bacteria.
(still under construction)
- +ecommendations
,) @tarting early is important. !he reactor can thankfully be restarted within (6
hours as new culture can be grow in less than (6 hours and inoculated into a
clean bioreactor.
() !o maintain high p" and reactor sterility with only the chosen bacterial strain
use feed medium containing ;)) mM urea.
5) 9nsure that amount of feed media is able to last the weekend. 'eduction of
reactor volume and starvation periods can hurt the bacteria.
6) Make sure when using the pumps that the same type of tubes with the same
diameters are used. @ome 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. lso try to have
sufficient reactor volume to distance the bubbling from the outflow tube, push
the tube as far down as possible this helps. >e warned this can result in
complete reactor drainage if the inflow fails. dd 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.
;) !ry to set up a periodic dripper using a small syringe with a needle to
continually add anti&foam agent. !his is needed for e#tended periods when the
chemostat is not observed like over the weekend as outflow removes the anti&
foam agent. 8ithout it foaming occurs and reactor volume is 0uickly lost.
. Conclusion
/ +eferences
Ralf ord!Ru"isc#. :ersonal %ommunication and dvice ())*.
$. %a#ns. mmonium<urea&dependant generation of a proton electrochemical
potential and synthesis of !: in &acillus Pasteurii. =ournal of >iotechnology
,.* pg. 6)5 &6)-. ,---.
P.A. 'arplus( ).A. Paerson. .) years of crystalline urease/ 8hat have we
learned. cc. %hem. 'es. Issue 5) pg. 55) H 55..
*ictoria S. +#iffin. Microbial %a%2
5
precipitation for the production of
biocement. !hesis paper for the @chool of >iological @ciences I
>iotechnology of Murdoch 7niversity ())6.
0 #ppendices
!rial ,
Oxygen Uptake Rate (OUR)
verage temperature, (*..;?%
c@ 4 6C* J ((*..; 35,.C)
4 ...; mg<L
kLa 4 ).)6C, h
&,
y 0.0461! - 0.2277
-0.04
-0.02
0
0.02
0.04
0.06
0.08
0.1
0 2 4 6 8
cS-c# (m"/#)
O
U
(

(
m
"
/
#
/
$
)
!rial (
27'
verage temperature 4 (-.C
c@ 4 6C* < ((-.C 3 5,.C)
4 ..C; mg<L
kLa 4 ).),.C h
&,
y 0.0176! - 0.1142
-0.15
-0.1
-0.05
0
0.05
0.1
0 1 2 3 4 5 6 7 8
cS-c# (m"/#)
O
U
(

(
m
"
/
#
/
$
)