The continued process of a chemostat for optimized growth
of Sporasarcina pasteurii, comparison to a batch culture, and
promotion of biocementation function through increased
urease function
Authors
Jack Weatherhead 32210638
Susan McPhee 31865152
Rachel Brewer 31853317
Tan Nguyen 32226147
Abstract
Construction Microbial Biotechnology, the production of ureolytic bacteria for the
use in biocementation is a relatively new technology being studied today. The method
of production using a selective growth medium to allow ureolytic bacteria
Sporasarcina pasteurii. To grow and proliferate in a bioreactor via an open system
chemostat. Whilst controlling and maintaining via a software computer program, the
chemostat was observed and changes in enzyme activity under various conditions,
such as airflow rate, stirrer rate, dilution rate and retention time at a constant
temperature of 28oC and pH of 10 together with batch like culture implementation
during the chemostat running of 15 days were tested for levels of enzyme
activity. High enzyme activity was achieved and maintained with its harvest proving
successful in producing a rock like sample through the implementation of a
biocementation method.
1
Introduction
Bioreactors are incredible living reactors that combine aspects of engineering, biology
and chemistry, used in many industries globally. Industries use bioreactors for
treatment for disposal of wastewater, in medicine for production of antibiotics such as
penicillin (Pirt et al., 1967), for production of biofuels (Temudo et al., 2007), as well
as in beer-making (Tata et al., 1999). These reactors can be sterile or non sterile
environments dependent on the product required, in some industries such as beer
brewing (Tata et al., 1999) a non-sterile environment is suitable; whereas in medical
bioprocesses (Pirt and Righelato, 1967) a sterile environment is required (Cheng et al.,
2013). There are many types of bioreactors, in this report there will be a focus on
chemostat and a fed-batch reactor.
The conditions of a batch culture change over time (Meyer et al., 1985). in conditions
that require different parameters, it is therefore difficult to automate. As conditions
change in a batch culture, the concentrations of solutes in the solution changes and
product build up inevitably occurs which can lead to product inhibition (Novick et al.,
1950). As no solution is either added or removed, the batch culture relies on time and
therefore does not run continually, leading to a definitive end where a condition
prevents further change. Thus, the batch culture experiences the phases of growth that
lead to its variable growth rates and an overall decreased productivity when compared
to the chemostat which is fixed in the exponential phase of growth giving a constant
high productivity (Stanbury, 2013). However, as the batch culture goes through
multiple phases of growth, it is possible to synthesise secondary metabolites in the
stationary phase within a batch culture, which is not possible in a chemostat, as the
microbes are fixed in the exponential phase (Meyer et al., 1985).
2
A chemostat is an apparatus used to cultivate bacteria in continuous and constant
conditions in order to produce a specific primary product; such as in the production of
some pharmaceuticals (Whiteley, 1997).
Alternatively, the chemostat can be used to study microbes where specific
enviromental conditions can be maintained, allowing such appropriate studies
(Whiteley, 1997). A chemostat is a continuous bioprocessor with static conditions
which include dissolved oxygen (ppm), pH, temperature, cell density, and nutrient
substrate; this steady state is achieved by maintaining inflow and outflow at constant
volume (Novick et al., 1950). Specific conditions can be set and adjusted to suit the
micro-organism of interest at optimum growth, but result in harsher conditions
(>9.5pH) to keep undesired contaminants at (Novick and Szilard, 1950). In order to
maintain the media concentrations lost from the catabolism by the microbes, a
constant volumetric flow of fresh medium enters the culture vessel, driven by a pump
at a constant rate; termed the inflow (Makri et al., 2010). With simultaneous removal
of the homogenous culture removed from the vessel at the same volume and rate in
order to collect the product and remove the equivalent volume added by the inflow
pump; termed the outflow (Makri et al., 2010). The chemostat facilitates automation
and is therefore regulated by a computer program which maintains the inflow pumps
and regulatory pumps at a constant rate. The chemostat will eventually reach steady
state where biomass concentrations remain constant at a given concentration; the
concentration at which the biomass reaches steady state depends on the microbial
species and the parameters of the chemostat (Makri et al., 2010). When one or more of
3
the operational variables are changed, the chemostat reaches a new steady state (Makri
et al., 2010).
In contrast to a batch culture, the chemostat is a highly productive, time-redundant,
constant culture of a microbe (Burton et al., 2001). Variables such as concentrations
of solutes in the solution, pH changes and biomass concentration changes are negated
(Meyer et al., 1985), allowing the ability to problem monitor. The chemostat also
selects for the fastest growing strain (Meyer et al., 1985), which is based on the
parameters set; thereby allowing the experimenter to artificially select a desired strain
by favouring conditions for the microbes growth. However, genetically modified
microbes might cause contamination from the inside due to back mutation to the wild
type(Meyer et al., 1985), which favours a faster growing microbe that does not
synthesise the desired product; as a compromise, a shorter run time is usually
implemented.
An understanding of the principles behind operating these bioreactors is required to
exploit the micro-organisms full potential in these systems. Parameters such as
airflow, feed input, reactor outflow, media type, stirrer rate, dissolved oxygen
concentration, temperature, and pH must be considered and their relationship
understood. Knowledge of these factors allows the exploitation of the specific
functions of the bacteria, such as optimising activity of an enzyme for conversion of a
desired
product
that
can
be
used
to
produce
urease
enzyme.
4
In this report we used knowledge of these parameters to increase productivity and
specific activity of the enzyme urease, by cultivating the gram positive
bacteria Sporasarcina pasteuriis. This microbe is able to survive in high pH
conditions and within liquids containing high concentrations of ammonia (Cheng and
Cord-Ruwisch, 2013). This enabled targeted growth of these bacteria within the
reactor through these selective pressures. The use of an open system (non sterile)
chemostat was employed to produce urea enzyme (amidohydrolase) within an
environment with a constant temperature of 28 degrees C and with a pH10 that is
optimal for increased activity of urease. The urease produced by these bacteria
catalyses the hydrolysis of urea to carbon dioxide and ammonia as follows:
UREASE
H2N-CO-NH2+2H20 -------------------> 2NH4+ + CO32-
The ammonia increases the pH of the surroundings, which in turn induces calcite
precipitation. S. pasteurii can precipitate calcite and solidify sand in the presence of
calcium and urea through a process known as biocementation (Siddique et al., 2011).
The binding strength of the precipitated calcium carbonate can be increased by
regulating the rate of carbonate formation by S. pasteurii. A CaCO3 product can be
used in many industrial applications such as reinforcing cement and solidifying soil in
earthquake prone areas (Sarayu et al., 2014). A new type of building material known
as biobricks have been developed by a North Carolina company in the USA
bioMASON, which were produced through the biocementation process. This method
enables huge reductions in CO2 emissions normally produced in traditional brick
making methods, which in term contribute to the reduction of the carbon footprint.
5
The aim of this experiment was to operate, test and, document the daily running of a
chemostat using the microbe S.pasturii to cultivate and maintain a high specific
productivity by varying certain parameters for optimum growth and quality,
harvesting a sample to be used to create a rock like sample using biocementation
technology.
Method
Bacterial strain
S. pasturii was provided by Murdoch Teaching with a recorded activity of 1.55
mS/10min; in conditions of temperature of 28-30 degC, pH 10, stirred at 400 rpm. at
an airflow rate of approximately 100L/h. The culture was grown in yeast extract feed
media.
During the chemostat process this strain was grown in yeast extract media at 28degC
at different airflow rates and differing stirrer rates (as indicated in results), at pH10.
Feed media
Yeast extract (provided by Murdoch Teaching) 1L volume (20g/L yeast extract,
10.21g/L Urea (0.17M) 20g/L sodium acetate and 2ml of 50mMstock/L
NicL2(0.1mM)
Chemostat set-up, biocementation set-up, and computer program
An open chemostat system, and all equipment required was provided by Murdoch
University teaching (as shown inFiguress 1 to 3 below) and the computer program
provided for this experiment was LabView software.
6
Figure 1:The chemostat set up adapted from Murdoch teaching hand out and presented past report.
Figure 2:A photograph of the inflow and outflow pumps, and the pH regulating pump.
7
Figure 3: A photograph of the waterbath containing the reactor.
Dissolved oxygen readings
A dissolved oxygen HANNA instruments conductor was calibrated prior to each use.
Use of conductor as per HANNA instruments instruction sheet.
pH
A pH metre was used as per provided instructions. The calibration was checked prior
to use, and checked throughout the experiment. The computer program performed
constant control of the pH and 3M NaOH was added into the reactor when required by
this program.
Biomass assay
Biomass assays were performed using a spectrophotometre at 600nm, using the feed
media as reference. Absorbance readings were converted into biomass concentrations
using C (biomass concentration, g/L) = 0.44 x O.D (600nm) equation. Dilutions using
feed media were performed when the initial absorbance readings were above 2.
8
Urease assay
Urease assay was performed as per instructions provided by Murdoch University
teaching in which biomass, 3M urea and deionised water were added at a
2ml:10ml:8ml ratio; at room temperature. Conductivity readings over 10mins were
recorded in mS/10mins to determine activity of urease in the reactor
Producing cement sample through biocementation
The biocementation process was performed as per instructions provided by Murdoch
Teaching in which 30ml of reactor outflow and with an activity rate of 2.3 mS/10min,
was passed through a 60ml PVC cylinder containing SiSo2. This was followed by
slowly adding 10mL of cementation solution calcium chloride/urea (CaCl2 H2O/Urea
1M ) allowing time (8 hours) for adhesion of bacteria to SiSO2, and for excess
bacteria to wash out. Three 20mL additions of cementation solution was added with a
time lapse of seven hours between each addition until the desired strength and solid
appearance was achieved.
Figure 4:A photograph of the biocementation setup.
9
Results
Dissolved Oxygen mg/L
The effect of the change of stirrer speed on dissolved oxygen levels
8
7
6
5
4
3
2
1
0
0
10
20
30
40
50
Time (hours)
60
70
80
Figure 5: An aeration curve of the dissolved oxygen concentration (mg/L) over time during the changes in
the stirrer speed test, at constant airflow of 250L/h. From 0-35 hours the stirrer speed was set at 350rpm,
from 53 hours the stirrer speed was set at 650rpm.
An increase of the stirrer rate from 350rpm to 650rpm was performed to increase the
dissolved oxygen level in the reactor. As per figure 5. this did not occur, the level
remained constant after the change took place. As seen on the figure 5 between time 0
to 35 hours the stirrer speed was at 350rpm noting the dissolved oxygen was
decreasing during this period to a stable level, which then remained after 35 hours
once the stirrer speed had been increased.
The effects of doubling dilution rate on biomass concentration, productivity, and
enzyme activity in the chemostat.
A decrease in enzyme activity rate by 23.21% was observed however an overall
increase in productivity was seen as productivity had increased to 0.06955, a overall
increase of production rate by 65.67%. Table 1 contains the relevant data for
determination of the enzyme activity rate change.
10
Table 1: Effects of Doubling Dilution Rate on enzyme activity, biomass, and productivity in the chemostat
trial. Dilution (D) was calculated by D(h-1) = F(L/h)/V(L) and Productivity (R) was calculated (R)(g/L/h) =
X(g/L)*D(h-1).
Parameters
Dilution Rate (D) (h-1)
0.03985
0.0797
Air flow (L/h)
150
150
Stirrer (rpm)
404
405
pH
10.2
10.7
D.O (mg/L)
5.69
0.77
O.D Biomass (600nm)
0.675
Average
Enzyme
Activity
(1:10
0.754
Dilution)
Dilution)
2.8
2.15
0.02367
0.06955
(1:10
(ms/10mins)
Productivity (R)(g/L/h)
The effect of using a batch culture to revive biomass concentration and
productivity, and enzyme activity
Biomass concentration and productivity were observed to be low, so forcing a batchtype culture by preventing inflow of feed and outflow of the reactor was performed to
increase the biomass to allow for increase in productivity and therefore increase in
enzyme activity.
Variations in pH of the bioreactor during a batch culture
Data for the pH of the batch culture solution was recorded between hours 0, 8 and 25
which included the starting and finishing times of the batch culture. Over the 25 hour
period of the batch culture, the pH of the solution seemed to increase; however, with
limited data, it is unsure what the pH reading was during the intervals between the
11
measurements. At time 0, the pH was measured at 10.2; after 8 hours in batch, the pH
increased to 10.9. Finally, at the completion of the batch culture, the pH of the
solution reached its highest value of 11.
Figure 6: Relationship between biomass concentration and the activity of the enzyme urease throughout the
process of an unsterilized batch culture containing S. pasteurii.
The results from the provisional batch culture showing the relationship between the
relative biomass concentrations within the bioreactor culture vessel and the enzymatic
activity, and therefore the relative amount of urease produced by S. pasteurii, indicates
there is a relationship between the two. As shown in figure 6 when biomass
concentrations decreased by 0.38 g/L during the first 8 hours of the batch culture,
there was a significant decrease in the enzymatic activity by a difference of 0.01
g/L/h. After 25 hours as a batch culture, both the biomass and enzymatic activity
reached a culmination, with biomass reaching a concentration of 0.774 g/L and
enzyme activity increasing 63 fold to 0.04 g/L/h, this is shown in figure 7.
12
Figure 7: Variations in urease activity within an unsterilized chemostat culture containing S. pasteurii. Both
trials 1 and 2 are shown, with enzyme activity relating to the activity of urease in the presence of urea.
Variations in doubling time of the biomass during a batch culture
The doubling time was calculated in relation to the specific growth rate of the culture
during the batch process. Data for the doubling time of the batch culture was recorded
at 52.35 hours at time 0 hours. At 8 hours, a decrease in the doubling time 2 fold was
seen with a result of 26.33 hours generation time. At the 25th hour, an increase in the
doubling time by 6 fold was seen, with a result of 156 hours per generation.
13
Biocementation process
Figure 8 shows the process of biocementation overtime after multiple additions of
cementation solution, made up of 1M Calcium Chloride and 1M Urea. The initial
product was a grainy sand, which after addition of the chemostat harvest and
cementation solution, became solidified.
Figure 8: The biocementation process overtime. A) prior to addition, b) after the first addition, c)
after second addition, d) after final addition.
14
Discussion
Doubling Dilution Rate
Doubling the dilution rate refers to the amount of feed entering the chemostat is
increased two fold, therefore more food available for the culture to use for growth and
production, also simultaneously increasing the outflow by two fold. Since productivity
is dependant on the dilution rate, it is possible to increase the productivity by
increasing the dilution (Nakashimada et al., 1998). Increasing dilution rate increases
productivity because the amount of feed that is being provided to the reactor has
increased, this means more feed to use by the bacteria for growth and product
production. More feed in the reactor allows the bacteria to take up more of the feed
that can be used for the growth of biomass. More biomass means the enzyme
production is increased meaning enzyme activity also increases (because there is
enzymes working) and this increases productivity of the overall bacteria in the
chemostat (Van Hoek et al., 1998). This is evident in Groups 2 data as when their
chemostats culture had increased in biomass there was also an increase in enzyme
activity, with the highest activity recorded at 1.6ms/10mins when the biomass
concentration was at 2.8864g/L (the highest biomass concentration recorded for group
2). Group 1’s data does support the theory of increase in biomass concentration
increases enzyme activity, as their highest activity was recorded at 2.1ms/10mins at a
biomass concentration of 4.2768g/L (not the highest but biomass concentration but it
is one of the highest recordings they had). So it can be concluded that a change in
biomass concentration does change enzyme activity.
Increasing dilution rate should be able to increase productivity infinitely but this is
only in theory, as test has shown that there is a limit to how much the dilution rate can
15
be increased by, for once a certain dilution rate is reached the bacteria in the reactor
will simply not be able to grow as fast as they are being pumped out (Van Hoek et al.,
1998). This value is known as the Critical Dilution value or ‘Dcrit’. When the Dcrit is
reached there will be ‘washout’ of the bacteria in the reactor. ‘Washout’ occurs
because the bacteria is unable to grow as fast as they are being pumped out of the
system, to avoid ‘washout’ of the bacteria the dilution value must not be higher than
the growth rate of the bacteria (Meers, 1971). The Dcrit value is different for each
chemostat, depending on the bacteria that are being used in the chemostat. The Dcrit
of our chemostat was not achieved nor known, as simply doubling the dilution rate did
not cause ‘washout’ of the bacteria.
The chemostat achieved a very high enzyme activity from the bacteria S.pasturii
however are unable to ascertain how the high activity was achieved it was proposed
that a test be carried out to see the high level of activity could be maintained whilst
increasing the production rate over time. The dilution rate was increased and the
chemostat run for a further three and a half days to allow changes to establish. After
24 hours the control variables were still the same with only slight to minor changes
that did not affect the overall system. The first test done on enzyme activity after 48
hours showed that the enzyme activity had decreased from 3.1ms/10mins to
2.5ms/10mins, another test that was done was biomass concentration. There was an
increase in biomass concentration. This decrease is correlated to the decrease in
biomass concentration that was also discovered. Though the enzyme activity had
decreased slightly the overall chemostat was still running twice as fast as it was before
the change meaning that the overall productivity had increased. Therefore we were
able to increase productivity as we had hypothesised by increasing the dilution rate but
16
we were not able to maintain the high levels of enzyme activity as we had before the
dilution rate change was made.
The revival through a batch-culture setting
Over the incubation period the pH of the solution seemed to steadily increase to a
maximum of 11 at 25 hours. The pKa of ammonia is 9.24 (Korner et al., 2001),
indicating that half of the ammonia is unionised and half is in the ionised form,
ammonium. The increase in pH could be explained by the increase in urease activity;
as urease breaks down the compound urea into NH3 and CO2, the increasing
concentration of NH3 in the system would have led to an increase in the pH of the
solution. Due to the lowest pH of the batch culture being 10.2, it can be said that most
species would be in the form of NH3 and not in the ionised form NH4+. NH3 is toxic
to most cells (Korner et al., 2001) and its small size and non-polar nature allow it to
cross the cell membrane by simple diffusion (Korner et al., 2001). Thus, the
conditions in the culture vessel would have increasingly harshened as pH increased,
leading to increased NH3 and a decrease in NH4+ concentrations causing further
cytotoxicity. However, Bacillus pasteurii is an alkalophile , suited to basic pH levels,
most suitably at 9.5 (Burton and Prosser, 2001). The high pH allowed for the selection
of S. pasteurii and the inhibition of microbes sensitive to the higher pH levels of 10.2
to 11. This was seen in the results; between 8 to 25 hours, biomass increased with
increasing pH, however, despite being a small change the activity increased
additionally, indicating a higher ratio of S. pasteurii in the bioreactor with respect to
the other microbes after the increase in pH.
It has been shown that pure enzyme hydrolyses urea approximately 20 times faster
than ureahydrolysed by S. pasteurii. The ureolytic rate of bacteria is dependent on a
17
number of factors, including the concentration of urea, cellular levels of urease and the
transport rate of urea in the cell. In this experiment, urea was in excess; thus, the
urease activity was dependent on the rate of urea diffusion across the plasma
membrane, which is determined by the concentration of urea in the solution. At time 0
hours, high potential energy for urea to enter the cells would have occurred, leading to
the influx of urea. However, the rate of synthesis and relative abundance of urease
might have been low, due to the microbes lacking sufficient urea media, in contrast
with the modification of the solutes in the solution by the addition of 450ml feed
media, the bacteria would therefore be adjusting to the conditions causing the lag
phase and the lower activity. In comparison to a chemostat with limiting urea
concentrations due to feed limitation, the batch culture has the capacity to process urea
faster, increasing urease activity; that is if sufficient concentrations of intracellular
urease have been synthesised. Interestingly, urease constitutes about 1% of the dry
weight of S. pasteurii, with urease being expressed constitutively. This explains the
rapid increase in enzyme activity during time 8 and 25 hours, which was caused by the
high concentrations of urease intracellularly due to the absence of urease synthesis
regulation. However, the increase in activity and therefore the increase in urease
synthesis would only occur in the exponential phase of the batch culture, as lack of
growth would occur in both the lag and stationary phase (Burton and Prosser, 2001).
At time 8 hours, the doubling time was most efficient at 26.33 hours as compared to
time 0 and 25 hours at 52.35 and 156 hours respectively. As the batch culture was
inoculated previously as a chemostat, the culture was in a starved state as the urea
media was the limiting factor, the generation time would therefore correlate with the
speed of the media inflow, which explains the higher doubling time at time 0 hours
18
compared to time 8 hours. As the culture was diluted in feed media, the feed was in
excess, allowing for rapid growth after the lag phase, which explains the higher
doubling time of 26.33 hours at 8 hours. At 25 hours, the doubling time seemed to
have increased dramatically; this could possibly be due to lack of feed in the solution
causing a proportion of the microbes to enter dormancy. This shows that there was
6.95 mg per litre of dissolved oxygen in the solution at time 25 hours, indicating
minimal oxygen uptake compared to time 0 with a dissolved oxygen reading of 0.46
mg per litre. The resultant growth might have been due to endogenous respiration.
However, if feed was still available in the culture vessel, growth might have been
reduced by the build-up of end products and secondary metabolites, which could have
resulted in product inhibition. Investigating the relative concentrations of urea in the
solution would need to be tested in order to identify the exact reason for the increase
in doubling time during time 8 and 25 hours.
The process of biocementation
Biocementation is a technology whereby biological agents are used as catalysts for
increasing stiffness and strength, where the carbonate from microbial hydrolysis of
urea where excess calcium ions together with calcite (CaCO3) precipitate.
Calcium carbonate in the form of solid crystalline known as biocement is produced by
microbial urease activity in a calcium rich environment. Cementation is a calcium
carbonate precipitation forming natural rocks in marine, freshwater and soil
environments.
The technology has potential to be used to strengthen or improve materials such as
cement products, soil and possibly timber through the precipitation reaction of calcite
19
and sand in the presence of calcium and a urea (Siddique and Chahal, 2011). Possible
applications could include stabilizing embankments, repairs to concrete that have
developed cracks, countering soil liquefaction, potential use as an alternative for
bitumen for walkways/cycle paths (more environmentally and aesthetically pleasing)
more suitable for structures built on sandy environments such as on Rottnest Island.
Tests performed on timber have shown that it becomes petrified and fire resistant
when a solution of biocement was applied and absorbed this may be a potential
treatment for termite resistance, though further studies are required (Bang et al., 2001;
Ramachandran et al., 2001; Cheng and Cord-Ruwisch, 2013).
Running and operating chemostat and problems encountered
Prior to running chemostat on day one tests using plain water were carried out to
determine the flow rate and calculate the on/off times for retention of 600mL over 24
hours. The pH probe, which was linked to the computer program for automatic
adjustment with NaOH pump for correction, was calibrated and antifoaming solution
was applied to reactor cover.
Several issues were encountered including the shut down of the automotive
monitoring computer program as well as non-functioning regulators for the air supply
and detachment of tubes from pumps. After these issues were resolved the bioreactor
was inoculated by adding the batch culture supplied. All parameters were checked and
adjusted and left overnight with a air flow rate of 175 L/h and stirrer rate of 263 rpm
and feed (dilution rate 0.039850 p/h) and outflow bottles surrounded with ice (to keep
feed fresh).
20
On day two it was discovered that the pH probe had not been placed in the reactor,
which had caused the automated addition of NaOH to such a high concentration, the
culture had died. Culture (250mL) was obtained from group 2 outflow (replacement
to enable a restart of the reactor) and brought up to a volume of 600mL with feed
media (350mL). Again a computer shutdown occurred over night and by day 5 the DO
was recorded at zero and it was decided to reduce the reactor volume to 150mL then
bring volume up to 600mL with feed media (450mL) to create a batch like culture to
be left 48 hours with just stirrer running with the view to increasing the activity rate.
Unfortunately after the 48 hour period the reactor was found to have only 150mL
volume, no explanation for this was ascertained as evaporation could not be attributed
to this about of lost volume. The chemostat was left to run for another 24 hours to
bring the volume back to 600mL. It was proposed that we reproduce these past events
to test whether the batch like conditions were indeed the reason for the following
increased activity readings but conditions could not be reproduced as there were
too many variables and sample culture to be tested had died.
After a further 24 hours it was discovered that due to the low volume in the reactor,
the pH probe had not been sitting in the liquid and needed to be re calibrated as the
computer was recording a pH > 11 a recalibration showed that the pH probe was
reading at least 2 levels higher than actual pH of the reactor.
From this time forward tests continued to show a consistent high activity reading with
varied DO readings (may have been due to faulty meter). Two hypotheses were tested
and results recorded and shown here in this report. On the fifteenth day the chemostat
21
was stopped due to a power failure during the night which stopped the program which
resulted in the death of the culture.
Group comparisons
The urease activity of group three chemostat had increased to approximately 32.00
umol/min/ml over a period of 5 days (Monday to Friday), after the dilution of the
original reactor volume from 600ml to 150ml together with 450ml of feed media (to
bring the total volume back to the original to 600ml). The newly diluted reactor was
run as a batch-like culture over a 48 hours period, with only the stirrer at 240rpm (no
inflow, outflow or airflow), However the volume of the reactor had decreased to
approximately 150mL cause unknown, therefore the reactor volume was gradually
increased to the original 600ml volume over a 24 hour period. It is proposed that the
afore mentioned actions over the 72 hour period may have contributed to the increased
enzyme activity values. Group Two operated their chemostat without achieving a high
activity rate compared to group three, even with a change in feed concentration and
pH change. Group one’s chemostat did however start to achieve an increase in activity
rate by increasing the rate of food supplied over time whether this would have
continued if the chemostat had run for a longer time is unknown.
22
Conclusion
The non-sterile chemostat working process was found to be successful in growing,
producing and maintaining a high enzyme activity from a culture of S.pastuerii in
conditions chosen to provide selective pressures for targeted growth. The use of a
batch culture to revive enzyme activity and biomass growth was successful, and
caused an increase in the urease activity. Though problems with reliability of this data
and inability to reproduce this batch-like system must be acknowledged. The doubling
of dilution rate showed the high enzyme activity rates were maintained, but not at the
same rates as with the batch culture, concluding that it may be possible to have a
quicker productivity over time whilst maintaining a high activity rate. The change in
the stirrer rate had no effect on the system, and did not increase in dissolved oxygen as
expected. Other groups did not achieve a higher enzyme activity through their changes
the chemostat; increasing feed media concentration by group two did not achieve a
higher activity production, nor did an increased stirrer rate change as seen in group
one.
Acknowledgements
We would like to acknowledge Murdoch Teaching staff for proving the set up for this
project, and for the supply of material, and assistance. We would also like to
acknowledge the other class members of BIO301 for use of their data for comparison
of chemostat functions.
Overall the report shows excellent understanding and good literature research in
23
addition to good lab work. The few parts where the report could be improved, and I
am saying this from the position of mini-research honours project, or mini publication:
Shorter by avoiding some info that is not essential to bring your points across.
More emphasis on explaining what your thinking is (hypothesis) how you tested it and
what it means. Less emphasis on generic literature background of chemostats.
More format of publications (like the one we gave you): short clear paragrphs that
have a clear point at the end.
8/10
References
Bang, S.S., J.K. Galinat and V. Ramakrishnan, 2001. Calcite precipitation induced
by polyurethane-immobilized bacillus pasteurii. Enzyme and microbial
technology, 28(4-5): 404-409. Available from
http://www.ncbi.nlm.nih.gov/pubmed/11240198.
Burton, S.A. and J.I. Prosser, 2001. Autotrophic ammonia oxidation at low ph
through urea hydrolysis. Applied and environmental microbiology, 67(7):
2952-2957. Available from
http://www.ncbi.nlm.nih.gov/pubmed/11425707. DOI
10.1128/AEM.67.7.2952-2957.2001.
Cheng, L. and R. Cord-Ruwisch, 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.
Available from http://www.ncbi.nlm.nih.gov/pubmed/23892419. DOI
10.1007/s10295-013-1310-6.
Korner, S., S.K. Das, S. Veenstra and J.E. Vermaat, 2001. The effect of ph variation
at the ammonium/ammonia equilibrium in wastewater and its toxicity to
24
lemna gibba. Aquat Bot, 71(1): 71-78. Available from <Go to
ISI>://WOS:000171326600006. DOI Doi 10.1016/S03043770(01)00158-9.
Makri, A., S. Fakas and G. Aggelis, 2010. Metabolic activities of biotechnological
interest in yarrowia lipolytica grown on glycerol in repeated batch
cultures. Bioresource technology, 101(7): 2351-2358. Available from
http://www.ncbi.nlm.nih.gov/pubmed/19962884. DOI
10.1016/j.biortech.2009.11.024.
Meers, J.L., 1971. Effect of dilution rate on outcome of chemostat mixed culture
experiments. J Gen Microbiol, 67(Aug): 359-&. Available from <Go to
ISI>://WOS:A1971K658600012.
Meyer, H.P., O. Kappeli and A. Fiechter, 1985. Growth-control in microbial
cultures. Annu Rev Microbiol, 39: 299-319. Available from <Go to
ISI>://WOS:A1985ARS4100014.
Nakashimada, Y., K. Kanai and N. Nishio, 1998. Optimization of dilution rate, ph
and oxygen supply on optical purity of 2,3-butanediol produced by
paenibacillus polymyxa in chemostat culture. Biotechnol Lett, 20(12):
1133-1138. Available from <Go to ISI>://WOS:000078348200009. DOI
Doi 10.1023/A:1005324403186.
Novick, A. and L. Szilard, 1950. Description of the chemostat. Science, 112(2920):
715-716. Available from
http://www.ncbi.nlm.nih.gov/pubmed/14787503.
Novick, A. and L. Szilard, 1950. Experiments with the chemostat on spontaneous
mutations of bacteria. Proceedings of the National Academy of Sciences of
the United States of America, 36(12): 708-719. Available from
http://www.ncbi.nlm.nih.gov/pubmed/14808160.
Pirt, S.J. and R.C. Righelato, 1967. Effect of growth rate on the synthesis of
penicillin by penicillium chrysogenum in batch and chemostat cultures.
Applied microbiology, 15(6): 1284-1290. Available from
http://www.ncbi.nlm.nih.gov/pubmed/16349736.
Ramachandran, S.K., V. Ramakrishnan and S.S. Bang, 2001. Remediation of
concrete using micro-organisms. Aci Mater J, 98(1): 3-9. Available from
<Go to ISI>://WOS:000166886200001.
25
Sarayu, K., N.R. Iyer and A.R. Murthy, 2014. Exploration on the biotechnological
aspect of the ureolytic bacteria for the production of the cementitious
materials--a review. Applied biochemistry and biotechnology, 172(5):
2308-2323. Available from
http://www.ncbi.nlm.nih.gov/pubmed/24395694. DOI 10.1007/s12010013-0686-0.
Siddique, R. and N.K. Chahal, 2011. Effect of ureolytic bacteria on concrete
properties. Constr Build Mater, 25(10): 3791-3801. Available from <Go to
ISI>://WOS:000292664700001. DOI 10.1016/j.conbuildmat.2011.04.010.
Stanbury, P.F., Whitaker, A. Hall, S.J., 2013. Principles of fermentation technology.
2 Edn., Amsterdam: Elsevier.
Tata, M., P. Bower, S. Bromberg, D. Duncombe, J. Fehring, V.V. Lau, D. Ryder and P.
Stassi, 1999. Immobilized yeast bioreactor systems for continuous beer
fermentation. Biotechnology progress, 15(1): 105-113. Available from
http://www.ncbi.nlm.nih.gov/pubmed/9933520. DOI
10.1021/bp980109z.
Temudo, M.F., R. Kleerebezem and M. van Loosdrecht, 2007. Influence of the ph
on (open) mixed culture fermentation of glucose: A chemostat study.
Biotechnology and bioengineering, 98(1): 69-79. Available from
http://www.ncbi.nlm.nih.gov/pubmed/17657773. DOI
10.1002/bit.21412.
Van Hoek, P., J.P. Van Dijken and J.T. Pronk, 1998. Effect of specific growth rate on
fermentative capacity of baker's yeast. Applied and environmental
microbiology, 64(11): 4226-4233. Available from
http://www.ncbi.nlm.nih.gov/pubmed/9797269.
Whiteley, M.B., E. McLean, R.J.C., 1997. An inexpensive chemostat apparatus for
the study of microbial biofilms. Journal of Microbiology Methods, 30: 125132. Available from http://www.dzumenvis.nic.in/Physiology/pdf/An
inexpensive chemostat apparatus.pdf.
26
Appendix
Bioreactor volume was 600mL retention time was 24 hours
RT =600ml/24hr = 25mL/hr = 0.416mL/min
The excel spreadsheet containing the calculations for results is supplemented as an
attachment upon submission. Additional data such as kLa and OUR values are found
within this attachment.
Additional figures
Table 2: Comparison of test readings at different temperatures, showing how temperatures affects
the results obtained.
Time (mins)
Activity at room temperature (mS) Activity in ice (mS)
1
5.6
5
2
5.8
5.3
3
6.1
5.4
4
6.4
5.5
5
6.6
5.6
6
6.9
5.6
7
7.1
5.7
8
7.4
5.7
27
9
7.6
5.7
10
7.8
5.8
Activity (ms/10 min) 2.2
0.8
Temperature
Time (mins) Dissolved oxygen (mg/L) O2 change/2 min
22 (addition of ice) 0
5.64
0.2
15
2
5.84
0.25
13
4
6.09
0.2
12.5
6
6.29
0.26
11.5
8
6.55
0.2
10.5
10
6.75
28
Figure 9: The overall process of the chemostat, including batch culture, for group three.
Figure 10: The overall comparison of biomass productivity vs. specific activity of the chemostat,
including batch culture, overtime, for group 3.
29