BIO 301
Chemostat Report
Group 2 BIO301
LeesaGoodsell 31557693
Guanchen Zhu 32046123
Zane Greaves 31014945
BIO 301
Abstract
Ureolytic bacteria are capable of CaC03 production via by product precipitation, occurring
naturally in the environment. This has the potential to be commercialised under non-sterile
growth for different applications, such as soil stability. A 1200ml chemostat reactor with a
HRT of 1200ml/10h was established and optimised to yield high productivity ions
(>0.2ms/min). Variables such as thestirrer rate, feed and air flow rate were changed and
regulated in order to increase the urease production to an optimum level, whilst the pH was
kept at a constant value of 10. The Specific activity was shown as the success rate. Therefore
from these results it is clear that it is possible to runSporosarcinapasteuriiunder a non-sterile
chemostat and get high urease production. started to read like a good abstract by saying
what was done. What also belongs into the abstract is what was found.
Introduction
Biotechnology is the technological application of biological processes in everyday life. Beer
making, pharmaceuticals, agriculture and waste management are all applications of
biotechnology. All of these processes utilize a certain microorganism’s ability to convert a
substrate to a product. An example of this would be a lactic acid bacterium converting the
substrate, a sugar source such as grapes, into ethanol in wine making. As a result these
bacteria are of profound importance to the industry in which their metabolic procedures are
employed.
Biocementation is becoming an increasingly more studied field as it is of interest for the
purpose of soil strengthening (Cheng and Cord-Ruwisch 2013). Biocementation is not a new
field; it has been of interest for some time in terms of the maintenance or repair of various
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materials (Jonkers and Schlangen 2007). Biocementation is a means of restoration to sites
where heritage is of importance. It was also put forward that the use of microbes in the
process of biocementation would be a cost effective way to repair cracks in concrete due to
the durability and strength of the “biological cement”. (Ramachandranet al 2001)
Biocement is created by the precipitation of calcium carbonate in the form of calcite
(Siddique and Chahal 2011). The precipitation of calcium carbonate relies on the ureolytic
bacteria producing the urease enzyme. This precipitation of the calcium carbonate crystals is
brought about by the heterogeneous nucleation on bacterial cell walls once supersaturation
is achieved (Siddique and Chahal 2011).
One such bacterium which has the ability of producing biological cement through this
mechanism is that of Sporosarcina pasteurii(which was previously known as Bacillus
pasteurii)(Achalet al, 2009).This bacterium is an endospore-forming, alkaliphilic,ureolytic,
soil bacterium. It generates microbial urease which then catalyzes the hydrolysis of urea to
ammonium and Carbonate (Achalet al, 2009). This is illustrated by the equation:
NH2CONH2 + H2O → CO32- + 2NH4+
The resulting increase in pH causes the ions Ca2+ and CO32- to precipitate to form CaCO3; a
process known as biocementation (Ramachandranet al 2001). Alkalaphilic bacteria have a
tendency to grow in high alkaline areas, for example an environment with a pH of 10 or
above. This high pH is toxic to most commonly found bacteria in the environment and hence
assists in contamination control. Yes it starts getting towards the point of the current
experiment now. Would be nice to connect the alkaline running conditions of the
chemostat with the idea of non sterile production of Bacillus pasteurii.
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In this study the bacteria (S.pasteurii) will be grown in a chemostat in order to control the
growth and various conditions needed to achieve the aim of biocemetation production. The
name chemostat is associated with a laboratory device used for growing microorganisms in
a cultured environment.Nice attempt of defining chemostat but you missed out on pointing
towards the continuous inflow and outflow reaching steady chemical conditions In this
chemostat the medium or feed that was used contained: sodium acetate, yeast extract,
urea, and nickel chloride.
There is not need in the intro to say what experiments were done. Introductions explain the
background before the experiments and terminat with deriving the aim.
Therefore, the aim of this study was to optimize the conditions for the urease enzyme
production under non-sterile conditions.
Materials and methods
Operating the chemostat
The chemostat consisted of a 1L beaker in a temperature waterbath (28 degrees Celsius). It
was continuously fed with fresh medium at a hydraulic retention time (HRT) of 80 hours,
initially and then at 16.6 hours. goodHRT calculated based on flow speed of 42 ml/h. the
decanting pump was set at a faster rate than the influent pump to ensure constant volume.
good Also, the decant tube was set at a length of 5cm whereas the influent was triple that in
measurement. Not clear to new readers Stirrer speed was initially set at 400 rpm. Air flow
was set to 150h-1. The pH probe was trigger set to a computer program that pumped NaOH
as required to a pH of 10.
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Biomass measurement (OD 600nm)
The biomass was measured in a spectrophotometer set at a wavelength of 600nm. The
spectrophotometer was blanked with either deionised water or with the feed medium
which was used in the chemostat. If the deionised water was used to blank the
spectrophotometer, then the feed medium absorbance was then subtracted from the
biomass absorbance.good If the biomass’s absorbance measured at a wavelength of greater
than or equal to 2 then the stock was diluted by either a 1 in 3, 1 in 6 or a 1 in 10 dilution to
get the absorbance value down.
Urease activity
Urease acts as an enzyme that converts urea into NH4+ and CO32-. This reaction converts a
non-ionic substrate into ionic products thus the rate of change in conductivity could be
measured to determine the urease activity.
Thus 2ml of culture was added to 10ml of 3M urea and 8 ml of deionized water. The
conductivity was recorded over 5 minutes in 30 second intervals under room temperature.
According to Whiffin, 1mS cm-1 min-1 corresponds to 11.11µmol min-1 ml-1 of urease activity.
Because of the dilution factor of 10, the urease activity of the culture is 111.1µmol min -1 ml1.
Dissolved oxygen
Oxygen concentration was measured by using the Dissolved Oxygen probe. Oxygen
concentration affects the setting of airflow rate. To make sure the accuracy of the readings,
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Dissolved Oxygen probe needs to be calibrated before use. The procedure of calibration and
measurement were provided by the manufacturer. During the measurement, feed/harvest
bung in the chemostat was removed and the probe was carefully introduced through the
aperture.
pH Controls
The pH was kept at a constant rate of 10, if the pH dipped below 10 the sodium hydroxide
pump would commence and the pH would be brought back to 10. The pH probe was
calibrated with the computer when the bioreactor first commenced and also during the
project to make sure that the pH probe was giving the correct readings. Another pH probe
was also employed to check the integrity of the probe in the bioreactor. pH probes were
calibrated when used at all times to ensure correct readings were given. The calibration
procedure followed was according to the device’s instructions. Could explain what algorithm
was used to administer NaOH (ml added per min if pH was low) how oftern was the pH
copared with the setpoint?
Cementation.
The highest productivity harvest was used to produce a sand column. A large spin column
was altered at the base with a sponge filter and tube to filter out flow through. Silicon sand
was filled into the modified system. 10 ml of the bacteria was added and every seven hours
the cementation mixture was added (calcium carbonate and urea mixture). After 14 days of
this treatment, the product was extracted as shown by figure @!@!@
technically this figure is a result and does not belong to methods section
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Figure 1: The before and after product of silicon sand and
biocementation, respectively
Results and Rationale (looking forward to the first report with rationale)
Continuously producing urease activity
Could start by explaining what the starting postion was (batch culture with certain activity)
The environment of the chemostat was kept at PH 10. Initially there was a drop in both
biomass and urease activity (enzyme activity in the graph) in the first 24 hours. However
after continued operation for two more cycles of retention time.,there was a increase in
both biomass and urease activity. In Day 6 measurement, we could clearly observe a decline
in urease activity(in Figure 2). This decrease will bediscussed in the next session. Rather
than describing what happened it would be good to apply the scientific method here.
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Figure 2: Graph shows the specific urease activity, enzyme activity and biomass
concentration over days. give units for enzyme activity and specific activity
The decrease in enzyme activity
As mentioned in the above session, there was a dramatic decline in enzyme activity in Day 6.
With its decrease, the biomass was also decreased. This is because 200mL of culture was
‘borrowed’ from the other group. Unclear, why was there a need to obtain inoculum from
other groups? The decrease on Day 5 1400hr measurement was expected why, what
happened there? but the following day which Day 6. We expected an increase of both
biomass and enzyme activity.why? However, only biomass increased. Enzyme activity
continued dropping. Since our finding was that the air flow rate dropped to 50L/h from
175L/h, our hypothesis is that this decline could be due to either lack of oxygen in the
solution or competitive growth from other species. Ok, good to see the first causal
relationship
From our routinemeasurement, we found that even though air flow dropped to 50L/h, the
oxygen concentration change from 5.65mg/L to 4.84mg/L, which is supposed to be enough
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for microbe to grow. But biomass concentration and enzyme activity caught our attention,
enzyme activity declined from 15.33 to 3.33 mol/L/min show example calculation of mS per
min convert to mol/L/h. Biomass concentration however, increased slightly from 0.83 to
0.86mg/L (Table 1).
biomass
Enzyme activity
DO
Day 5 1400hr
0.83 mg/L
15.33 mol/L/min
5.65 mg/L
Day 6
0.86 mg/L
3.33 mol/L/min
4.84 mg/L
Table 1. Biomass, enzyme activity and D.O in Day 5 1400hr and Day 6
Thus it was suspected that contaminant species was growing in the culture which competed
with the urease producing species. In order to eliminate the contaminants and keep the
high PH, the following means were tried.

Double the concentration of Urea in the substrate
from 10.21g/L, which could be utilized and transferred to ammonium in the reaction
and increase the PH in the culture.

Retention time
Shorter retention time means faster flow of feeding. We expected to wash out the
contaminants by increasing the medium flow rate. Is that based on the assumption
that contaminants grow slower than B. pasteuri? Thus the medium flow rate was
adjusted from 1200mL/24hrs to 1200mL/10hrs. In retention time (reactor volume
600mL), it is from 12 hours to 6 hours (Table 2). I don’t understand the logic of this.
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You more than doubled the flow rate and claim a halving in retention time. Table 2
does not show retention time.

Air flow rate
In this project, high PH pH was kept in two ways. PH was monitored every 60
seconds and NaOH solution was added if PH was lower than 10. But the major way
to keep this high PH was by high ammonium concentration in the culture. The
ammonium was produced by enzyme urease which was produced by the ureolytic
bacteria and was also the product in this project. Good There was equilibrium
between ammonium and ammonia. Ammonia is highly evaporative in the air. Thus
faster air flow rate stimulates more ammonia evaporated into the air and drags the
equilibrium from ammonium to ammonia. The PH in the solution will decrease.
What we found in Day 6 measurement was that NaOH solution for pH adjustment
was consumed dramatically faster than ever. That proved the declined ammonium
concentration. Evidence? In order to decrease the ammonia evaporation and
pushed the equilibrium to ammonium. We decreased flow rate from 175L/h to
150L/h (Table 2). Good thinking. Almost usage of the scientific method.
Urea
Retention time
Air flow rate
Enzyme activity
concentration
Day 6
10.21g/L
1200mL/24hrs
175L/h
3.33mol/L/min
Day 7
20.42g/L
1200mL/24hrs
150L/h
15.33mol/L/min
Day 8
20.42g/L
1200mL/10hrs
150L/h
18.66mol/L/min
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Table 2. Urea concentration, retention time, air flow rate and enzyme activity on Day
6, 7 and 8 Table title should be on top of table. Retention time is in hours but you
seem to give it in mL/h. Also very unusual to give mL/ 10 h.
As the result we could observe in Figure 2, enzyme activity raised after 24 hours and
continued growing in the following days of the project which meant that the growth of the
wanted bacterial species. Did you change more than one parameter at a time?
Overall growth of the bacteria
The overall growth of the bacteria (biomass) was reflected by the optical density (OD) in the
project. As we can see in Figure 2, after a short period of decline in the first 24 hours, the
biomass kept going up till Day 5 @1400 when 200mL of culture was taken out and the OD
was decreased which was expected since the shrunken population of the bacteria. Even
though we expected a growth on the following day (Day 6) and indeed the biomass grew
from 0.83mg/L to 0.86mg/L, the growth does not satisfy us (stick with objective
statements) . The reason was that the measurement of declined enzyme activity (Table 1)
warned us the changed ingredients of the culture which was the suspected existence of the
contaminants.
With different means of eliminating unwanted species in the culture (Refer previous
session), biomass concentration grew steadily and the increase in enzyme activity supported
that the species was the one that we expected (shown in Figure 2). English could be
improved but thinking is good.
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Overflow of the culture
During the project, there was an overflow happened on Day 7. The overflow was not caused
by the overfilling but was told due to the foaming. As the contamination noticed in Day 6
and means of elimination of contaminants had taken place, the retention time was adjusted
from 1200mL/24hours to 1200mL/10 hours in order to wash out the contaminants. With the
faster solution exchange speed, the anti-foam solution was also exhausted faster. Thus by
Day 7, anti-foam solution ran out in the culture and the bubbling of the airflow caused
foaming and overflowing of the culture. By adding the anti-foaming in the following days,
this problem was solved.
Discussion
Selective growth under non-sterile conditions
Testing was based on a method that favours the production of urease enzyme by ureolytic
bacteria from a selective environment under non-sterile conditions. Many industrial
applications of bacteria production (biofuel production, bio-gas production) are done under
sterile conditions that are costly; this has led to eco-technology. One could argue that
having non-sterile conditions may lead to other expenses such as contamination control
which could be just as expensive as operating under high tech sterile conditions. However,
eco-technology plays on the physiology of the bacteria to favour its growth. Valid point
(van loosdrechtet al, 2007). Understanding the physiology and metabolic process involved
with the growth and products of the target organism is imperative to the success of this
type of culture growth.
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Sporosarcina pasteurii favoured environmental conditionals are relatively understood. One
of its most distinguishable feature, apart from utilising urea, is its preferred pH level. This
bacterium is able growth in a pH of 10. With this and its NH4+ by-product, the growth
environment is hostile to most common bacteria in the environment.
Important Biochemical processes attributing to cell growth and product formation
Ammonium and urea affect the overall proton electrochemical potential(Dp) shown in
figure 3. The electrochemical gradient can be seen as important in establishing the high
energy molecule, ATP. This was demonstrated by Peter Mitchell. Proton gradients are
essential to life forming the mechanism that synthesises ATP. Ammonium regulates
intracellular pH and under certain growth conditions replaces potassium which is important
in electro and chemical gradients in cells. An alkalinisation of the cytoplasm occurs with the
addition of ammonium or a precursor (urea). This increases the electrochemical gradient
and activates ATPase as it remains mostly inactivated at pH of below 6.8. Not clear how you
link the Mitchel hypothesis of membrane potential and proton gradient to the role of
ammonia under alkaline conditions.
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a.proton motive
, Source is
Urease enzyme
Urease is an enzyme that catalyzes the hydrolysis of urea to carbon dioxide and ammonia.
Specifically, the enzyme hydrolyses urea to produce ammonia and carbonate. The carbonate
is degraded by hydrolysis to produce another ammonia and carbonic acid (Zimmer, Marc
(2000). Molecular mechanics evaluation of the proposed mechanisms for the degradation of
urea by urease.J BiomolstructDyn.17 (5); 787-97). Urease activity increases pH of the
environment from the basic molecule produced, ammonia (environmental selectivity,
above). Optimising this enzyme will result in an increase in the reaction it catalyses. This
would lead to an increase in the bacterium’s ability to convert sand material into cement
like structure and to increase the environment pH.
As explored above, the proton gradient is important in the biochemical functioning of this
organism which is extended to urease activity (with ammonia and urea having strict affects).
Another important limitation to urease is nickel.Nickel is required for active sites
functioning. Manganese and cobalt can be substituted as a co-factor for the functioning of
the active sites, but each trace elements affinity, and overall affect needs to be studied and
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better understood to compare to each other to infer the most effect towards urease
activity. (interplay of metal ions and urease. Carter, E; Flugga, N; Boer, J; Mulrooneya, S;
Hausinger, R.) You discuss too much the theory of the metabolism. Rather focus on
discussing your results.
Temperature effect on bacterium growth and product formation
It has been shown that the optimum temperature for s. Pasteurii growth and urease
production is 28 degrees Celsius (Benini et al., 1991). Changes in temperature with affect
the reactor volume through evaporation. Evaporation rate would need to be monitored and
could be measured by recording the air humidity and general turbulence (penman
equation)The room temperature and the reactor temperature mixing and transferring heat
would need to be assessed as this would also play a role in bioreactor volume loss.
However, the rate of volume loss may not be too important if the continuous restocking of
fresh medium counteracts this volume loss. The volume loss would be more important in
standing cultures (batch cultures).
ammonia/ammonium equilibrium
ammonia and ammonium exsist in equilibrium under optimal conditions. pH has a direct
influence on ammonia forming to ammonium. A low pH results in more ammonia being
changed into ammonium. On the other hand, a high pH results in more of ammonia being
produced. This affects the cytoplasm of the cell as well as the bacteriums environment; an
equilibrium exsists not only in the outside environment, but inside the cell (influenceing the
proton electrochemical gradident, above).
NH2CONH2 + H2O → CO32- + 2NH4
This reaction:
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Shows the production of carbonate and ammonium. Once ammonium reacts with air,
ammonia (NH3) is the product and evaporates from the system. This is detrimental to the
harsh environment that the bacterium creates for its own growth and deterrent of other
microorganisms.
Commercial and Industrial uses
This bacterium can have a significant role in the application of industrial construction and
soil stability. Examples of its use include:

Soil stability (land construction, compacting earthquake prone areas)

Bioconcrete

Dust control Meyer, F., Bang, S., Min, S., Stetler, L., and Bang, S. (2011)
Microbiologically-Induced Soil Stabilization: Application of
Sporosarcinapasteurii for Fugitive Dust Control. Geo-Frontiers 2011: pp.
4002-4011.
doi: 10.1061/41165(397)409
Recommendations

Have a humidity control

Set group equipment

Run over weekend

Give HD to students who produce biocement
Overall, some good thinking that could have been used to demonstrate the
application of the scientific method in your report.
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Presentation could be more focussed
Discussion should concentrate on discussing the impact or significance of your
results.
7/10
References
Achal, V., Mukherjee, A., Basu, B. C., Sudhakara Reddy, M. (2009). “Strain improvement of
Sporosarcinapasteuriifor enhanced urease and calcite production”. Industrial Microbiology.36:pp981–988
Cheng, L., Cord-Ruwisch, R. (2012). “In-Situ Soil Cementation with Ureolytic Bacteria by Surface
Percolation”.Ecological Engineering. Pp64-72
Jonkers,H. M., Schlangen, E. (2007). “Crack repair by Concrete-Immobilized bacteria”. Proceedings of the First
International Conference on Self-Healing Materials.pp1-7
Ramachandran, S. K., Ramakrishnan, V., Bang, S. S. (2001). “Remediation of concrete using
microorganisms”.ACI Materials. (98):pp3-9
Rao, V.S., Rao, P. R. (2009). “Basic chemostat model revisited”. Differential Equations and Dynamical Systems.
Pp3-16
Siddique, R., Chahal, N. K. (2011). “Effect of ureolytic bacteria on concrete properties”.Construction and
Building Materials. (25)10: pp3791-3801
Meyer, F., Bang, S., Min, S., Stetler, L., and Bang, S. (2011) Microbiologically-Induced Soil Stabilization:
Application of Sporosarcinapasteurii for Fugitive Dust Control. Geo-Frontiers 2011: pp. 4002-4011. doi:
10.1061/41165(397)409
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Benini, S., Rypniewski, W.R., Wilson, K.S., Miletti, S., Ciurli, S. and Mangani, S. 1999. A new proposal for urease
mechanism based on the crystal structures of the native and inhibited enzymes from Bacillus pasteurii: why
urea hydrolysis costs two nickels. Structure 7: 205-216
interplay of metal ions and urease. Carter, E; Flugga, N; Boer, J; Mulrooneya, S; Hausinger, R.)
Zimmer, Marc (2000). Molecular mechanics evaluation of the proposed mechanisms for the degradation of
urea by urease.J BiomolstructDyn. 17 (5); 787-97
vanloosdrecht MCM, Kleerebezem R (2007) mixed culture biotechnology for bioenergy production.
Curropinbiotechnol 18:207-212
Whit'lln VS (2004) Microbial CaCO3 precipitation for the production of biocement. Ph.D. thesis, Murdoch
University, Perth
Cheng L, Cord-Ruwisch R (2013) Selective enrichment and production of highly urease active bacteria by nonsterile (open) chemostat culture, Journal of Industrial Microbiology and Biotechnology, issn 1367-5453
Appendix
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