BIO 301

Chemostat Report

Group 2 BIO301

LeesaGoodsell 31557693

Guanchen Zhu 32046123

Zane Greaves 31014945

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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.

In this study a culture of S.pasteurii was inoculated in a bioreactor and kept at a constant

temperature of 28oC and maintained at an approximate pH of 10. The chemostat was kept

under non-sterile conditions, with the conditions of: stirrer rate, air flow rate, harvest and

feed retention time (and as a consequence what was in the feed) were changed and

regulated in order to increase the urease enzyme production to an optimum level. 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.

Further, at the end of the experiment the CaCO3 precipitation was tested to determine the

success of optimization. An underlying purpose to this project was the minimization of

contamination which could be seen through results that produced a high biomass and low

enzyme productivity. Without the enzyme being produced or, with the enzyme being

produced in small amounts the CaCO3 precipitate would not form.

Therefore, the aim of this study was to optimize the conditions for the urease enzyme

production under non-sterile conditions.

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Materials and methods

Operating the chemostat

The chemostat consisted of a 12L 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.

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

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Urease acts as an enzyme that converts urea into NH4+ and CO3

2-. 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 ml-

1.

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,

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

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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

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

Figure 1: The before and after product of silicon sand and biocementation, respectively

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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., Tthere was a dramatic

increase in both biomass and urease activity. In Day 6 measurement, we could clearly

observe a huge 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.

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

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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 routinely measurement, 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

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

In this occasion, we decided to doubled the urea concentration from 10.21g/L,

which could be utilized and transferred to ammonium in the reaction and increase

the PH in the culture.

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Retention time

Faster Shorter retention time means faster flow of feeding the harvest. 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. 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 environment 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.

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Urea

concentration

Retention time Air flow rate Enzyme activity

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

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.

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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.

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

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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.

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 highly

exergonic 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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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

Figure 3.Biochemical process of ureyolitic bacteria.proton motive force (Dp), the membranepotential (Dc) and the proton gradient (DpH), Source is missing!

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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

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

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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). This reaction:

NH2CONH2 + H2O → CO32- + 2NH4

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

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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.

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

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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

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 non-sterile (open) chemostat culture, Journal of Industrial Microbiology and Biotechnology, issn 1367-5453

Appendix

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