Kristen Redmond Patricia Tay Katheren Rumbold Caroline Chua

27/10/14Chemostat culture of Sporosarcinapasteurii Group 6

Introduction

A chemostat is a bioreactor where the chemical and biological environment – dissolved oxygen concentration, pH, cell density and nutrient concentration – remains static (Novick. A. 1950). The unique steady state is created by constant and equal volume of inflow and outflow, of which can be adjusted to set the specific growth rate of the micro-organism. In this experiment, a small 500mL bioreactor was maintained in chemostat conditions by the simultaneous pumping in and out of equal volumes with Dema pumps and continuous stirring, air pumping, and pH monitoring (Figure 3).

A chemostat can either be a closed sterile environment or an open, non-sterile environment. In an open chemostat the desired culture is selected for and maintained by creating an environment that is most suitable to the desired micro-organism and less hospitable to other organisms. In this experiment the chemostat was maintained at a pH of 10 by the periodic pumping of 10 molar NaOH with a micro-pump and a high urea concentration (<5M), which ensured that the environment was inhospitable to most air-born infectants.

Chemostats are used widely. In research applications they are primarily used to determine steady state data on particular organisms and to model metabolic processes. (Herbert. D. 1956) In industry, chemostats are used in industrial manufacturing of products such as ethanol (Purwadi. R. 2007) or enzymes, such as amylase production (Perderson. H. 2000) or urease production (Cheng. L. 2013).

In this experiment the chemostat has been used to cultivate the urea amidohydrolase enzyme from Sporosarcinapasturii type alkaliphilic bacteria. Sporosarcinapasteuriiis a Gram positive bacteria which can thrive under extreme conditions of high ammonia concentrations and pH 10 where few bacteria thrive (Cheng 2013). The economic importance of its ability to produce CaCO3 to reinforce cement and solidified soil in earthquake vulnerable areas, from its urease activities has made it a highly valuable commodity in the construction industry (Sarayu et al. 2014).

301 group 6 culture P22 9 14 jpg

Figure 1: Sporosarcinapasteurii of group 6. Photo by technician Carol Tann.

Urea amidohydrolase (urease) is an enzyme which catalyses the hydrolysis of urea. Traditionally this has been isolated from Jack beans (Canavliaensiformis) and crystallised for research use. With the capacity to industrially process urease from S. pasturii, the possibility of utilising the microbial urease to catalyse calcium carbonate precipitate in the presence of calcium and urea to bio-cement or soil strengthen has arisen (Al-Thawadi. S.M. 2012).

Sporosarcinapasteurii has been shown by Al-Thawadi and Cord-Ruwisch (Cord-Ruwsich. R. 2012) to be selectable in an environment consisting of high pH (~10) and urea concentrations up to five mole per litre. Following these guidelines Cheng (2013) subsequently developed achemostat with hydraulic retention time (HRT) of 10h-1 that could maintain urease production at 60umol min-1 mL-1.

The purpose of this experiment is to optimise the production of urease under aerobic and non-sterile chemostat conditions. This is done by modification of feed concentration of yeast extract, airflow rate and HRT of the S. pasturii culture. The experiment is also set-up for selective cultivation via high ammonia concentration, 0.17 M urea in yeast extract and pH 10.

Materials and Methods

The chemostat was taken over in fully functioning operation from Group 4 on 18/09/2014. The chemostat had been running for approximately 10 days at the time of take-over and had been operating with a HRT of 23.4h-1.

The chemostat consisted of a stirred glass 1L beaker that was maintained at a volume of 500mL by the positioning of the removal pump pick up hose. The set-up of the chemostat and control network can be seen in Figure 3.

The stirring rate was initially set at 400 rpm, the airflow at 50L/min and the pH at 10. The pH was controlled at the set point and dosed with NaOH (10M) as required to maintain the pH. The pH meter was calibrated daily to ensure accuracy. Dissolved oxygen, urease activity, biomass, airflow and stirrer rates were monitored and recorded daily. DO was measured with a polarographic oxygen electrode. The urease activity was measured by the change in conductivity over a ten minute interval using a conductivity probe. The biomass was determined by spectrophotometric absorption at 600nm using the feed media as blank and multiplying the optical density by 0.44 as per Cheng (2013). The airflow rate and stirrer rate were read directly from the meters controlling flow or drive as required.

The feed bottles were placed in ice to avoid contamination of the feed flowing into the reactor and consisted of 2L glass bottles containing yeast extract (20g/L), Urea (0.17M), sodium acetate (20g/L) and NiCl2 (0.1mM). At time of take over the feed had contained yeast extract at 40g/L. All other parameters were left to determine the steady state operation for the new feed.

The chemostat was placed in a water bath to maintain the temperature at 28 deg C, however overnight from 18/09/2014 to 19/09/2014, the water bath temperature controller broke and the temperature rose to 36 deg C for approximately one day.

No variables were changed on 19/09/2014. At 4pm, the system was shut down completely, with no stirring, no air supply and no pumping in or out and no dosing with NaOH for the weekend as no data could be obtained over this time.

Due to the time constraint, the group was given a week to alter variables for maximising urease production. Therefore altering variables such as pH were decided against as pH alterations had been shown to lead to cross species contamination (Cheng 2013). The pH has been maintained at 10 throughout the experiment. This selects specifically for S. pasturii as there are other urease positive organisms such as P.vulgaris which can thrive in fairly high pH (9.5) and high ammonia concentration (Cheng 2013). The variables which have been altered are the feed media yeast concentration, HRT and airflow rate.

Materials

Chemostat Feed media containing (Figure 1):

Yeast Extract : 20g/L

Urea: 20g/L

Sodium Acetate: 20g/L

NiCl2: 2mL of 50mM stock/L

Spectrophotometer

pH probe

pH buffers (pH 4, 7, 10)

Dissolved Oxygen probe

Conductivity probe

Biomass determination

Calibrate the spectrophotometer and use the feed media as a blank
Obtain sample from chemostat and measure the absorbance at 600nm
Optical density (OD) should not be over 2. Dilution will be done if necessary
Biomass = 0.44 x OD (Cheng L. 2013)

pH calibration

Calibrate the pH probe with the use of the buffers
Take pH measurement of the chemostat with the use of the Labview software

Oxygen concentration

Calibrate oxygen probe
Submerge the probe into the chemostat and ensure that there are no bubbles on the probe
Allow the probe to stand for 5mins before taking the reading.

Urease Activity

Calibrate the conductivity probe using the Urea buffer
Obtain 2ml of sample from chemostat and mix it with 8ml of 3M Urea and 10ml of DI water.
Immediately immerse the probe and start a timer for 10mins.
Record the reading shown every minute
(Reading @ 10mins - Reading @ 1min) / 10mins = Urease activity (mS/min)

(Much better presented than in the original version of the chemostat)

Figure 2: Overview of chemostat setup.

Figure 3: Chemostat diagram courtesy of Ralf Cord-Rudwich

Results and discussion

Effect of SR (substrate reservoir) yeast extract concentration on urease activity produced from S. pasturii.

Yeast extract concentration was halved to determine the effect on urease activity, under a hydrolytic (hydraulic) retention time of 23.44 hours and an airflow rate of 50L/hr. A urea concentration of 0.17M and pH 10 was maintained to select for the alkaliphilic bacterium, S. pasturii. The substrate yield co-efficient would predict a halving in the yeast extract substrate reservoir (SR) concentration would halve biomass concentration and therefore urease activity. (Good) After 20 hours of incubation at the new yeast concentration (20g/L) from day one to day two (figure one) specific urease activity dropped and biomass increased slightly – an indication of contamination. Urease catalyses the conversion of urea to ammonium, the latter existing in equilibrium with ammonia in solution: NH4+ = NH3 + H+. Correct. (Could point out how then alkaline pH causes more toxicity in liberating the toxic NH3. )

With a sudden decrease in urease activity by 67% (table one) and less ammonium - more ammonia - in the system, a sudden increase in acidity provided the opportunity for contamination. (Good) A drop in pH from 10 to 9.5 in Cheng et al., 2013 study caused a significant decrease in specific activity. (Also good to relate this to literature) The urease activity reasonably decreased by from day one to day two 67% (table one) given an expected halving in activity and potential contamination.

The feed pump was turned off on day two and after 71 hours of starvation, a slight decrease in biomass and stable urease activity were expected, due to elimination of carbon source. The 37% increase in urease activity (table one) indicates bacterial cell lysis and release of urease enzyme from cells (Cheng et al., 2013) and the increase in biomass can be associated with acidification driven contamination, as the NaOH pump was disabled.

After day five, the bacteria were given 24hours to readjust to the onset of substrate availability (20g/L) (figure one). Specific activity increased from day two (before weekend) to day six (24 hours after pump was activated) (figure one) indicating a decrease in contamination. ( Good) Comparison of day one data to day six data (table one) provides support for the original hypothesis, with a 54% decrease in urease activity. A19% decrease in biomass rather than 50% is due to contamination, indicated by the decrease in specific activity from day one to day six (figure one).

It can be concluded that a halving in the substrate concentration will cause a halving in urease activity and contamination due to a sudden upset in the balance of ammonium and ammonia, leading to a decrease in pH. Contaminating bacteria will compete for substrate and push the concentration of S. pasturii below the minimum 50% biomass decrease. Prolonged pH regulation should solve the issue of contamination. Conditions on day six should have continued for another 24 hours to see if continued pH regulation after the weekend, could increase the specific activity back to the value before substrate halving. Cheng et al., 2013 showed that subtle pH change was critical for selective urease production.

In Cheng et al., 2013 study, a urease activity of 19.1 umol/min/ml was attained under conditions of 0.17M urea, 20g/L yeast extract, 40g/L sodium acetate concentration and a steady state oxygen concentration of 5-6mg/L. The highest urease activity attained under the same urea and YE concentration in the current investigation was 9.11umol/min/ml. It has been reported that sodium acetate can enhance the total urease activity by increasing specific urease activity (Cheng et al., 2013). In the current experiment, half the sodium acetate concentration was used. Dissolved oxygen stabilized at a concentration of 3.71mg/L during urease activity of 9.11umol/min/ml (figure one) – a concentration below the required 4.5 mg/L. These factors and contamination can explain the significantly lower urease activity attained in the current experiment, compared to Cheng et al., 2013. Good to see comparison of own results with literature

Table 1: Changes in urease activity and biomass concentrations under certain yeast extract, substrate reservoir (SR) conditions, in terms of percent

Effect of aeration and dilution rate on urease activity produced from S. pasturii.

KLa was altered by changing air flow rate, to determine the effect on urease activity. On day six, air flow rate was decreased from 50L/hr to 25L/hr and HRT was decreased from 23.44 hours to 12 hours. The effect of aeration on urease activity was therefore monitored with day seven (time zero in figure two) as the superficial starting point. Increasing airflow only would lead to an effect if the DO was close to zero before. I can’t see whether this is the case.

The reactor was run for 24hours after a doubling of the air flow rate at time zero, in an attempt to increase urease activity. Specific urease activity increased by 42% (table two). This change can be associated with a greater KLa and lower D.O content (table two) resulting in a greater oxygen transfer rate, equal to the oxygen uptake rate during steady state bioreactor condition. Biomass also increased, leading to a prominent 70% increase in urease activity (table two). Conditions were continued for another 24 hours to see if the steady-state urease activity could be prolonged. Decreases in urease activity, specific activity and biomass are not significant (figure two) and are associated with a temporary disconnection of the air pump.

The results show that air flow rate has an effect on specific urease activity under oxygen limitation. Despite the increase in urease activity, only a maximum of 4.33 umol/min/ml was attained (figure two).

Under conditions of 50L/hr airflow and HRT of 12 hours, a urease activity of 4.33 umol/min/ml was attained (figure two) whereas 9.11umol/min/ml was attained under the same air-flow rate but a HRT of 23.44 hours (figure one). As dilution rate increases, biomass concentration decreases due to slight washout, as demonstrated by figure 3. Fair explanation. The decreased specific activity due to further contamination, also results in a significantly lower urease activity. The positive effect of an increased dilution rate on productivity is not sufficient enough to accommodate for the difference in total urease activity between day six and day eight (figure three) leading to an overall decrease in productivity (table three).

If the specific activity could be increased, productivity may be increased by increasing dilution rate. Cheng et al., 2013 demonstrated that specific activity is stimulated by increased urea concentrations, however, biomass decreases simultaneously.

Table 3: Effect of HRT on urease productivity under SR: 20g/L and air flow: 50L/hr.

Enhancement of urease productivity of S. pasturii bioreactor

In the current study, a maximum urease activity of 9.11 umol/min/ml was attained. This activity failed to meet the threshold – 10umol/min/ml – required for direct application in biocementation (Cheng et al., 2013). When the chemostat was handed over, a yeast concentration of 40g/L was already in place. To determine the effect of SR on urease production, the SR had to be lowered (by half) resulting in a halving of urease production. These results demonstrated an important relationship between SR and urease production, but with a consequential decrease in urease production, of which the aim was to maximise.

Contamination was a major hindrance in the production of urease activity after yeast extract reservoir concentration was halved. Contamination was believed to be caused by a subtle decrease in pH. PH regulation appeared to stimulate specific urease activity, however the original activity prior to SR change could not be reached.

An increase in air-flow was shown to increase specific urease activity, biomass and consequently, urease activity, under oxygen limitation. However, only very low urease activities could be attained under a HRT of 12 hours, airflow of 50L/hr and SR; 20g/L. The low urease activity was associated with a low specific activity indicating contamination. Due to contamination, an increase in dilution rate, failed to increase productivity.

It can be concluded that a combination of high yeast extract concentration, air-flow rate and greater control of contamination have the potential to stimulate high urease production from S. pasturii in an open, continuous, non-sterile operation. Longer operation of the chemo-stat would have allowed a more thorough investigation into this potential. Completely different report, demonstrating understanding a purpose and the effective use of literature findings. This report would be top of class but must be marked down to allow for the extra time used for the second revision. 8.5/10  7.5/10 which is much higher than the original 4.5/10

References

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Master Thesis. Univeristy of Montana.

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Appendix

Photos

Left photo: on the left, bottle of feed media on the right: outflow collection bottle.

Bottom left: Stirrer set at 400rpm and at the bottom of stirrer is the culture in a beaker.