BIO301 INDUSTRIAL BIOPROCESSING AND BIOREMEDIATION

Chemostat ReportGroup 1

Bryce Hobbs

Fang Zhi Chen

Patricia Tay

Syaza Mohd Salem

Table of Contents

Introduction

Materials and Methods

Results

Discussion

References

Appendix

Introduction A batch culture is a closed environment offering a sterile environment, a limited running time with a set amount of growth medium. Here the bacteria go through stages of growth, production and senescence in the terminating system with limited influence and controls in the system (Perderson 2000). Batch culturing systems have benefits in the production of secondary metabolites which happens after the growth phase. The limitations in the batch culture area a limited supply of nutrients as well as toxic product inhibition (Perderson 2000).

Chemostats have a wide variety of uses. In the industry, chemostats are used in industrial manufacturing of products such as ethanol (Purwadi. R. 2007) or urease production which then can be used to produce bio-cement (Cheng 2013). The chemostat is a continuous culture system where the bacterial culture is maintained in the growth phase. Here the specific growth rate in the culture is influenced by the dilution rate (D) (Novich 1950). The dilution rate in the chemostat vessel is maintained by pumps set with the out flow extraction of enriched culture and product harvest set at a desired height and minimally faster than the inflow rate of the substrate growth medium of a specific concentration (Elswarth 1956). The chemostat regulates the bacterial culture with environmental controls that can be varied and monitored for changes in conditions like growth and productivity (Novich 1950). The regulation of the growth medium feed rate, oxygen levels, stir rate, temperature, and pH level in the vessel affects the outcome in growth and productivity of the culture (Novich 1950; Perderson 2000). This growth phase culturing of the chemotat allows the focus on primary metabolites produced from the steady state growth rate achieved (Elswarth 1956). The chemostat system may also be used to culture larger amounts of bacteria in an open system than could be achieved by a batch culture system, this is done by harvesting the outflow of the chemostat culture vessel (Cheng 2013).

The potential of bio-cement to achieve production levels can be found in a few species of ureolytic bacteria. These ureolytic bacteria demonstrate a pH 10 cell growth selectivity that is dependent on urea as a food source and identifiable by urease enzyme activity (Al-Thawadi 2012). This ureolytic bacteria species can be enriched in a chemostat operation (Cheng 2013). The pH 10 ureolytic bacteria tolerance acts as a form of selective enrichment of the culture where the bacteria is selected through a feed medium hydrolysing urea to produce ATP (Cheng 2013). Nickel within the urease enzyme assists in the urea hydrolysation to carbon and ammonia (Cheng 2013). Microbial urease has demonstrated the ability to catalyze Ca+ and urea to produce CaCO3, a process called microbial induced carbonate precipitation (MICP)(Cord-Ruwsich 2012;Cheng 2013). There exists an potential industry for the development of eco technology that is cheap, effective and environmentally friendly in the area of bio cements for

purposes like stabilisation of embankments (Sarayu et al. 2014). Presently the sterile conditions and the low productivity of batch culture systems for MICP make these bio-cementation projects too costly to be viable. While recent experiments in the open systems of chemostat systems where the bacteria are regulated by physiological tolerances by adjusting environmental conditions like pH to 10 is producing bacterial cultures in sizeable quantities to make bio-cementation a potential industry (Cheng 2013; Purwadi 2007).

A chemostat is used in this experiment is a 600ml bioreactor where the growth rate of a specific bacterial culture is maintained by a dilution rate introducing a steady state inflow of growth medium and a outflow of culture harvest and enzyme product with the use of pumps which can be seen in figure 1 below. Parameters such as dissolved oxygen concentration (DO), stirring rate, temperature, pH, cell optical density (OD) and the nutrients provided via the feed line is kept as a constant so that culture growth rate can reach a steady state is equal to the dilution rate of the chemostat (Novick. A. 1950). The chemostat for this experiment was kept in an open and non-sterile environment which may lead to a rise of contaminants present in the chemostat. This is countered by adjusting parameters such as pH to harshen the conditions in the chemostat to prevent the growth of airborne contaminants that can be introduced into the chemostat while favouring the growth of the desired bacteria. The aim of the experiment is to demonstrate that an open chemostrat is a viable method of culturing ureolytic bacteria as a pH 10 physiological selective species in open systems. Urease activity is used as an indicator of the culture density along with optical density (OD) as to the success of the species cultivation. This experiment also aims to demonstrate that a substantial amount of urease could be produces demonstrating a viable cultural enhancement using chemostat with a pH 10 selectivity. This result could be used to produce bio-cement and give support to the research paper of Cheng 2013. The setup was run over 3 weeks and on the third week the hypothesis was tested.

Materials and Methods

Chemostat

Materials Setup is done as shown in Figure 1 with the following equipment:

1. Chemostat Feed media2. Harvest Vessel3. Inflow and outflow pumps1. Spectrophotometer2. pH probe3. pH buffers (pH 7, 10)4. NaOH pump5. Dissolved Oxygen probe6. Conductivity probe7. Electronic Stirrer

Methods

1. Biomass Concentration based on ODThe Biomass was determined by measuring the optical density using a spectrophotometer. A culture with a higher biomass would have more cells per unit of space hence having a higher population density. As such, when light attempts to pass through the solution, less of it will get through than the same light attempting to pass through a less dense solution due to the reduced amount of cells blocking the path of light.

a. A sample from the chemostat vessel and the feed vessel was obtained and transferred to a cuvette.

b. The absorbance was measured and recorded at 600nmi. 1:10 Dilution was done if the reading was above 1

c. Biomass is then calculated by multiplying the absorbance by 0.44 (Cheng 2013)

2. pH Calibrationa. With the use of the computer, calibration was done by synchronizing the pH probe with

the use of pH buffers to the computer. b. The NaOH pump which is connected to the computer helps to maintain the pH when a

drop in pH is detected.

3. Urease Activitya. A 2mL sample was obtained from the chemostat.

b. It was then mixed with 8mL of 3M Urea and 10mL of DI waterc. The conductivity probe is then used to measure the Urease activity by recording the

readings at 1 minute intervals for a total of 10minsi. Urease activity is calculated by the difference obtained from the first and last

reading (mS/10mins).

4. Oxygen Concentration (DO)a. Using a calibrated oxygen probe, the dissolved oxygen from the chemostat was

measured and recorded to ensure that the amount of oxygen supplied is sufficient for bacterial growth.

5. Other Parametersa. The temperature of the water bath was maintained at 28-30℃b. The stirring speed was constant at 400rpmc. The oxygen airflow was constant at 50L/h

6. Growth medium (feed)a. The feed of the Urease positive bacteria (S. pasteurii) was composed of 20g/L Yeast

extract, 20.42g/L (0.34M) Urea, 20g/L Sodium acetate and 2mL of 50mM stock/L NiCl2 (0.1mM).

b. The pH of the solution was adjusted to 10 with NaOH after being made up to a volume of 1L with distilled water

Biocementation

Materials

Biocementation container with sand 50ml flow through system with unused fluid capture.Culture from chemostat harvest bottle 50mlCalcium Chloride and urea feed medium

Method

Poor chemostat harvested culture slowly through sand in biocementation container let settle.

Then slowly drip 10mls feed calcium chloride and urea feed medium using 1ml plastic pipette through biocementation container as feed for the bacteria settled between the pores of the sand.

Repeat feed process after 7 hrs with 20mls of feed medium.

Figure 1: Overview of chemostat setup.

Figure 2: Chemostat diagram courtesy of Ralf Cord-Rudwich

Results

Chemostat

Effect of variant feed regimes on the productivity of Urease from feed mediaChemostat operations producing continuous

Figure 3: Results obtained when the chemostat was left with a constant pH of 11.3. HRT = Hydraulic Retention Time

The data from the above table was obtained from the first bacterial culture obtained in the first week. The bacteria that was used is able to withstand a high pH of 10. This is one of the parameters used to reduce the growth of foreign airborne bacteria. When the pH was increased to 11.3, a significant decrease was seen, which also caused the death of the bacterial culture.

Figure 4: Comparison of results obtained between the chemostat set-up and batch culture over the weekend.

As the equipment for the chemostat was switched off during the weekends, the environment in the vessel would then behave as a batch culture from the conditions set by the chemostat. The highlighted yellow regions indicate the period whereby the vessel undergoes batch culture (Fig 4.). There are 5 stages in total between the chemostat set-up and batch culture throughout the experiment. The first chemostat stage takes place within the first 25 hours. At this stage, Specific Urease Activity and Enzyme Activity increases from 12.69 mmol/min/g biomass to 18.6 mmol/min/g biomass and 10878 umol/L/min to 13320 umol/L/min respectively.

From the graph, it can be seen that Enzyme Activity shifted from 8000 umol/L/min to 17000 umol/L/min and Specific Urease Activity increased from 12 mmol/min/g biomass to 17 mmol/min/g biomass during the first batch culture stage. The optical density increased in a relatively small amount, from 2.275 mmol/min/g biomass to 2.389 mmol/min/g biomass.

During the chemostat stage of HRT 91H to 185H, there is quite a variability in the data. The urease activity had a major decline from 17.3 mmol/min/g biomass on the 91st hour to 4.8 mmol/min/g biomass within the next 20H, then continued to decrease to 3.74 mmol/min/g biomass towards the end of the phase. Enzyme Activity decreases from 17316 umol/L/min to 12210 umol/L/min on 137H, then increases to 15540 umol/L/min on 160H before a slight decrease to 14763 at 185H.

Between 185H to 255H in batch culture stage, all three data points seem to decrease over the weekend. Enzyme activity has decreased significantly from 14763 umol/L/min to 2553 umol/L/min. Optical Density decreased from 8.97 to 7.46, corresponding to a decrease in

biomass concentration of 3.95 g/L to 3.28 g/L, and Specific Urease Activity decreasing from 3.74 mmol/min/g biomass to 0.78 mmol/min/g biomass.

In the final stage of chemostat between 255H to 279H, all data points have slightly increased, with Enzyme Activity, Optical Density, and Specific Urease Activity measurement of 2553 umol/L/min to 3552 umol/L/min, 7.46 to 9.82, and 0.78 mmol/min/g biomass to 0.82 mmol/min/g biomass respectively.

Optical Density on the other hand generally increases throughout the experiment, but there is a slight decrease between 150 and 250 hours. As the optical density relates directly to the biomass concentration, the biomass also follows the Optical Density trend, up to 4.3208 g/L when the OD is at 9.82.

Figure 5: Results obtained when testing the hypothesis of increasing the feed rate by 100%

As seen from the data above, there is a significant increase in optical density and enzyme activity when the dilution rate is increased. Increase in optical density indicates that there is an increase in biomass concentrationThe Optical Density is at about 3.58 at the start of the experiment, and has gradually increased to about 5.6 and 6.9 at 23rd and 47H respectively before reaching to 9.72 at 71H.

Optical Density is used to calculate Biomass Concentration following the equation Biomass concentration = 0.44*OD. This works out to 1.5752 g/L of biomass at the start of the experiment and 4.2768 g/L on the 71st hour.

The Specific Urease Activity is an indication of the efficiency of urease production by the bacteria. The Specific Urease Activity is 9.58 mmol/min/g biomass at the start of the experiment, decreasing to 5 mmol/min/g biomass at the 23rd hour then increased up to 5.45 mmol/min/g biomass on the final reading. Increasing feed rate has resulted in a decrease in Specific Urease Activity within the first 20 hours, then it slowly increased until 70H.

The Enzyme Activity is 15096 umol/L/min at the start of the experiment, decreasing to 12321 umol/L/min at the 23rd hour, then increases gradually to 16095 umol/L/min and 23310 umol/L/min at 47H and 71H respectively.

Biocementation

After first initial placement of culture and feed medium the sand medium was stirable using the 1ml plastic pipette with a consistency of wet sand. The following day the sand appeared to be set in a cementitious form when applying force with the pipette, this was most prevalent on the sand around the edges while the centre of the sand mix in the container had not set.

Discussion

The group’s hypothesis was tested by increasing the dilution rate by 100% to determine if there would be an increase in urease activity which would indicate a culture enhancement for use in the production of biocement. Hence, it can be deduced that there is an increase the desired bacteria in the chemostat.

The setup induces a flux between a batch system and a chemostat system. This is induced when the dilution rate as the inflow feed medium and the outflow culture was turned off at 5pm on Friday and on again at 9am on Monday. A new condition is observed on each of the Mondays and this was dependant on the factors affecting the chemostat when turned off causing the system to switch over to behaving as an open non sterile batch system. A fresh cultural enriched bacteria from a sterile batch system supplied from the technicians was placed into the chemostat three times during the experimental operation time over three weeks as a result of three operational failures that was observed.

The effects of change made on the culture supplied from a closed batch system to a open chemostat then a open batch system and back again to a open chemostat was observed as changes in OD, DO, pH, Temperature, and urease activity.

The first experimental operation - the effect of high pH

In the first experimental operation, the effect of high pH was observed due to a system failure. In order to reduce the growth of foreign airborne bacteria, the pH of the chemostat vessel was set at a pH of 10. This enables inhibition of the growth of foreign bacteria while not affecting the desired bacteria. In the system failure event the pH increased to 11.3 and the growth of the desired bacteria was inhibited as seen in Figure 3. At pH level of 11.3, the Optical Density, Specific Enzyme Activity, and Specific Urease Activity significantly decreased as compared to that at pH level of 10. The DO increased from 5.78 to 6.65 shows how the oxygen use decreased along with the urease activity that decreased from 1.0 to 0.1. The high pH change exhibited strong influences on the culture and caused culture death.

In the second experimental operation - chemostat v’s batch culture

Within the first stage of chemostat, which is in the first 25H, OD has decreased, whereas Enzyme Activity and Specific Urease Activity level both increase both increase before decreasing in the last 3H. A decrease in OD indicates that there was a decrease in biomass concentration (g/L). As the stirring rate, airflow rate, inflow feed, and temperature were kept constant, the decrease in OD may be due to bacteria adjusting to the pH level and removal or death of foreign bacteria that can not withstand the pH level. The same reason is applied to the decrease of Specific Urease Activity and Enzyme Activity in the last 3H of the first stage of chemostat culture.

The second stage of the experiment, which is within the open non-sterile batch culture stage at 25H-91H, the bacteria consumed the feed medium, multiplied and was were not washed out and produced waste buy products. Here the OD, Specific Urease Activity, and Enzyme Activity have all increased until the system reaches its inhibition limit. This may be because in batch culture, cell growth is limited by accumulation of waste products and not by nutrients. Hence, there is a higher cell concentration of the bacteria and the cell viability is maintained for a longer period of time.

In the third stage of the experiment the chemostat was turned on again (91H-185H). The Enzyme Activity decreased and then increased while the OD showed a steep stepped growth the Specific Urease Activity has decreased within the five days. These changes relate to the bacterial culture biomass reaching a steady state after the dilution rate is reintroduced into the system. The plateauing of the biomass mid-week and the decrease Enzyme Activity decrease followed by an increase of the biomass and Enzyme Activity shows that the pH level had reselected for the wanted bacteria.

The fourth stage from (185H-255H) where the system was turned off again for the weekend showed again adjustment changes to the open non sterile batch system where the slight decrease in biomass is matched by a rapid decrease in Enzyme Activity and a substantial lowering of Specific Urease Activity to the stage as to consider the selected bacterial culture and non-viable as the culture was dominated by unwanted bacteria.

In the fifth stage 255H-279H the chemostat was turned back on and a slight increase in activity was seen in biomass and Enzyme Activity but this was low so the experiment was terminated to establish a new culture for the hypothesis testing. Observations drawn from the experiment

was that pH strongly influences bacterial selection and that chemostat processes allow for this process if it is not interrupted to perform as an open non-sterile batch system which can be seen in figure 4.

In the third experimental operation - the effects of an increased dilution rate

The hypothesis tested stated as a positive null for the effect of an increase in the dilution rate by 100% from the dilution rate established in the setup methodology. This experiment demonstrated a positive null after allowing the chemostat to settle into a steady state by showing an increase in OD, Enzyme Activity as well as Specific Urease activity in an open chemostat system operation. The increase in the measurement of OD represented an increase in the biomass of the selected bacterial culture, this which was verified by the measurement of the increase of the Enzyme Activity of this particular bacterium as seen in figure 5. The results demonstrated that a bacterial enrichment process using pH selectivity in a chemostate system is possible.

Bio-cement formation from chemostat culture enrichment harvest

A final experiment was performed using the chemostat bacterial culturally enriched harvest for bio cement formation. This was demonstrated with the hardening and setting of the sand as indicated by the results and it is proposed that the sand would have achieved more thorough hardiness had it been left for a greater period of time. This indicated the viability of the chemostat to supply active culture in sufficient quantity for the formation of calcium carbonate between the sand pores.

This positive null gave support our hypothesis and also gave support to the paper of Cheng 2013 that an open chemostat system is a viable economic eco-technology method in industry for the cultivation of ureolytic bacteria for the purpose of creating bio-cement and is worthy of further scientific investigation.

7.5 + 0.5 (cementation trial) = 8 /10

This is a mixed quality type report with excellent intro and some good paragraphs but also major parts of text of lesser quality. The results are not easy to read and understand and seem mainly descriptive.

References

Al-Thawadi. S.M., Cord-Ruwisch. R. “Consolidation of Sand Particles by Nanoparticles of calcite after concentrating ureolytic bacteria in situ.” International Journal of Green Nanotechnology 4 (2012): 28-36.

Cheng. L., Cord-Ruwisch. R. “Selective enrichment and production of highly urease active bacteria by non-sterile (open) chemostat culture.” Journal of Industrial Biotechnology 40, no. 10 (2013): 1095-1104.

Cord-Ruwsich. R., Al-Thawadi. S.M. “Calcium Carbonate crystals formation by ureolytic bacteria isolated from Australian soil and sludge.” Journal of Advanced Scientific Engineering Research 2 (2012): 13-26.

Herbert. D., Elsworth. R., Telling. R.C. “The Continuous Culture of Bacteria; a theoretical and experimental study.” Journal of General Microbiology 14, no. 3 (July 1956): 601-622.

Novick. A., Sziland. L. “Description of the Chemostat.” Science 112 (1950): 715-716.

Perderson. H., Beyer. M., Nielson. J. “Glucoamylase production in batch, chemostat and fed-batch cultivations by an industrial strain of Asperigillus niger.” Applied Microbiology and Biotechnology 53, no. 3 (2000): 272-277.

Purwadi. R., Brandberg. T., Taherzadeh. M.J. “A Possible Industrial Solution to Ferment Lignocellulosic Hydrolysate to Ethanol: Continuous Cultivation with Flocculating Yeast.” Journal of Molecular Science 8, no. 9 (2007): 920-932.

Sarayu K, Nagesh R, Ramachandra (2014) Exploration on the Biotechnological Aspect of the Ureolytic Bacteria for the Production of the Cementitious Materials—a Review. Applied Biochemistry and Biotechnology Volume 172, Issue 5, pp 2308-2323.

Appendix PhotosUpper Left: Stirrer set at 400rpm, stirring the bacterial culture in the beaker. Upper Right: Harvest Vessel on the left, Feed Vessel on the right.Bottom: Biocementation Setup.