Cost Effective Continuous Urease Production by Bacillus Pasteurii Under Non-Sterile Conditions

Cost effective continuous urease production by Bacillus pasteurii under non-sterile conditions by a chemostat.

Amanda Edwards

Ednara da Costa Sampaio Alvino

Nicole Elliot

Shin Lau

Abstract.

Many technological applications utilise microbes, such as bacterium to covert a substrate to a particular desired product. Two systems that can be set up to exploit such bacterial processes are a batch culture and a chemostat. In this experiment a non-sterile continuous chemostat containing a culture of B. pasteurii was set up and run for the duration of 11 days, with the aid of computer software. This was done in order to produce the product urease and attempt to optimize such production. The chemostat was also run to observe what issues emerged and to overcome such issues. It was found that various technical issues with pumps (controlled by the computer software) arose. Likewise various chemostat parameters, such as pH and dissolved oxygen and dilution rate had to be adjusted throughout the experiment. Such issues had detrimental impacts on productivity and unease production, however once these were overcome the productivity and urease production increased. (This abstract does not give any of your findings (e.g. urease could be produced under non sterile conditions provided the pH…))

Introduction:

A batch culture is a contained environment with a finite running time, whereas a chemostat is a continuous culture with inflow and outflow. The disadvantages that affect batch culture such as nutrient depletion and toxic product inhibition can be overcome by using an open system with continuously growing culture. A chemostat can be either a sterile or non-sterile system and is characterised by a production vessel housing a microbial culture, with an inlet through which fresh media is pumped, and an outlet through which excess cells and product leave; through this design steady chemical conditions can be maintained. The growth rate of the culture is controlled by the dilution rate, which can be calculated by dividing the flow rate by the volume of the culture vessel and altered for optimal growth. The chemostat’s main advantage over batch culture is that through constant dilution with fresh media the growth rate as well as biomass, substrate and product concentrations can be kept constant throughout the process, achieving high productivity (Najafpour, 2007). Maintaining optimal conditions for the particular culture strain, such as pH is extremely important in most non-sterile chemostat cultures as any deviation from this may result in contamination by other microbes, which can greatly affect the desired outcome, such as the production of a specific enzyme.

The pH of the culture vessel must be maintained at a level appropriate to the species of microbe grown. The structural and functional integrity of a microbe’s internal proteins rely heavily on it’s cytoplasmic pH. If the microbe cannot maintain it’s internal pH then such proteins cannot function and growth and replication can be compromised. Consequently other microbes may colonise the culture vessel and compete with the desired microbe.

The microorganism used for this experiment was the alkalophilic bacterium Bacillus pasteurii, which requires a pH of 10 (Padan et al., 2005). B. pasteurii is an a soil bacterium that grows in alkali conditions containing high ammonium salt concentrations. It has been shown that ammonium is converted to free ammonia inside the cell for amino acid transport, namely glutamine. It has been assumed that through the direct activation of a sodium-translocating ATPase, an electrochemical sodium potential is produced, which the cell requires for the transport of substrate. This stimulation is caused by ammonium, which requires an alkaline pH to function. (Jahns, 1995). When ammonium salts are substituted for urea, B. pasteurii produces urease, an enzyme that hydrolyses urea to ammonia and carbamate (Bang, 2000).

Urea is a substance ubiquitous throughout nature, and large amounts are excreted into the environment in the waste of mammals, birds, reptiles and most terrestrial insects through many biological actions and its release is involved in the biodegradation of nitrogenous compounds such as arginine. The hydrolysis of urea is catalysed by the enzyme urease, which is synthesised by many eukaryotic and prokaryotic microorganisms. Microbial ureases are a significant substance both medically and ecologically. Bacterial ureases are associated with the pathogenesis of many bacteria species and have been implicated in clinical conditions such as urinary catheter encrustation, peptic ulceration, ammonia encephalopathy and the formation of infection stones. Conversely microbial ureases have a significant role in the rumen and gastrointestinal tract of sheep, cattle and other ruminants. Animal derived urea is a major source of nitrogen for commensal bacteria colonising the rumen and is recycled there where its enzymatic activity releases ammonia. The hydrolysis of urea also occurs in humans, mice and other monogastrics however its significance is such animals is far less important than that of ruminants (Mobley and Hausinger, 1989)

In this experiment a non-sterile chemostat was run for the duration of two weeks containing a culture of B. pasteurii. The bacteria were fed on a media comprised of yeast extract, urea, sodium acetate and nickel chloride. The chemostat was run at 28°C and a pH of 10; this was maintained throughout the experiment using a sodium hydroxide pump which tested the pH every 20 seconds and adjusted it accordingly. The culture was continuously aerated and stirred using a an electronic stirrer to ensure the culture was well oxygenated. The feed, harvest and sodium hydroxide pumps were all controlled via a computer program designed for running a chemostat.

The main aim of this experiment was to run a non-sterile chemostat under continuous conditions through reproducing a suitable environment for the bacterium B. pasteurii in order to maximise its urease production over the two-week period. The urease activity of B. pasteurii was tested and recorded daily, along with the optical density and dissolved oxygxx en concentration. A second, equally important aim of this experiment was to become familiar with the setting up and operating of a continuous, non-sterile chemostat while overcoming the various issues that arose, such as contamination, adjusting flow rates to maximise productivity and dissolved oxygen concentrations.

(Introduction is very well written and it provides in depth background to what is needed to understand your project. Towards the end of the intro where it is supposed to lead to the objective and the purpose of this experiment there is less focus such that in fact the objective is not clearly enough specified. The fact that it is an open culture in which harsh conditions are intended to be used to favour the desired organism B. pasteurii over contaminants, is not coming out clear.)

Materials and method:

Chemostat Set Up:

A 600ml bioreactor was placed in a 280C water bath, the water level to be kept at the same level as that in the bioreactor and water to be kept at constant temperature. Attached to a stand and clamp, a drill stirrer was placed into the bioreactor from above. An airflow tube was inserted into the bioreactor; the tube was connected to an airflow source. Both feed and harvest bottles were connected to separate pumps via tubes; the tubes then ran from the pumps to the bioreactor. In the bioreactor, the harvest tube was shorter than the feed tube to ensure that the bioreactor was never fully drained into the harvest bottle. The feed and harvest pumps were also connected to the labjack data card, which was then connected to the computer, which controlled the Chemostat and recorded data with the LabView software. A pH probe was inserted into the bioreactor, which was connected to a pH pump responsible for pumping NaOH into the system automatically when LabView recognised a change in pH. (Diagrammatic set-up of entire chemostat pictured below, figure 1.1).

Chemostat Inoculation and Running:

At the beginning of the experiment, 600ml of Bacillus pasteuri was inoculated into the bioreactor. The culture was provided with feed for the duration of the experiment with varied retention times; harvest was also pumped out of the system. The harvest tube was placed at the 600ml mark of the culture vessel to ensure that 600ml of culture was always present. The harvest pump was set at a higher flow rate than that of the feed pump to ensure that the culture did not overflow into the water bath. The temperature of the water bath was constantly kept at 280C and the pH kept at close to 10. The stirring rate was kept at 400rpm.

Chemostat Feed:

Lab technicians prepared feed media daily, which had a pH of 10. 1L of feed was made up using 20g of yeast, 10.21g of urea, 20g of sodium acetate and 2ml of 50mM stock solution of NiCl2. Deionised water was then added to make up the 1L volume. The feed was stored in the fridge and then in a tray filled with ice while being used for the Chemostat to avoid contamination (pictured below, figure 1.2).

Urease Activity:

A conductivity assay was used to determine the cultures’ urease activity. This involved adding 10ml of 3M urea and 8ml of deionised water to a 50ml centrifugation tube. The conductivity meter was then turned on and set to mS to 2 decimal places. The conductivity probe was calibrated to 12.88mS before being rinsed with deionised water. With 10 mins on the stopwatch, 2ml of culture was added to the centrifugation tube, immediately followed by the conductivity probe. The probe was used to gently mix the solution, ensuring that all of the holes on the probe were fully submerged and filled with solution. The initial conductivity reading was recorded and the stopwatch started. The conductivity reading was recorded every minute over 10 minutes at room temperature. The overall change in conductivity was measured over the 10-minute period. The conductivity of the culture indicated the urease activity within the culture and, more specifically, how fast the Bacillus pasteuri converted H2N-CO-NH2 into NH4+. It was expected that the activity would increase over the 10-minute period. (How did this essay allow you to derive the units showing in figure 2.1?)

Optical Density (O.D.):

Approximately 2-3ml of feed was transferred into a cuvette and 2-3ml of culture was transferred into another cuvette. Once the spectrophotometer was switched on, the wavelength was set to 600nm. Using the feed sample as a blank, the spectrophotometer was ‘Auto Zeroed’. Replacing the feed sample with the culture sample in the carrier the optical density was taken and the absorbance displayed on the screen was recorded. If the O.D. was above 2, the culture sample was diluted with feed media and the process was repeated until an absorbance of less than 2 was recorded. The true optical density could be calculated by multiplying the absorbance reading by the dilution factor.

Dissolved Oxygen:

The dissolved oxygen probe was turned on and left to initialise on the desk for 5-10 mins. The probe was then held in the air to be calibrated, after which the reading should have read close to 100%. The range was then changed to mg/L (ppm) before being placed in the culture, ensuring that it was fully submerged. The reading from the screen was then recorded.

pH:

The pH was measured using a pH probe. The probe was calibrated and left submerged in the culture for the duration of the Chemostat experiment. When the LabView software detected a deviation from 10, the pH was adjusted by pumping in 10M NaOH.

Figure 1.1: Relative diagram of entire chemostat set up (sourced from “Chemostat Projects- Student Notes”).

Figure 1.2: Feed media used for chemostat is placed in tray full of ice.

Results

Results always start with text that introduces the figures not with un-introduced figures.

Figure 2.1: Urease activity over the time of experiment. The dilution rate was 0.035 h-1 from day 1 to day 8, and 0.075 h-1 from day 8 to day 11.

Urease Activity:

During the experiment time the urease activity went up and down as can be seen in figure 2.1. On day 3 and day 8, which were after the weekends (when the feed and harvest pumps were turned off), it was observed that the urease activity dropped. Day 3 and 8 had the lowest recorded urease activity throughout the experiment. After both decreases on day 3 and 8 the activity increased again steadily. In the case of the day 8, in order to increase the urease activity, it was decided to double the dilution rate (from 0.035 to 0.075 h-1). As can be seen on the graph 1, the urease activity increased after day 8.

Figure 2.2: pH over the days of Experiment.

Figure 2.2 shows the pH for the duration of the experiment. As can be seen the pH started lower than required, around 9. On day 5 the pH increased to the appropriate level of 10 and remained here for the duration of the experiment.

Optical Density:

Another measurement taken was the optical density (OD 600), which was used to measure the concentration of bacterial cells. From the value obtained of OD the biomass was calculate by the following equation:

Figure 2.3: Biomass productivity over the time of experiment. The dilution rate was 0.035 h-1 from day 1 to day 8, and 0.075 h-1 from day 8 to day 11. The biomass was calculated from relation of biomass and dilution rate.

Figure 2.3 shows the biomass productivity. As can been seen biomass productivity changed slightly between day 1 and day 8; however after doubling the dilution rate (from day 8) the biomass productivity doubled as was expected.

Figure 2.4: Dissolved Oxygen.

Dissolved Oxygen (D.O)

Figure 2.4 shows the dissolved oxygen concentration for the duration of the experiment. During the experiment the measurement of oxygen was not as expected. However it was found the oxygen probe not work properly and almost every time the oxygen concentration was measured, it was not reaching a stable value. Once a new oxygen meter was acquired more stable and suitable oxygen concentrations were recorded.

(Your results section has compiled all findings in figures and then displayed the figures without demonstrating intent. You don’t say what the purpose was of particular periods of the chemostat operation, what parameters were varied and what the effect was of changing certain parameters.)

Discussion:

As an ureolytic organism, B. pasteurii has the ability to produce urease enzymes when urea is given as an energy source (Sarda et al., 2009). Urease is produced in order to hydrolyse urea for ATP production for microbial growth. The increase in both urease and biomass productivity shown in figures 2.1 and 2.3 confirmed that urease was readily being produced and as a result, more biomass was also produced. (Correct. You were able to continuously run the chemostat. What about the key objective of the experiment to improve the urease production rate (productivity)?)

It is important to note that B. pasteurii is a spore-forming organism, which sporulates under stressful conditions for survival (Prescott et al., 2005). As recorded on Day 3, which was followed after a weekend of non-feeding, it is possible that the microorganisms had sporulated under such stressful condition. In its spore form, urease is not produced. This theory could explain the massive drop in urease activity but not in the biomass level shown if figures 2.1 and 2.3, respectively. (Could be correct but it is speculative as you have not evidence to back up this claim).

The dilution rate was set to 0.035 h-1 on Day 1 to 8, then increased to 0.075 h-1 on Day 9 onwards by altering the pumps on/off times. By increasing the dilution rate, it is only natural that productivity increased according to the equation - “ Productivity = Dilution Rate * Biomass Level”. The results shown in figures 2.1 and 2.3 confirmed the increase in productivity after the change in dilution rate after Day 9. (This could be a key part of your report (trying to increase productivity). However in this particular case you don’t want to produce bacteria (OD) as that may well be contaminants that overgrow your producing strain, but you want to produce the enzyme.)

B. pasteurii is an alkalophile and its optimum pH is around 10 (Wiley & Stokes, 1962). Controlling the pH to around 10 with the help of LabView, f high pH-tolerant bacterial strains can grow. This restricts the growth of unwanted strains, which is especially important in open (non-sterile) system. (So did you change the pH ? Did you feel there was contaminants growing as well for example by dividing the enzyme activity by biomass to obtain specific activity?)

Several problems were encountered during the eleven-day span of this experiment. The biggest problem was the technical issue with the computer software, where the pump would be stopped for many hours, sometimes all night before it was fixed. This irregularity of feeding and harvesting surely had impacts on the biomass and usearse production of the B. pasteurii culture. Other problems include the malfunction of the oxygen probe and the wrong set up for pH pump. As seen in figure 2.2, pH reached 8.95 on Day 1, due to no sodium hydroxide being pumped into the system. (What effect did you expect this to have and did it have the expected effect?) The pump was found to have a tube diameter too small for the pump. This meant that although it was registering a lower pH thean required and was pumping, no sodium hydroxide was reaching the culture vessel. These issues contributed to the fluctuation in results.

Overall, the continuous open (non-sterile) chemostat proved to be effective in growing a selective strain, if grown under with the suitable condition. It is useful and economical in industrial processes that do not require sterility such as biogas digestion and wine making (Cheng & Cord-Ruwisch, 2013). Under the optimum condition, high productivity can be achieved with low cost.