(1 M). Power Outage and Oxygen Limitation Conditions Limited the Determination of Maximum

Biocement

Jo-Anne Tan 30382633

Chier Ai Na 30384126

Robert Syme 30271254

Alex Yong 30383694

Matt Tye 30385839

Mark Robert 30407165

Cielito Marbus 30229879

Peter Zeller 30385866

abstract

The production of urease by Sporosarcina pasteurii in a chemostat was investigated. The specific urease activity was on a decreasing trend from day 2 to day 12. The maximum specific urease activity was on day 2 (0.621 mM urea hydrolysed/min/OD). A dramatic drop in specific urease activity occurred after a power outage on day 10-11. The maximum productivity (of what? If the product is urease it should be given as urease (in mM/h) produced per L of reactor per hour). was obtained on day 15 (0.365g/L/h). The oxygen uptake rate (OUR) and the kLa increased when the stirring rate was increased. The ammonium productions were constant throughout the experiment. Cement was successfully produced using urease (the product), calcium chloride (1 M) and urea

(1 M). Power outage and oxygen limitation conditions limited the determination of maximum activity of urease. In conclusion, even though there was product formation, this experiment was not deemed sustainable under non-sterile condition over 3 weeks. (Conclusion is clear, however the aim should be explained first)

Introduction

Sporosarcina pasteurii, formally known asBacillus pasteurii, is a Gram-positive alkaliphic microorganism that requires high ammonium concentration of around 40mM for growth. (Leejeerajumnean et al. 2000, Mörsdorf et al. 1989)It is a widespread soil bacteriumthat is unique among the Bacilli because it produceslarge amounts of urease. (Ciurli et al. 1996)

Urease is an enzyme that is designed to hydrolyse urea to its smaller components to free ammonium for nitrogen use by the bacteria.

H2N-CO-NH2 + 2H2O ------ 2 NH4+ + CO32-

When combined with water and urea, urease breaks down the urea into carbon dioxide and ammonium. As the carbon dioxide and ammonium are in the water the carbon dioxide becomes carbonic acid (hydrogen ions and carbonate) and the ammonium undergoes an equilibrium with ammonia (Gibson, 1934). This is useful for maintaining a pH as the excess hydrogen ions can be taken up by the free ammonia that hasn’t left solution via evaporation (Hausinger, 2004). The carbonate produced in this process can then be combined with Calcium ions to produce calcium carbonate. In order to function effectively urease requires that nickel ions are present in order to act as a cofactor. Each molecule of urease requires 2 nickel ions to work effectively as shown by figure 1.

Ureases are homo-oligomeric proteins which contain 90 kDa and it has multimers of two or three subunits complexes (Follmer et al., 2004) and it is commonly found aerobic and anaerobic in some of microbial species (Hammes et al, 2003). Urease requires at least 4 accessory protein such as UreD, UreE, UreF and UreG for its maturation (Lee at al., 2002). UreE is the only nickel-binding protein among all the accessory proteins and a metallochaperone is use to fit in the nickel ions to urease (lee et al., 2002). (This is an example of more detail included than what is necessary to introduce the objective of the project. While it is correct it somewhat takes away focus from the report. And focus is important.)

S. pasteurii was selected for this experiment because of its ability to grow at ammonia concentrations greater than 500 mmole.L-1.(Leejeerajumnean et al. 2000) The significance of this for the experiment is that it has been shown that very few strains of bacteria can tolerate highly alkaline environments around pH 9.(Leejeerajumnean et al. 2000) This is advantageous to the experiment as the high pH and ammonia concentrations inside the chemostat should inhibit the growth of any contaminant strains.

The purpose of producing the biocement(you made a logical jump here from urease to biocement without explaining the link between the two, meaning the reader may get confused) in the way proposed by this experiment is to preform in situ cementation (currently not practical with other methods). This can enable a variety of uses though they are limited to specific situations such as hardening the dykes in Holland. There are several advantages to using this method namely; lack of time required for labour (just spray on and the bacteria do all the work), hardening of present structures (like the dykes in Holland) and production of highly porous cement (easy to drill and good for drainage).(To many gaps in the presentation to allow the reader to understand)

This research plans to expand on previous papers by examining more than just the optimum conditions of bacterial growth(be specific with the goals). Alternatively, this research aims to investigate the feasibility of producing ‘biocement’ from the degradation of urea to ammonium and carbonate via the urease enzyme formed by Sporosarcina pasteurii enzyme. From this research we can ascertain relevant industrial or commercial applications of this product.

Materials and Methods

Organism.S. pasteurii obtained from R. Cord-Ruwisch, Murdoch University.

Growth Media. S. pasteurii strains were grown in a complex media containing: yeast extract at 20g/L, ammonium sulphate at 75mM, sodium acetate anhydrous at 100mM, urea at 330mM and Ni2+ at 5 µM. The media pH was adjusted to 9.25 using 5M NaOH. The growth media was not autoclaved.

Growth Conditions. A 2.5mL inoculum of S. pasteurii was grown in 1L of growth media as a batch culture for 24 hours in a shaking water bath at 25°C. After initial growth had been confirmed, (by what)the bacteria were transferred into a chemostat reactor with a 500mL working capacity. Growth medium was added to the reactor at a dilution rate of 0.0449 h-1 unless otherwise specified using a Chemaster™ peristaltic pump. The culture was stirred at a variety of stirring rates due to mechanical failure. The reactor was oxygenated with unfiltered air first at 250 qnL/h and then at 500 qnL/h when the reactor was found to be operating under oxygen limitation. The product was collected and sampled at least three times per week.

Sampling Procedures.

1. pHThe pH of the chemostat outflow was measured using a Hanna Instruments HI 8424 pH meter.
2. Optical Density The absorbance of the product was measured at 600nm in a Hitachi U-1100 spectrophotometer.
3. Urease Activity Assay Urease activity was calculated by the increase in conductivity elicited by a 5mL sample of product in 30mL of 1.5M urea using a Hanna Instruments HI8733. An increase of 1mS in the urease solution is equivalent to the hydrolysis of 10mM urea (Cord-Ruwisch, personal communication, 2007) better refer to written notes such as in Salwa’s thesis).
4. Cell Dry Weight 1.5mL of product was centrifuged, the supernatant discarded and the pellet dried at 37°C overnight. The mass of dried cells was measured the next day.
5. NH4+ Tests The ammonium concentration was measured using a method from Lee Walker (2007, personal communication). 1.5mL of chemostat product was centrifuged and 100µL of supernatant was added to a 4mL cuvette. 1900µL of DI water, 25µL of mineral stabilizer, 25µL of polyvinyl alcohol and 25µL of Nessler’s reagent were added to the cuvette. A spectrophotometer was blanked with DI water and the absorbance of the sample at 425nm was measured 60 seconds after the addition of Nessler’s reagent.
   

Figure 1 Chemostat setup

In the text you need to first refer to the figure in brackets (fig.1).

Cementation

A 60mL syringe was filled with dry sand and packed under compression. The syringe shaft was removed and the top fitted with a bung that allowed liquid to flow out of the top of the sand column and into the feed vessel (Figure 2). 500mL of product was left to settle overnight and 400mL of the supernatant discarded to give 100mL of concentrated biomass. The concentrated solution was added to the feed vessel and pushed through the sand column by a MasterFlex™ L/S peristaltic pump(give an approximate pump rate to enable other people to reproduce the experiment).

Figure 2 Chemostat product was pumped though the column in a loop for 24 hours to immobilize the bacteria on the sand.

After 24 hours, the product in the feed vessel was replaced by a 1M urea, 1M CaCl2 solution. Urea/calcium solution was fed through the system for 24 hours, after which the column could be cut out the syringe.

Results

Introduce individual experiments with goals, findings and conclusions.Only after explaining the goal and the reason for it will the reader be interested in what results were obtained.The culture was run under batch condition for 24 hours. Below are the data obtained from the batch culture(table1):

Table 1 Batch Culture data

Date / Day / Activity (mS/min) / Activity (mol urea hydrolysed/min) / pH / Optical density
27-Sep / 1 / 0.196428571 / 1.964 / 9.21 / 1.198

A table with only 1 line is not a table it is a list which is better reported in the text. There is no point of having columns for that.

Dilution rate

It is not a good idea to structure the report chapter according to individual parameters. It is better to cover individual objectives and experiments.The reactor flow rate was kept at 415 mL/h on day 2 to day 20. The timer was set to run for 2s and stop for 58s. From day 21 to day 23, the flow rate was 5.61 mL/h. The timer was set to run for 2s and stop for 238s. Hence, the dilution rates were 0.0449 h1on day 2 to day 20 and 0.0112 h-1 on day 21 to day 23 respectively (Appendix). What is the purpose of doing this or of listing these details. It is not useful for the reader to understand what how the experiments went.

Temperature and pH

The temperature and pH were kept at a fairly steady state throughout the experiment.(There is not info in this statement and it is better left out.

Average temperature of the reactor = 21oC (not a result but an insignificant part of the methods)

Figure 3 change of ph over days of the experiment

Optical Density (OD)

OD was used to measure the concentration of bacterial cells (Figure 4). A fluctuating trend was seen in the bacterial growth. The bacterial growth was generally slow, (how fast was it then? Doubling time? Specific growth rate? )with a 4 fold increase in OD on days 13-14. The power failure during the weekends (day 10-11) does not seem to affect the optical density on day 12. The OD was on a downward trend after the peak.

In conclusion, the optical density was observed to be on an upward trend with increasing time. What does it mean? You could at least say that this signifies an increased yield coefficient (which is actually not given).

Figure 4 Changes in OD of the S. pasteurii chemostat culture. Sample size was 1 mL, diluted accordingly (Appendix). Bacterial culture was grown in batch conditions for 24 hours. The dilution rate WAS 0.0449h-1 (day 2-20) and 0.0112h-1 (day 21-23) respectively while the stirring rates were 262 rpm (day 1 to 21) and 1282 rpm (day 22 to 23). Please don’t use capitals. It is unusual, has no point and is established to be more difficult to read. Text content is useful.

Specific activity of S. pasteurii

The specific urease activity (SUA) is an index of the efficiency of urease production by S. pasteurii.The specific activity of S.pasteurii was on a downward trend with increasing time. Explain how it is measured, what the units are and what its relvance is. Then the reader is more interested in reading how it developed.

Also you need to refer to the figure (fig 5

At the start of the experiment, there is a slight decrease in SUA, followed by a slight increase on days 8-9. After that, SUA sharply declined before becoming stable from day 14 to the end of the experiment. The change of stirring rate from 262 rpm to 1282 rpm on day 22 and the change of dilution rate from 0.0449 h-1 to 0.0112 h-1 on day 21 caused a slight decrease in SUA. (too descriptive, not really explaining the purpose or outcome of experiments but just listing details)

Figure 5 Specific activity of urease (mM urea hydrolysed/ min/ OD) ON THE days of the experiment. The dilution rate WAS 0.0449h-1(day 2-20) and 0.0112h-1(day21-23) respectively while the stirring rates were 262 rpm (day 1 to 21) and 1282 rpm (day 22 to 23).

Activity of S.pasteurii

The urease activity can be used as a measure of the total activity of the bacterial culture.

The activity was shown to be a steady downward trend except for occasional peak in activity. The highest activity was recorded on day 7, and the lowest was on day 23. The stirring rate impacted the urease activity negatively, as shown on day 23. Change of dilution rate from 0.0449 h-1 to 0.0112 h-1 on day 21 caused a decrease in activity of S.pasteurii.

Figure 6Urease activity (mM urea hydrolysed/min) of the S.pasteurii under chemostat condition

The increase in OD caused an increase in urease activity (Figure 4 and Figure 6Figure 4), but not specific urease activity (Figure 4 and Figure 5)

Productivity

In the beginning of the experiment (day 6 to 10), the productivity was seen to be on an upward trend. The power failure during the weekends caused the lowest productivity to be recorded on day 12. After day 12, the productivity was observed to be on an upward trend until the peak on day 15. As seen from Figure 7, the highest productivity was obtained on day 15. After day 15, the productivity drops sharply on day 16 and continued its downward trend until day 20. Change of dilution rate from 0.0449 h-1 to 0.0112 h-1 on day 21 increased the productivity. (it reads like the person describing rather than explaining the experiments has not been part of the experimental planning: too descriptive not explanatory or conclusive)

Dilution (h-1) * Biomass (g/L) = Productivity (g/L/h)

Figure 7Productivity over the days of experiment

The productivity data don’t seem to be in line with the OD data and the fact that the dilution rate was constant over days 2-22. It seems like a calculation error.

Oxygen Uptake Rate (OUR)

The OUR was calculated from the dissolved oxygen (Appendix).

At low stirring rate (262 rpm), the OUR calculated was very low, which was 2.98 mg/L/h while the mass transfer coefficient (kLa) was 0.68 h-1. (This kLa look too low. I hope I can see you r kLa calculation somewhere)

At high stirring rate (1282 rpm), the OUR calculated was 46.6 mg/L/h while the kLa was 10.77 h-1.Somewhat unclear to me hwo it was determined . If you determined the kLa and derived the OUR from the steady state concentration, then show the measurements. It seems you determined the OUR by stopping the airflow though, and it is not clear to me at all why the bacteria take up the oxygen faster when they had been grown at a higher kLa. These things would be worthwhile explaining not just listing.

Figure 8Comparison of dissolved oxygen at high and low stirring rate vs. time

NH3-N Analysis (Nessler’s)

Figure 9Standard curve for ammonium concentration

Good to see a standard curve.

Ammonium concentration of the product was derived from the standard ammonium curve (Figure 9).

Figure 10 Ammonium concentration of the products determined from the standard curve (Figure 9).

The ammonium concentration of the products remained constant throughout the experiment. It’s a shame your have done excellent work in this project but the write-up lets you down. Why was NH3 measured? What was expected. It seems normal that all urea added to the medium is being converted to NH3. From that aspect it would be expected and it seems not much point of monitoring the NH3.

Cement production

The urease activity was concentrated by mixing the bacterial cells with calcium and urea. (I don’t understand the logics of this sentence)

After a day of continuous flow of the product and another day of continuous flow of Ca2+ ions and urea, the biocement was produced.

Mass of cement = 112 g

Volume of cement = 50 mL

Density of cement = 2.24 g/mL

Figure 11Our very own biocement made from S.pasteurii.

Experimentally you have accomplished more than other groups on this topic. It would be nice to explain how the enzyme assists in the production of cement.

the next step (glass cement)

Figure 12: Biocementation of glass beads using the same setup as with the biocementation of sand (Figure 11).

After the success of the biocementation of sand, we decided to perform the biocementation of glass beads instead. (Again, what is consistently missing in this report is explaining the objectives and purpose of experiments)The experiment is still going on and no results have yet been collected.

Discussion

S. pasteurii is an alkalophile; it requires high pH for growth. On other bacteria, high pH causes disruption in plasma membranes, denaturation of proteins, and decreased availability of nutrients (Prescott 2005). In this experiment, the pH was kept above 9 to prevent contamination. This is also the optimum pH for this bacterial strain (Salwa, 2003).

Alkaline pH was needed to convert NH4+ to free NH3 which is necessary for the transport of substratesat low concentrations across the cell membrane. High ammonium concentration was needed due to the absence of an ammonium transport systemand of a glutamine synthetase in S. pasteurii (Jahns, 1996). The addition of ammonium at concentrationsabove 20 mM, which are in the physiological range for growing cells of S. pasteuriiresulted ina net increase of proton gradient due to the alkalinization of the cytoplasm (Jahns, 1996).(Interesting but not quite clear to me).During normal conditions, H+ ions will naturally diffuse out of the bacterial cell.(there are actively transported out. It is not diffusion but part of the respiration chain.In alkalophiles such as S. pasteurii, it needs to drive H+ back into the cell to generate ATP. This can be done by increasing pH in its cells which cause alkalinization of its cytoplasm or increasing its membrane potential (∆ψ)by the efflux of a cation (NH4+) via ATP synthetase rather than H+. As a consequence of increasing the charge separation across the membrane (∆ψ); the proton gradient drives back the protons into the cell against the concentration gradient (Whiffin, 2004). As a conclusion, high NH4+ concentrations are needed for the growth of this bacterium. The mechanism described also caused a decrease in pH with time (Figure 3). You had a good try at explaining this. It is not quite clear to me.Amittedly it is a difficult topic. I would hope that after this excursion into the biochemistry that this background is used for explaining the purpose and outcomes of your experiments.