Continuous production of urease by Bacillus pasteuriiunder non-

sterile conditions in a chemostat

Abstract

The enzyme urease has a great deal of commercial potential, especially in its ability to

produce ‘bio-cement’. As such, optimizing its production is an important factor in achieving

viable production levels. In this experiment we attempted to find the optimum conditions

under which Bacillus pasteurii could produce urease by adjusting flow rates, feed

components and pH levels. Our results, however, proved inconclusive as we were unable to

achieve good levels of enzyme production until the last day of the experiment and thus

couldn’t determine how sustainable the conditions were. (The last statement is not useful.

Instead you should list whatever effects you have measured and what is explainable and wht

is not. I would discourage students to use as the key outcome of a project to write it was

inconclusive)

Abstract should contain what was attempted, what was found not just what was concluded,

in particular if the conclusion was considered to be “inconclusive”.

Introduction

In general all bacteria require the presence of some inorganic chemical

components for growth, such as, potassium, phosphorus, and sulphur. Moreover,

most bacteria also requires an energy yielding carbon source, such as glucose or

lactate for example. In addition to these elements different kinds of bacteria require

more complex compounds, for instance, amino acids, which they are not capable to

synthesizing. (Novick, et. al. 1950) Not incorrect, but not sure how it helps

introducing this particular topic.

Basically, the growth rate of a bacterial strain may be within wide limits of the

chemicals concentration, organic or inorganic growth factors, but since at zero

concentration, the growth rate is also zero, in other words, there are necessities at

low concentrations of the growth factor. I could not find any sense I this sentence.

On the other hand, it should be possible to maintain a bacterial population

over an indefinite period of time growing at a normal growth rate simply by controlling

growth factors at a sufficient level to cultivate a bacterial strain, as is done, for

example, in a Chemostat. (Novick, et. al. 1950) ) (The literature you use is very

outdated)

Urease

(There is a sudden jump from chemostat to urease which could be lessened

by making a transition between the two topics) Urease (EC 3.5.1.5), is a nickel

dependent metalloenzyme which catalyzes the hydrolysis of urea to yield ammonia

and carbonate and the latter compound spontaneously hydrolyzes to form carbonic

acid and another molecule of ammonia (Andrews, et. al. 1984). The reaction occurs

as follows:

(NH2) 2CO + H2O → CO2 + 2NH3

Functionally, ureases belong to the super family of amidohydrolases and

phosphotriestreases (Holm & Sander, 1997). Urease activity tends to increase the

pH of the environment because it produces ammonia as a product and ammonia is a

basic molecule. Ureases are found in numerous bacteria, fungi, plants and some

invertebrates, as well as in soils, as a soil enzyme (Krajewska, et.al. 2012). The

urease activity can be used in the biocalcification process of soils (Mahantyet. al.

2013), and some clinical studies related to the virulence of pathogenic bacteria

(Collins et, al. 1993). The jump from chemostat to urease is sudden and there is not

link that helps the reader understanding what the introductory background is

Bacillus pasteurii

Bacillus pasteurii, also called as Sporosarcinapasteurii is a bacterium that

carries out cementation via the precipitation of calcite and solidifies sand with urea

and calcium. This bacteria is a highly urease active alkaliphile that can thrive in high

ammonia environment. (Lee et al. 2000)

The focus of this project is to investigate the production of urease via

alkalinicBacillus pasteurii in a non-sterile condition, and maximize the productivity of

this process for stable urease production. (As background info, the paper provided to

you by Cheng et al.) should have been used. It is essential background that B.

pasteurii can be grown in an open chemostat)

Materials and MethodsThe Chemostat was set up by the help of the Lab technicians.The following diagram illustrates this:

Figure 1: Set up for the chemostat BioreactorNice drawing. You could indicate the author.

Culture conditions- Temperature: 30o

- pH 10- Mixing rate: Approx. 400rpm- Airflow rate: Approx 50 L/H

The set-up of the BioreactorOn day 1, the Bioreactor was set up as shown in Figure 1. The volume of the reactor was made up to 600ml. This was made by using 552ml of Inoculum and 48ml of the feed as the volume of the inoculum provided was not exactly 600ml. After this, 2 drops of anti-form was added to bioreactor.

Preparation of the enrichments for the Micro-organismsOn day 1 to Day 13, the feed of the Urease positive bacteria was prepared as follows:-  20g/L of yeast extract was used- 10.21g/L of 0.17M of Urea was used- 20g/L Sodium acetate was used- 2ml of 50mM stock/L of 0.1mM of NiCl2 was used.And the contents were shaken. The pH of the feed was adjusted to 10 by using NaOH.

On day thirteen the yeast extract concentration was increased to 40g/L and the urea concentration was increased to 20.4g/L. . (This would be an experimental test and goes into results section.)

pH ControlsThe pH was constantly being monitored by a pH meter which was calibrated (you mean to say that the setpoint was pH 10, not the calibration) at a pH of 10, if the pH dropped below 10 the sodium hydroxide pump would commence and the pH would be brought back to 10.NaOH pump was kept at a constant rate of 1sec and with the set point of 60 sec .Statement could be clearer. 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 Good. In general use full stops at the end of all sentences. However, on the day 13, pH was measured at 11.7 due to error in calibration or the computer system. Then the whole system was analysed again and it was found that the Ph probe was not working so the PH probe mistakenly underestimated the exact PH and so the pump would falsely pump NaOH into the culture so as to restore the pH to what it believed to be 10 again. After this, the problem was solved state exactly how the problem was rectified, most likely the probe was not “broken”. and the pH was back to normal again.

Operation of the systemThe chemostat consisted of a glass bioreactor with an operating volume of 864mL for the first seven days that the chemostat was running and then reduced to 670mL for the remaining seven days. This culture was continuously fed with fresh medium for the 2 weeks at a hydraulic retention time (HRT) of 32 hours initially and then at 16.5 hours from day 8 onwards. Nice and clear. The decanting pump was set at a faster rate than the influent pump to ensure constant volume. The decant tube was set at a length of 5cm whereas the influent was triple that in measurement. So, as of day eight, we instead used the harvest pump tube to pump feed into reactor and the feed tube for the harvest bottle. The stirring speed was set at 400 rpm and the airflow was a constant 50 L/h throughout the experiment. The pH of the reactor was controlled to a set point of 10 by computer program that pumped NaOH, which was changed to 10.2 on the seventh day and lowered to 10.1 on the thirteenth.  The temperature of the culture was kept constant at 30oC. The culture was then tested regularly for the urease activity and optical density and hence enzyme activity and biomass concentration were recorded.Normally the daily changes whether on purpose or not belong to the results section as they are not methods.

Monitoring methods

Biomass measurement (OD 600 nm):On a daily basis, the biomass concentration was recorded with the feed being used as the blank. If the biomass’s absorbance measured at a wavelength of greater than or equal to 2 then the stock was diluted to get the absorbance value down to a level at which the spectrophotometer could measure it acurately.This was calculated for converting the absorbance reading into biomass concentration is as follows:

C ( Biomass concentration. g/L) = 0.44 x OD (600 nm)(Liang.Cheng& Ralf Cord- Ruwisch.2013)

Urease activity:

Urease is a constitutively expressed enzyme that hydrolyzes urea to NH4+ and CO3

2 The urease activity was determined by measuring the rate of change of conductivity over time due to the conversion of non ionic substrate Urea to ionic products NH4

+ and CO32 -

(Liang Cheng and Ralf Cord-Ruwisch, 2014). Thus, 2ml of the culture was added to 10 ml of 3M urea and 8 ml of deionised water and the conductivity was recorded over a period of 10 minutes in 1 minute intervals at room temperature (25oC). The mS per minute obtained from this was converted into units of enzyme activity per litre by multiplying the value obtained by 11100. Sounds good this part. Could say what the definition is of one unit in terms of urease converted per time.

Dissolved oxygenOxygen concentration was measured by using the Dissolved Oxygen probe. Oxygen

concentration needs to be monitored as it is important to ensure that the oxygen

concentration does not become a limiting factor. To ensure the accuracy of the readings,

the dissolved oxygen probe needs to be calibrated before use.. During the

measurement, feed/harvest bung in the chemostat was removed and the probe was

carefully introduced through the aperture.

Results

Initial conditions

The initial culture used to inoculate the chemostat had a pH of 10, an optical density of 1.463 and an activity of 4051.5 units of enzyme per litre. 600mL of this culture was added to the chemostat vessel and made up to 864mL with feed. The pumps were set to turn on for one minute out of every 20, giving a flow rate of 27mL/h. From Thursday the 4th (day 1) through to Monday the 8th (day 4) the pH was not regulated and was measured at about 9.5. Good start for results section. What the reader wants to know now is the purpose, aim of your experiments and the hypothesis you are going to test.

Results of Initial running conditions

Between the beginning of the experiment (day 0) and day 4 the conditions were maintained as described above for days 0, 1 and 4, with the pumps being turned off for days 2 and 3. Over this time an increase in biomass productivity was observed (figure 1 and 3). This was determined by measuring the optical density and multiplying the value obtained by 0.44 to give the g of biomass/L (Cheng, L., Cord-Ruwisch, 2013). There is an inconsistency between your explanation leading to g/L and the units used in the figure (g/L/h). It would be good to list your dilution rate such that the calculation can be verified. (Your calculation does not seem to take into account the dilution rate. So is it really the biomass level in g/L that you record?)

In addition we observed a decrease in the enzyme activity and production (Figure 2 and 3). Enzyme activity was determined by measuring the rate of change in the conductivity of a urea solution when exposed to the culture (see materials and methods above) and multiplying the rate by to give activity in Units of enzyme/L No need to repeat, it was explained in the method (Cheng, L., Cord-Ruwisch, 2013).

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Biomass Productivity (g/L/h)

Biomass Productivity (g/L/h)

Figure 1: Biomass productivity over the first four days showing a sharp increase in biomass by day 4. Always write the units next to the axes.

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Enzyme Productivity (U/L/h)

Enzyme Productivity (U/L/h)

Figure 2: Enzyme productivity over the first four days, showing a marked decrease between day 0 and day 1, with a smaller decrease by day 4.

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Biomass concentration (Xg/L)Enzyme activity (U/mL)

Figure 3: Biomass concentration and enzyme activity, showing the decrease in enzyme activity in relation to the amount that the biomass increased.

This increase in biomass coupled with a drop in enzyme activity implied to us that the culture had become contaminated. Correct conclusion. However to the reader it is a bit surprising as you have not lined out in the intro, aims, methods or results that this is an open chemostat subject to contamination when the pH and ammonia don’t generate “extreme’ conditions.

It is evident that this was occurring as of the first day as the enzyme activity dropped markedly while the biomass concentration remained relatively steady, indicating a higher proportion of non-urease producing bacteria in the culture. The sharp decrease in enzyme activity over the first day is likely due to the dilution of the original culture with the feed media Dilution would result in both OD and enzyme activity to decrease. So I am not sure whether

this explanation is backed up by data. but the slight increase in biomass over this time, despite the dilution, implies that bacteria were growing. The increase in biomass and decrease in enzyme activity over the next three days implies that non-urea producing bacteria continued to be dominant and the number of active Bacillus pasteurii continued to decrease. Our next goal was to correct this by removing the contaminant organisms. Yes, you are leading to the objective for the next period. That is good. It should be done by formulating a hypothesis and testing it. It is not so much removing contaminants but favouring the growth of Bacillus relative to that of contaminants.

Removing Contaminants

On day four the NaOH pump was turned on and the chemostat was set to be regulated at 10pH. Where are the pH recordings ver time and what was the pH in the previous period? This was maintained until day 7, when the pH pump was recalibrated and the setting changed again. The purpose of the increased pH was to create an environment in which Bacillus pasteurii could survive but which would inhibit or prevent the growth of other micro-organisms. Should have explained in the intro what the specific extromphilic nature is of Bacillus p. Between day four and day 7 there were problems with the computer program such that the feed and harvest pumps were not running for significant periods of time. Other conditions such as feed rate, airflow rate, feed concentration and reactor volume were maintained during this time. Despite the increase in pH we continued to experience an increase in biomass concentration and a decrease in enzyme activity during this period (figure 4).

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Biomass concentration (Xg/L)Enzyme activity (U/mL)

Figure 4: Biomass concentration and enzyme activity between days 4 and 7 of the experiment, showing further increase in biomass and decrease in enzyme activity despite the increased pH. Vague. where is the precise pH monitored?

Increasing Enzyme Production Rate

Despite our lack of success in removing contamination it was decided that our focus should shift to increasing the enzyme production rate. In order to do this we increased the flow rate to 40.5mL/h and decreased the reactor volume to 670mL from day 7. This meant that our

dilution rate changed from 0.03125h-1 to 0.060448h-1, almost doubling. Reads OK, but you should explain here why an increase in dilution rate is expected to improve the situation, without that it would seem it is trial and error rather than using the scientific method. Also, avoid excessive number of digits which imply precision that was not realistic. This had the effect of initially increasing our theoretical enzyme production rate (figure 5) although attempts to actually harvest this were frustrated by further problems (specifically what? feel free to admit that operator errors were made too) with the computers, leading to the feed and harvest pumps again not functioning for much of the time. In addition, the pH setting was increased to 10.2 and the pH probe recalibrated. We believe that at this point the pH probe was mis-calibrated and the actual pH achieved was closer to 11.7. All other conditions were maintained as stated above and these were maintained until day thirteen with the exception that the reactor was turned off on days 9 and 10. (That is not too bad as you can now discuss the expected and found effect of pH11.7 on this culture. Will it kill the culture or the enzyme activity, will it kill more contaminants than alkalophilic strain?)

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Enzyme Productivity (U/L/h)

Enzyme Productivity (U/L/h)

Figure 5: Enzyme productivity between days 7 and 13, showing the increase in production between days seven and eight and the subsequent drop in enzyme productivity.

Following this both the enzyme activity and biomass concentrations dropped drastically (figure 6). The initial assumption would have been washout due to the increased harvest rate It is not initial assumption, it is the most likely explanation if the biomass concentration decreases. Correctly spotted but then the issue is confused by referring to pump problems. but the failure of the computer program and subsequent lack of harvest discounted this possibility. On day 13 the pH metre was recalibrated and the actual pH (which had been measured at 10.1) was shown to be 11.7. (Still? Is this an accidental repeat writing, or has the pH not been able to be corrected since the last observation of 11.7?) Assuming that this error had occurred when the pH metre was recalibrated on day 7 this would explain the drop in both biomass and enzyme activity as such a high pH would kill off both contaminant organisms and Bacillus pasteurii. Well done, this is nice evidence based explanation what is likely to have happened.

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Biomass concentration (Xg/L)Enzyme activity (U/mL)

Figure 6: Biomass concentration and enzyme activity between days 7 and 13 showing the decrease in both. We believe that this was due to a high pH induced by a pH metre mis-calibration. The slight increase in enzyme activity on day 13 may imply that Bacillus pasteurii performs better at these extreme pH levels than other microorganisms and had begun to recover.

Recovery of Culture

When the pH problem was discovered on day 13 it was immediately reset to a pH of 10.1 in an effort to allow the culture to recover. To aid this the feed media was also changed, increasing the yeast extract to 40g/L and the amount of urea increased to 20.4g/L. These changes were intended to increase the growth rate of Bacillus pasteurii and decrease the stress on it so that it could recover to useful levels. This was achieved as the levels of both biomass and enzyme activity increased (figure 7). The enzyme activity in particular increased more than fivefold, while the biomass increased by about a third, possibly indicating that there is still contamination in the culture. (No, it indicates regrowth of the target strain only, as would be evidenced by plotting the specific enzyme activity). Well done, you improved your report quality from measurements on the last two days.

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Biomass concentration (Xg/L)Enzyme activity (U/mL)

Figure 7: Increase in biomass and enzyme activity between day 13 and 14, after the pH was reset and feed changed. This indicates that the Bacillus pasteurii is recovering and able to produce significant levels of urease enzyme.

Monitoring of Oxygen Concentration

Throughout the experiment the O2 concentration was also measured to ensure that it was not a limiting factor. The oxygen levels clearly responded to the biomass concentration, decreasing as the biomass increased and rising again as the biomass dropped (Figure 8). Because we never achieved a true steady state we were not able to calculate an accurate bacterial oxygen uptake rate or kLa for this culture.

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Oxygen concentrationBiomass Concentration

Figure 8: Oxygen concentration and biomass concentration over the course of the experiment, showing the approximate inverse relationship between the two.

Enzyme Production Results

Overall we were able to increase our enzyme productivity from our initial rate of 126.6094U/L/h to 168.4135U/L/h (figure 9). Given that our flow rate at this point was 40.5mL/h this means that we were able to harvest 6.8207 units of enzyme each hour.

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Enzyme Productivity (U/L/h)

Enzyme Productivity (U/L/h)

Figure 9: Enzyme productivity throughout the experiment showing a general decrease until

the last day at which point enzyme productivity was increased over the original value.

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Optical Denisty vs Time

OD

Figure 10: Graph showing the overall pattern of change in Optical Density at 600nm of the

Chemostatover the course of the experiment.

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Enzyme Activity vs Time

Activity (Units of enzyme/L)

Figure 11: Graph showing the overall pattern of change in Enzyme activity over the course of the

experiment.

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RX vs Time

RX (g/L/h)

Figure 12: Productivity rate of bacteria over the course of the experiment. This is not necessarily a

good indicator of the success of our chemostat as it also measures the production of contaminant

bacteria. (Very true. Why not plotting the R-Enzyme then?)

Strangely the pH plot (a key parameter) and the dilution rate were not plotted.

Discussion

The chemostat project experienced a number of different setbacks over the fourteen day period,

however ultimately we were able to derive some meaningful results and achieve a level of level of

bacterial production and enzyme activity which we felt was an achievement.

From day zero to day eight the optical density of the culture in the chemostat grow from an initial

reading of 1.463 to a high of 5.706 (digits!?!)on day seven. Following this the optical density of the

culture then went into decline from days eight to 13 due to a mis-calibration of the pH reader that

will be discussed further on. Optical density fell from 5.595 to 3.556 on day 13 as much of the

biomass in the culture was killed off. By day 14 the optical density had once again risen to 4.782

under favourable conditions. These patterns are shown in figure 10.

The enzyme production performance of the bacteria was measured over the course of the

experiment by taking readings of the conductivity of the chemostat culture and converting this to

the enzyme activity of the bacteria. When given our starter culture enzyme activity was determined

to be 4051.5 units of enzyme per litre. This activity fell for the major part of the experiment,

dropping from the starting value to 1143.3 units of enzyme per litre by day eight. From day eight to

day 13 the activity dropped sharply again due also to miss-calculation of the pH probe. Here enzyme

activity fell to a low of 388.5units of enzyme per litre on day twelve and 455.1 units of enzyme per

litre on day thirteen before the pH was corrected. Under the morefavourable conditions of the

experiment enzyme activity rebounded strongly on day fourteen to a figure of 2786.1units of

enzyme per litre. This was our first significant rise in enzyme activity and was measured twice for

verification. The changes in enzyme activity are shown graphically below. (The discussion does not

need to describe the results in detail again but just focus on the overall implications and lessons

learned)

The first major problem experienced by the group was that the computer software program used to

run the experiment suffered a number of software crashes. For much of the first week and into the

second the program would crash over night and as a result the chemostat culture was not fed and

no harvesting was done. The effect this had on the experiment was that the culture was likely

undernourished for much of the fourteen days and may have gone into a slowed growth phase or

even a catabolic state where the desired bacteria reduced in numbers. Although the optical density

did increase for much of the experiment, the enzyme activity fell. This could be due to

contamination with an outside organism as the culture was an open one. This software problem was

rectified towards the end of the second week of the experiment and harvest volumes and regular

feeding was achieved. From an industrial stand point this type of situation would be highly

undesirable. The lack of harvest would signify a lack of production and as a result a production

company would not be making a return on investment during this time. In an industrial situation

these software errors could be picked up by staff working throughout the day or fixed earlier with

skilled operators of the equipment.

The second problem experienced by the group was that the pH probe was recalibrated incorrectly

on day eight. The group had made the decision to increase the pH of the chemostat culture in an

attempt to increase the rate of enzyme activity and reduce competition for resources from outside

contamination. Initially the culture had a pH of 10.0. This fell to 9.6 on day one and 9.5 on day 4 due

to the machine that pumped the NaOH into the chemostat not being operated correctly. The sodium

pump was changed and the pH returned to 10.0 by day five. The time in which the pH was below 10

may have provided the window in which airborne bacteria could have entered the system and

without the protection of such harsh condition favoured by our bacterial strain they may have out-

competed the preferred bacterium. This theory is supported by research conducted by Cheng and

Cord-Ruwisch (2013) where a similar chemostat experiment was overgrown with non-urease active

contaminants when pH values were in the range of 9.5 to 9.75. (Good use of literature) By

maintaining a pH of 10 Cheng and Cord-Ruswisch were able to increase urease activity greatly over

continued operation in excess of 140 hours. The miss calibrated pH probe was discovered by tech

staff on day 13 by which time the pH had risen to 11.7. The effect of this overabundance of NaOH

was that it severely limited the growth of any bacteria within the chemostat as seen by the falling

optical density during this time. However it may have had an unexpected positive effect on our

culture in that it may have been responsible for eradicating any outside contaminants which lead to

the increased enzyme activity on day fourteen as the bacteria had no competition for resources. In

an industrial setting this mistake would be unlikely to occur. Experienced staff would have more

experience with pH probes and be unlikely to make a novice mistake such as this. Also an industrial

setting would have more expensive and automated pH probes which would be more reliable. Even

by having a second pH probe on an industrial chemostat this could help to catch errors such as this.

(Paragraphs are a bit long and could do with some streamlining and focusing (1 point per paragraph),

but apart from that there is some good writing and understanding here)

A number of other decisions were made by the group over the course of the experiment to improve

productivity. The volume of the chemostat was reduced from 864 mL to 670 mL on day eight. The

purpose of this change was to reduce the hydrolytic retention time or the amount of time it takes for

the fluid in the system to be completely replaced. HRT was reduced from 32 h by this decision to

24.8 h. HRT was further reduced the following Monday on day 11 by increasing the feed media flow

rate from 27 mL per hour to 40.5 mL/h. The effect this change had on HRT was further reduction

from 24.8 h to 16.5 h. In an industrial setting this would mean that, assuming the feed media was

fully utilised by the bacteria, productivity could have been effectively doubled by halving the HRT

from 32 hours to approximately 16 hours.

Further indicators to the performance of the chemostat can be measured through the RX which is

grams of bacteria produced per litre per hour and the RP which is units of enzyme activity produced

per litre per hour. (No paragraph break needed here)

From figure 12 we can see that for much of the experiment RX increased with only a small fall from

days 11 to day 13. While optical density fell in this time, the increased flow rate and reduced HRT

offset this and in fact more net bacteria was produced. The final value for RX was highest on day 14

with the group achieving a best RX of 0.127 grams/L/h. (Biomass X is not what we are aiming for, the

enzyme is the desired product.)

Figure 9 shows much the opposite of figure 12. While bacteria formation increased over the course

of the experiment, the same could not be said for enzyme formation until the very last day. RP fell

from 60.7 U/L/h on day 1 down to 27.5 U/L/h on day 13. Whilst there were some minor increases

due to increasing the flow rate and reduced the HRT the trend was downwards as reflected in figure

11 which showed the fall in enzyme activity. It is however worth noting that the slump in RP was

finally rectified on day 14 with an RP of 168.4 U/L/h for the last day of the experiment. This last

positive reading is as a result of the group at last showing an increase in the enzyme activity as all

the mistakes of the past two weeks were finally overcome.

Overall it is felt that the experiment should be considered a success as the group was able achieve its

aims, were the aims clearly listed in this report ? (Success was already by preventing the enzyme

concentration to completely vanish, even though it was produced at lower levels) although at the

very last minute. Success can be measured in increased optical densities showing increased RX and

increased enzyme activity leading to increased RP. It is worth noting that a number of challenges

were overcome in the form of software problems as well as mistakes in equipment usage. The major

aim that was not achieved was that of maintaining a consistently high rate of enzyme production but

based on the results achieved towards the end of the experiment and by utilizing the lessons learnt

in overcoming the challenges that arose we believe that such a goal is readily achievable.

No References

No author list or group number. Assume this is group 1

Discussion is a bit wordy. Could have focused more on what was accomplished than on what

went wrong. Overall a good report 7.5/10.