The purpose of this investigation is to compare the ability of two different strains of yeast to respire, when using different sugars as respiration substrates.
Considering the lengths that have been reached to develop varieties of yeast with greater suitability and effectiveness for very particular fermentation purposes, it seems reasonable to suppose that two different strains of the same species of yeast, selected for their different fermentation properties, have developed requirements that are not uniform.
As a result of the selection and development process, yeast best suited to ferment in a given application, possess a range of different characteristics.
One such characteristic may be the ability to metabolise different carbon sources at different rates. This quality is important because in each application where different respiration substrates are available, a specific strain of yeast may be required. Yeast unable to utilise the available carbon sources will have undesirable fermentation rates, and therefore may not be selected for use in that application.
The two yeast here compared, have two such different applications.
The first yeast from the Saccharomyces cerevisiae variety is of the Hansen strain and is used in the baking industry, whereas the second also of the Saccharomyces cerevisiae family is the Narbonne strain, utilised in the brewing of wine.
The optimum ability of a variety of yeast to use a carbon source for respiration will depend upon the presence of certain enzymes within the yeast cells.
An enzyme is a biological catalyst, which speeds up metabolic reactions, and in the case of yeast is required in order to hydrolyse a particular carbon source.
Enzymes are globular proteins, which possess complex tertiary structures. The ‘lock and key’ mechanism is a theory, which explains enzyme-substrate complex formation. The globular nature of an enzyme’s structure gives rise to an area on the enzyme known as an active site. Due to the substrate being complimentary in shape to that of the enzyme’s active site, they fit together as a key would in a lock.
The binding together of the enzyme and substrate results in the substrate being hydrolysed. In the case of yeast respiration, this degradation turns the substrate into a form more usable in glycolysis, the first stage of cellular respiration. Read about anaerobic respiration in yeast experiment (temperature)
Enzyme + substrate ?enzyme-substrate complex ?enzyme + product
Respiration of yeast will be most rapid when glucose is available as a substrate, because it is the simplest usable carbon source, and will be used preferentially in its role as a precursor of glycolysis. Other simple sugar molecules will also facilitate respiration but at slower rates, because they will not form complexes with the yeast’s enzymes as readily as glucose.
During the bread-making process, yeast is added to the dough mixture to facilitate fermentation. The evolution of carbon dioxide as a result of this process is required in order to leaven (raise) the dough.
The substrates available for respiration during this process are largely derived from the complex sugar, starch. Two types of starch exist in flour, amylose and amylopectin. Although the Hansen strain of baking yeast contains neither the enzyme ?-amylase nor ?-amylase, required to degrade the carbohydrate chains which form these two types of starch, they are contained in wheat flour. Additionally, the flour milling process causes damage to these lengthy chains. This damage allows the yeast to start metabolising the starch, and is achievable through the presence of the enzymes, maltase and zymase, which degrade the sugars maltose and glucose respectively.
Generally, grapes used in wine production are comprised of sugar levels between 15% and 25% of their total mass. These sugars are mainly glucose and fructose, both simple saccharides. The Narbonne strain of wine yeast is best suited to the production of wine from grapes, rather than from other fruits, vegetables or grains such as elderberry or rice.
Another important constituent of grape juice is the acid content, consisting mainly of tartaric and malic acids. As a result of the environmental demands placed upon the Narbonne strain, a higher tolerance to acidic conditions is likely to be a characteristic which differentiates it from strains not specialised in the fermentation of wine.
The carbon source provided will ultimately be used within the cytoplasm of the yeast cells, during glycolysis. As the substrate is fermented, there is a build up of reduced electron carriers (NADH+H+). In order to prevent this build up causing glycolysis and therefore metabolism to stop, the reduced electron carriers must be re-oxidised. To achieve this, pyruvate is first converted to ethanal, through the removal of a molecule of carbon dioxide:
CH3COCOOH CH3CHO + CO2
(Pyruvate) (Ethanal) (Carbon dioxide)
The newly formed ethanal molecules combine with the hydrogen ions (H+) being transported by the reduced NAD+, thus re-oxidising the reduced carriers and producing the alcohol ethanol:
The re-oxidised co-enzymes are now available to accept and transport more H+ and glycolysis can continue in its cyclical nature.
This process is expressed diagrammatically below:
Pyruvate Ethanal Ethanol
During yeast fermentation in the brewing of wine, the desired effect is the production of ethanol (ethyl alcohol), rather than carbon dioxide as is the case in bread making. The process of winemaking takes a significantly longer time to reach completion than bread making.
The rate of respiration in baking yeast is faster than in wine yeast, when glucose is used as a respiratory substrate.
To test this hypothesis, the rate of respiration in baking yeast and wine yeast will be compared using four different carbon sources as respiration substrates.
During the respiration of the yeast cells, carbon dioxide will be produced. This carbon dioxide will dissolve within the liquid medium in which the yeast is growing, and carbonic acid will be formed. The volume of carbonic acid produced is influenced by the rate of respiration, therefore, the greater the rate of respiration, the greater the volume of carbonic acid produced.
After an eight-hour fermentation period at 25oC, the extent of anaerobic respiration that has occurred will be estimated using a titration against the alkaline 0.1 mol dm-3 sodium hydroxide solution. The volume of the sodium hydroxide solution required to neutralise the mixture will indicate the rate of respiration that has occurred. This will be indicative of the degree to which the yeast is able to use the particular carbon source as a substrate for respiration.
Equipment and materials
* Ten 500cm3 flasks
* Permanent marker
* Digital scales
* Measuring cylinder
* Glass rod
* Cotton wool
* Thermostatically controlled incubator
* Clamp stand
* Five beakers
* 25cm3 volumetric pipette and filler
* Pink colour chart
* Carbon sources:
* Culture nutrients:
* Ammonium phosphate
* Ammonium sulphate
* Distilled water
* Dried yeast granules:
* Saccharomyces cerevisiae – Hansen strain (Baking yeast)
* Saccharomyces cerevisiae – Narbonne strain (Wine yeast)
* Phenolphthalein indicator solution
* 0.1 mol dm-3 sodium hydroxide solution
Take ten 500cm3 flasks, and using a permanent marker, label five ‘baking yeast’, and the remaining five, ‘wine yeast’. On each of the flasks bearing the mark baking yeast, one of each of the following should be written: control, glucose, maltose, galactose and lactose. The same should be done for the flasks marked wine yeast.
Into each of the eight flasks, 4.00g of the respective carbon source should be added. Also added at this point are the culture nutrients, which will be utilised by the yeast in their various stages of growth. These nutrients comprise 0.50g of ammonium phosphate and 0.50g of ammonium sulphate. 200cm3 of distilled water should now be measured out with the use of a measuring cylinder.
With the use of a glass rod, the aqueous solutions should be stirred until the sugars and the culture nutrients have been dissolved and dispersed.
To the five flasks bearing the mark ‘baking yeast’, 2.00g of dried baking yeast should be added. Once made wet, the yeast is activated. Again the vessels should be stirred, in order to suspend and dissipate the contents throughout the flasks. Plug each flask with cotton wool, and place in a thermostatically controlled incubator. The activated yeast solutions should now be left undisturbed for eight hours, at a constant temperature of 25oC.
An hour is left between the activation of the two strains of yeast. This is arranged in order to prevent the activation period of one strain of yeast being extended, as it waited for the other to be titrated. This cannot be permitted, as it would allow more time for the waiting strain to ferment the sugar substrate, affecting the equality of the conditions and the reliability of the results obtained.
Once an hour has elapsed after the activation of the baking yeast, the wine yeast must be activated in exactly the same manner, and again allowed to incubate for eight hours at a constant temperature of 25oC.
Just prior to the removal of the baking yeast solutions from the incubator, the next stages of the investigative process must be organised.
Suspend the burette in a clamp stand, ensuring that the tap is closed. This should be done in order to prevent any 0.1 mol dm-3 sodium hydroxide solution (NaOH), from being able to escape from an open valve whilst the filling of the burette is occurring. To aid in the filling process, and reduce the potential risk of spilling, place a funnel in the top of the burette.
Arrange five beakers, four of which are for a 25cm3 sample of one of each of the four baking yeast containing sugar solutions, and one for the baking yeast control. Label each of the beakers clearly and appropriately with a permanent marker.
Once the eight-hour incubation period has elapsed, remove the baking yeast solutions from the incubator. Note that the five flasks containing wine yeast solutions still have an hour of incubation time remaining.
Using a glass rod, thoroughly stir each of the solutions before any samples are removed for titration. This will help to ensure that the solutions are well mixed and all samples taken are of equal consistency, and contain a proportionate amount of all suspended/dissolved components.
To ensure that accuracy is maintained, a 25cm3 volumetric pipette and filler are used to remove 25cm3 of each solution. These samples should be placed in the corresponding appropriately labelled flasks to hand. Between the removal of each sample, the pipette must be flushed through with distilled water to ensure that contamination is avoided.
To each of the five beakers, three drops of phenolphthalein indicator solution should now be added and stirred in, so to disperse throughout.
Fill the burette with the 0.1 mol dm-3 NaOH solution, using the funnel and caution so as not to spill any of this hazardous liquid. The volume of NaOH now contained within the burette should be recorded. (Note that with regard to safety, due to the nature of the burette and its contents, it should not be left full when not in immediate use, hence the time between setting it up and filling).
Place the beaker containing the 25cm3 of baking yeast control underneath the burette, and open the tap in order to titrate the NaOH into it, gently swirling the beaker all the while. Keeping one hand on the tap, as the solution begins to turn pink, slow the release of the NaOH by turning the tap towards the closed position. As a means of accuracy control, take the pink colour chart and continue to titrate slowly until the set point has been reached, i.e. the colour of the solution exactly matches the colour on the chart. From the burette, take the reading from where the reduced volume of NaOH lies. From that subtract the volume present prior to the titration. This will give the volume of the alkali required in neutralising the carbonic acid produced as a result of the respiration of the yeast cells. Record this figure in the corresponding column of the result table.
Follow this method for each of the five solutions, being careful to use exact volumes and measurements at all times. Once this is completed, empty each of the now neutralised pink solutions and clean the beakers with distilled water.
This entire process can now be repeated a further seven times for each flask, until a result table for the baking yeast, containing a total of forty titrations has been compiled.
By completing eight repeats for each carbon source, it would be possible to assess the degree of variability in the results and from that, determine whether the method is capable of yielding results which are reliable. Any anomalous results can also be identified and discounted, as after comparison they can be seen to differ from the other results obtained.
Having been activated from its dry state an hour after the baking yeast, once the eight hours have elapsed, the wine yeast solutions should be removed from the incubator. This must be done so that both strains have had equal activation periods of eight hours at 25oC.
Exactly the same titration process must now be carried out for the wine yeast, in order to achieve another set of highly accurate results, suitable for comparison, discussion and evaluation. Again, utmost care must be used at all times in order to ensure that personal safety is not threatened or compromised.
This method was devised following the need to modify the implemented procedure used in a pilot experiment. The pilot was carried out in order to assess the ability of the chosen method to generate results that are useful for comparison and analysis.
The method used during the pilot experiment differs slightly from the revised method. The pilot experiment was run using a greater range of respiration substrates. Along with the four carbon sources used in the revised method, the yeast’s ability to utilise fructose and sucrose as substrates was also determined. Due to the time-consuming nature of multiple titrations, it was decided that reducing the range of substrates for comparison was necessary, so that the experiment could be completed within the allotted time.
Only five repeats were made for each of the respiration substrates during the pilot experiment. This was considered to be insufficient to produce results fit for comparison because the grouping of the results was considered to be potentially misleading and possibly not representational. With a small number of repeats anomalous results are also harder to spot and discount.
A key modification made to the method in light of the pilot experiment, was the fermentation period. Only three hours fermentation time was allocated, and as a consequence, the results obtained seemed to be fairly insignificant. An increase in fermentation time to eight hours was settled upon in an attempt to make the results more revealing.
During the pilot, when titrations against the 0.1 mol dm-3 NaOH solution were being carried out, assessing the end point was difficult. It was hard to settle on a consistent pink colour. Comparisons were made between the sample being worked on and the one last finished. The fact that the pinkness of the mixture fades with time meant that it was hard to keep the end point constant and consistent for each sample. For the revised method, a colour chart was used so accuracy and equality could be ensured when judging at exactly what point neutralisation had occurred.
The results obtained through the running of the pilot experiment have been recorded and presented in table 1 and table 2. Any results that were considered to be anomalous have been highlighted in bold script to allow for ease of recognition.
Control of variables
During any investigation, variables must be controlled. This is in order to instil a degree of reliability into the technique employed, that otherwise would not be achieved. An investigation lacking the stringent control of variables would be incapable of providing results that the experimenter could feel confident in using as a basis for comparison, and from which conclusions can be drawn.
In this investigation the following variables were subject to controls:
The control of incubation time is an important factor. It is essential that each of the samples have precisely the same length of time (eight hours), to ferment the available respiration substrate. If care is not taken to ensure that this is the case and samples are afforded different lengths of incubation time, the degree to which the carbon source is used would be affected, leading to inequality in the resultant outcomes. Through making a note of the time which the samples entered the incubator, their due removal at the appropriate time can be established.
All dry mass components must be weighed using digital scales, in order to make sure that all the masses of the appropriate materials are the same for each sample, which will help to ensure equality.
The volumes of the various liquids involved in this experiment should be measured using appropriate devices i.e. a measuring cylinder for the distilled water, a 25cm3 volumetric pipette and filler for the removal of the yeast/sugar mixture samples and a burette for titrations, etc.
The incubation temperature must be controlled and maintained at a constant level (25oC), throughout the period of incubation. This should be done in order to prevent temperature change causing the rate of enzyme activity to fluctuate. The control of temperature can be achieved through the use of a thermostatically controlled incubator.
The concentration of the various elements must also be controlled. The use of the same source of pre-prepared 0.1 mol dm-3 NaOH solution will ensure that the concentration cannot be a factor which will cause variation in the results, as it will be the same each time.
The practical procedure in this investigation is of a low risk. However, the implementation around others means that in order to reduce potential risk of mistaken use, all containers should be labelled.
It should be noted that the alkaline 0.1 mol dm-3 sodium hydroxide solution is considered to be a mild irritant, and so care should be taken to avoid spillage. If contact is made with skin, the affected area should be washed with cold water. If ingested or contact is made with eyes, plenty of cold water should be used to flush it out and medical advice should be sought. As with any investigation involving chemicals, safety glasses should be worn at all times.
Through respiring anaerobically, the yeast will cause the medium in which it is growing to turn acidic. Although the acidity will be weak in its nature, care should still be taken in its handling.
The use of glassware means there is the potential for breakage and therefore should be handled with care. Any broken glass should be cleared away thoroughly and with caution to avoid cuts.
Phenolphthalein indicator solution is considered to be a minimal risk to users.
The yeast itself will pose no risk to the vast majority of users however, there is a very small chance of allergic reaction. There are no ethical implications to be considered with the use of yeast in this investigation.
The following pages show the tabulated results obtained through the close following of the documented method. Manipulated data and graphical representation of the results have also been recorded.
Table 5 contains the collation of all the mean volumes in cm3 of NaOH required to neutralise the 25cm3 samples of the yeast/sugar mixtures, displayed in tables 3 and 4.
The mean hourly respiration rates of the two strains of yeast can be found displayed in table 6. The results are presented in arbitrary units. The arbitrary units represent the mean volume of NaOH in cm3 required to neutralise the mean hourly volume of acid produced during the incubation period.
As with both strains of yeast, the results from the controls show a similar ability to respire despite the lack of substrate provision. If the control results are accurate and not influenced by the documented method limitations, the prospect that the yeast are using a carbohydrate store within their cells becomes a likely one. It is possible that if this is the case, the carbohydrate store is greater in size, or more readily used as a substrate for respiration in wine yeast than in baking yeast. This would help to explain the fact that the mean in table 5 and consequently the mean hourly respiration rate in table 6, are almost 10% greater than the respective results obtained from the baking yeast.
When lactose was provided as a respiratory substrate, neither of the two strains of yeast seemed very well able to use it as a carbon source. The baking and wine yeast strains have respective mean hourly respiration rates of 0.764 and 0.676 (arbitrary units).
Lactose is a disaccharide comprised of one molecule of glucose and one of galactose, and is found in mammalian milk. Of these two yeast strains, neither has an application concerned with the fermentation of milk. As a result, it seems reasonable to propose that these yeast do not possess the ability to produce the enzyme lactase, required in the hydrolysation of lactose.
The difference between the level of respiration of lactose by baking yeast and wine yeast as seen in tables 3 and 4, may again be as a result of a differing degree to which the yeast are making use of a stored respiratory substrate.
Another point of interest came to light when analysing the respiration levels of the two yeast. When provided with a carbon source containing galactose molecules (i.e. lactose and galactose), the level of wine yeast respiration that was brought about, was less than when no substrate was supplied, as in the control (see table 3 and table 4). This was also the case when the respiration substrate given to baking yeast was galactose. This is strange as it suggests that the presence of these two substrates had an adverse effect upon the ability of the wine yeast to respire, and the provision of the galactose also had a negative result on the baking yeast’s respiration rate. It may be possible to explain these results if the control data lacks accuracy as a result of experimental limitations. If this were the case, the use of the data from the control samples as a basis for comparison with the other saccharides, in an attempt to establish respiration rates may be flawed.
As can be seen in table 6, the mean hourly respiration rate for the wine yeast control mixture, stood at 0.790 (arbitrary units) compared to 0.676 and 0.733 when lactose and galactose respectively, were provided. From this it can be calculated that by providing the wine yeast with lactose, the respiration rate was reduced by 14.4% and when galactose was available for respiration the rate dropped by 7.2%. When galactose was made the available substrate for baking yeast, the rate of respiration fell from 0.716 (as with the control) to 0.686 arbitrary units, which is a reduction of 4%. When provided with lactose however, the baking yeast respiration rate increased by over 6.5%.
Again the yeast’s inability to hydrolyse galactose molecules indicates that the enzyme required to degrade this carbon source may not be present in the cells.
Maltose is a disaccharide comprised of two glucose molecules. When provided to the baking yeast, the mean volume of 0.1 mol dm-3 sodium hydroxide solution required to neutralise the post incubation mixture was 11.98cm3. This volume is more than double that required to neutralise the control. From table 6 it can be calculated that the respiratory rate has increased by almost 110% to 1.498 arbitrary units. This is a very significant increase, especially when it is compared against the statistics of the wine yeast, where only a comparatively marginal increase in respiration has occurred. Table 4 shows that the neutralisation requirement has changed from 6.32cm3 to 6.84cm3 of NaOH, a mere 8% increase by comparison.
The Hansen strain of Saccharomyces cerevisiae must contain the enzyme maltase. This fits in with the earlier assertion that the different strains of baking yeast that have been developed, contain maltase in order to metabolise starch. It seems apparent that the Narbonne strain of wine yeast lacks the maltase enzyme required to hydrolyse maltose.
When it is considered that there is no requirement to have the ability to metabolise starch during the production of wine from grapes, there is also no need to produce the maltase enzyme. It therefore follows that during the selective development of a highly specialised strain of wine yeast, the ability to produce maltase, and with it the ability to hydrolyse starch is not an objective. As a result, this ability is overlooked in preference of other characteristics more important to the desired fermentation objective.
Graph 1 shows a visual comparison between the rates of respiration of the two strains of yeast. The mean hourly respiration rates plotted were derived from table 6. The inclusions of error bars were facilitated through the use of table 7 and table 8. Table 7 documents the mean hourly respiration rates calculated from the highest volume of NaOH required to neutralise each of the post incubated mixtures, i.e. the result at the very top of each of the ranges, in tables 3 and 4. These two tables also provided the data for the lowest volume from each of the ranges, used to calculate the lowest mean hourly respiration rates found in table 8. At this point it should be made clear that any results considered to be anomalous have been excluded from the calculations used to formulate the tables beyond table 4, for reasons addressed in the anomalous results section.
As observable on graph 1, the baking yeast has the greatest respiration rate recorded. Standing at 1.498 (arbitrary units), it was reached when maltose was provided as the respiration substrate.
The ability of the baking yeast to respire the maltose substrate was closely matched by its ability to use glucose for respiration. The rate at which respiration occurred when glucose was the substrate was 1.479 (arbitrary units).
The respiratory activity of the wine yeast when respiring maltose is only 57% of the rate at which baking yeast respires maltose. A similar comparison based upon the respiration rate using glucose as the substrate, shows that wine yeast was respiring at nearly 96% of the rate of baking yeast.
Graph 1 gives more strong support to the fact that baking yeast is able to metabolise both glucose and maltose. The apparent similar ability of wine yeast to use glucose as a respiration substrate is also clear.
More impressionable evidence is presented on the graph of the inability of the two yeast strains to effectively metabolise galactose and lactose. Also the degree to which baking yeast is more able than wine yeast to hydrolyse the maltose carbon source is clearly illustrated.
Anomalies in results may be caused by failure to fully control one or more variables.
Results that appeared to fall outside of the general grouping, and as such did not fit the overall pattern, have been highlighted in bold script.
Table 1 and table 2 contain the results obtained through the running of the pilot experiment involving the two strains of yeast. Although the results in table 2 do not bear any influence upon forthcoming conclusions, it is interesting that the anomalies in the pilot experiment all fall within the column of repeat number 2. The reason for this may have been the lack of a fixed visual comparison when judging at what point the mixture had been neutralised.
All of the anomalies are of greater value than the other results obtained, and as such have led to an increase in range at the high end of the variability scale. These discrepancies may have been caused by the use of the previously titrated mixture as a completion point indicator. As previously discussed the colour of the post-titration mixture fades over time. It is possible that this had occurred and gone unnoticed, and so the mixture was still used as a basis for comparison, which may explain such an increase in variability. The use of the fixed colour chart during the main experiment meant that this could not be a recurring factor.
The results displayed in table 3 and table 4 show the volumes of NaOH in cm3 required to neutralise 25cm3 samples of the yeast/sugar mixtures after incubation. The results came about through the following of the method described. All but one of the anomalous results in tables 3 and 4 are on the high side of the mean, and lead to an increase in the range of the results for the carbon source concerned. This may be as a result of experimental error.
Failure to swirl the beaker sufficiently during titration may lead to too much NaOH solution being released from the burette because unless the yeast/sugar mixture is being agitated enough through swirling, the colour change will not take place evenly throughout the mixture. When mixed in, it may be the case that the neutralisation point has been overshot.
Failure to have proper control of the release of the contents of the burette may also cause the point of neutralisation to be missed.
Another potential influence on the obtaining of anomalous results may be the insufficient stirring of the post incubation mixture. If not well mixed prior to the removal of the 25cm3 sample, there is no guarantee that the sample taken will consist of equal proportions of what the flask contains. The mixtures must be stirred in order to dissipate the contents evenly, helping to ensure that all samples removed are homogenous.
To allow for comparison with unmanipulated data, the anomalous results found in tables 2, 3 and 4 have been included within the basic calculations, alongside the amended results. The anomalous results have been discounted from further calculations from which evidence will be drawn to support conclusions, in order to avoid them from being potentially misleading and resulting in wrong conclusions being proposed.
The appearance of the calculations involving anomalous results alongside those manipulated shows how these peculiarities may alter the way results appear. In this case other than increasing the range of result variability and therefore the significance of the error bars found on graph 1, there would be little other major impact. The mean results although affected slightly (see tables 2, 3 and 4), would probably not be altered to the extent that any conclusions made on their basis would be unsafe. None the less, the results deemed to be lacking in representational value are discussed no further.
With both the baking and the wine yeast strains, the results obtained from the control samples suggest a surprising degree of respiration. Discounting the anomalous 3rd result, the baking yeast control in table 3 showed a mean of 5.73cm3 of 0.1 mol dm-3 NaOH, was required to neutralise the carbonic acid produced as a result of the yeast’s cellular respiration. This may appear curious considering that no carbon source was present as a respiration substrate. When compared with the yeast control in table 4, the manipulated wine yeast mean is an even more significant 6.32cm3 of NaOH.
Limitations of method
Although a colour chart was used to assess the point of neutralisation, the comparison between the mixture and the chart is still dependent upon the opinion of the experimenter, and therefore is subjective.
Another problem with regard to colour matching, is the concentration of suspended/dissolved particulates. Although the mixtures are well stirred, and the contents suspended, each sample may have different amounts of solids within it. This variation can alter the appearance of colours, as light penetration will occur to different degrees depending on the amount of suspended material. By filtering the mixture through muslin, the removal of the visible particles, i.e. the yeast cells will make the judging of colour easier and more accurate for each sample as particulate density will no longer be a factor.
The reliability of the results obtained from each of the control samples may be questionable. As no substrate was provided, the amount of dissolved components were much less in the controls, and as a result they appeared a lot less cloudy then the other samples containing saccharides. This coupled with the other problems of end point determination, means that an undue amount of confidence should not be placed upon the control results.
By using a pH meter, readings could be taken directly from the post-incubated flask. This method would mean that titrations are not necessarily essential, however, in order to obtain a greater number of readings, the pH meter could also be used to determine when each 25cm3 sample has been neutralised by the 0.1 mol dm-3 NaOH. This method would mean that the phenolphthalein indicator solution and the colour chart would not be required, and more importantly reduce the reliance on subjective judgements.
A magnetic stirrer could be used in order to agitate the sample yeast/sugar mixtures, whilst pH readings are taken. This would ensure that the contents are not allowed to settle, and that all contents are evenly dispersed, allowing the pH meter to take accurate readings.
Where there is a large range between the highest and lowest results (discounting anomalies), it makes conclusion drawing difficult. A larger number of repeats may have ultimately helped to produce more clear-cut grouping, from which conclusions could be made with more confidence.
It is hard to judge the significance of the range of results obtained for both yeast strains, particularly those involving the glucose results. The error bars on graph 1 clearly show a degree of overlap between the results of the two strains of yeast. These overlapping results cannot be discounted or ignored, because from the number of repeats it cannot be concluded whether they are representational, or merely lacking in accuracy.
On this basis and with a lack of other conclusive evidence, the hypothesis stating that respiration in baking yeast is faster than in wine yeast, when glucose is used as a respiratory substrate, should be rejected.
Due to the tentative nature of single investigations, in order to realise a higher level of confidence in the eventual results obtained, further work could be carried out in this area.
Through employing the mentioned amendments to the method, any future extensions to this investigation may help to establish the reliability of the method used. Further investigative work could involve the re-testing of the lactose and galactose substrates to see whether they really had an adverse influence on the respiration rate, or whether they appeared to, due to unreliability in the results of the control solutions.
Another limitation of this method is that from the end results it cannot be determined to what extent the substrate had been used, or whether the rate at which it was used at different stages of the incubation period varied. It may be for example, that some of the carbon sources were only just starting to be metabolised at a reasonable rate at the end of the incubation period, giving the impression that the yeast was unable to effectively use them as respiration substrates. Another possibility may be that the majority of the respiration occurred within the first couple of hours, thereafter tailing off.
By taking pH readings every half an hour throughout the incubation period, the degree of respiration at each point could be estimated and plotted on a graph. Once plotted, the accumulated data would have the potential to show at what point of incubation the respiration began to occur, when the greatest rate of respiration occurred and whether respiration had stopped by the end of the incubation period. This information would allow more accurate conclusions to be drawn regarding the respiration rates, and the ability of the yeast to respire the particular carbon sources.