Production of Penicillin - Murdoch University



Group Number 3Group Members: Nemdharry Devashi Devi (31624575)Priyadarshinee Boojhawon (31668482)Hu Shi (32082998)ABSTRACTPenicillin is a group of antibiotics, a secondary metabolite from the fungus Penicillium and is produced when growth of the fungus is inhibited by stress. Penicillin G is the most widely used form of penicillin. It is produced in a batch culture. It has a bacteriostatic effect on spore forming bacteria. The aims of this experiment were to test whether the penicillin production occurred at higher rates during the idiophase and to determine the production of biomass and penicillin by the fungus Penicillium chrysogenum. This is not an abstract of your work but more like an intro.INTRODUCTIONPenicillin G, C16H18N2O5S, was the first naturally occurring antibiotic discovered (Tom and Lynne). Some microorganisms can form many types of metabolites, the primary and the secondary metabolites. Primary metabolism is the metabolism of energy production for the cell and for its own biosynthesis while secondary metabolism regards the production of metabolites that are not used in energy production (Tom and Lynne) (Stanbury, 1998). Microorganisms that produce secondary metabolites display two phases in a batch culture. Those are the trophophase and the idiophase. Trophophase is the growth phase of the culture and idiophase is when secondary metabolites are produced. A batch culture simply means that a fixed amount of substrate is added at the beginning of the process (Tom and Lynne). Penicillin G is a secondary metabolite and is produced mainly when growth has stopped during the idiophase (Tom and Lynne). MATERIALS & METHODSThe materials and methods are described in the Murdoch University BIO301 Industrial Bioprocessing and Bioremediation Laboratory Manual 2013, Practical on Production of Penicillin, pages 14-19.RESULTSData: A standard curve was plotted using the simple dilutions of standards supplied and then these were spread onto sterile disks. The dilution table is as illustrated on table 1:goodTable 1: Dilution for table for standard curve:Standards (U/30ul)Diffusion Diameter (mm)Sterile control00.0260.04100.1140.25180.5020124225326427628Figure 1: Standard Curve of penicillin Inhibition ZoneProblem 1:Table 2: Biomass concentration at different sampling times and the rate of penicillin production over time.Sample time (h)Biomass Dry wt(g)Biomass concentration (g/L)Inhibition zone(mm)Penicillin production rate(U/L/h)00.06510.65115-240.40424.042160.069444444401.086810.868210.833333333471.263212.632220.952380952632.09220.92230.208333333701.536215.362255.2380952381342.411124.11122-0.625You show more digits than what can possibly within the accuracy of your measurments. How was the very high rate at sample time 70 h obtained. Sample calculation would be usefu.2052320308610Iodiophasee00Iodiophasee680085550545Trophophase00Trophophase Figure 2: Linear plot of Biomass and Penicillin Concentration against time. Why trying to fit a linear equation to the biomass growth if in fact an exponential development is expected? The penicillin concentration is too high or the units are incorrect. How do the penicillin numbers in this plot relate to the ones in the table?Figure 3: Semi-log plot of Biomass and Penicillin concentration against time.From figure 2, the Linear plot of Biomass and penicillin concentration versus time, the separation between Trophophase and Idiophase can be seen clearly.Introduce all figures first with text.Problem 2: According to Figure 2, the highest concentration of penicillin produced was at the point where biomass concentration started to drop. This looks like over-interpretation of an outlier point to me. When Biomass concentration was 20.92 g/L, there was a rise in penicillin concentration. Thus, from figure 2, the slope of the curve for penicillin concentration will give us the rate at which penicillin was produced. The slope was steepest during the trophophase hence we can say that the penicillin production rate was highest in the trophophase. You were supposed to conclude whether there is a trophophase from the results.The graph of penicillin versus time will be expected to be an increasing curve reaching a peak value and then decrease after the peak value has been reached and then stays constant. Why is that expected?The graph Penicillin versus time is shown below.From the above graph, the penicillin production rate dropped dramatically. Where?This is because the secondary metabolite acted as an inhibitor for the cells at high concentration. You can’t base conclusions on one single point Problem 3:Using the formula: Specific growth rate = ln2 / td (doubling time)From the semi log plot, the doubling time, td, is approximately= 8.6 hoursTherefore the specific growth rate = ln2/ 8.6 = 0.081 h-1Whereas the specific growth rate for the linear plot is = 0.1821 h-1 which is a little bit higher than in the value obtained from the semi log plot but lies within the same range. Problem 4: Table 3: Penicillin production rate of each sampling interval.Sample Time (h)Penicillin production rate (Units/L/h)0-240.027777778400.833333333470.952380952630.208333333705.238095238134-0.625Figure 4: Graph of Penicillin Production Rate vs Time. The shape of this curve is almost identical to that of the figure above. Extra marks if you can convince me that this chart has taken into consideration the different biomass valuesConclusion: For the first 20 hours, penicillin production rate was zero. This is because at this phase, primary metabolites were present speculative and these metabolites are essential for the growth of the cells incorrect. When cells grow, they start producing secondary metabolites which secondary metabolites, (what is the evidence?) until they reach a maximum value at 5.2 unit/L/hr (Idiophase). Before the highest value was reached, the penicillin production rate dropped to zero at around time, T, = 63 hours. This (what?) can be due to the presence of primary metabolite which is competes for the active site of the enzyme. Very speculative. Which enzyme? As the primary metabolite start to decrease, cells reach the stationary phase; penicillin production rate is highest as there is no similar substrate analogue to compete for the active site of the enzyme. Then the production rate decreases until it reaches a value under zero; -0.625 unit/L/hour at time t= 134. This must be due to product inhibition (Stanbury, 1998) a negative rate is not explained by product inhibition. What does a negative rate really state?Problem 5:Table 4: Specific Penicillin production rate of each sampling interval.Time (Hr)Specific Production Rate (units/g/L)00240.017180714400.076677708470.075394312630.009958572700.340977427Figure 5: Graph of Specific Penicillin Production Rate vs Time. What is different her to figure 4?According to the graph, the specific production rate is at its highest at 47 hours. After 47 hours, the graph shows a decrease. This may be due to the fact of product inhibition.Problem 6: Penicillin G is being produced in response to the presence of gram-positive bacteria and is produced as a secondary metabolite (Tom and Lynne).No. The type of penicillin that is produced depends on the organism and medium conditions.Problem 7:A sterile control was included in the experiment so as to ensure that during the inoculation process, the culture was not contaminated.No How can contamination be avoided by adding a sterile control?ReferencesPeter.F. Stanbury. Fermentation technology.1998Ralf.C Ruwish. (2013). BIO 301 Industrial Bioprocessing and Bioremediation Laboratory Manual. Murdoch University, Perth, WA.Tom O’Hara and Lynne White. Penicillin Production incomplete reference While a lot of work has been done and graphs have been plotted, the key purpose is not achieved , which is to interprete from the data whether there are tropho phase and idio phase. Instead of concluding from your results you take for granted that there must be two phases .The over-interpretation of a single outlier point point has resulted in some speculation which is neither based on literature nor on own data.The report did not really convince of a good understanding. How do you data would a proper secondary metabolite look like like In future reports stick to what comes out and admit if there are inconsistencies of data.Less speculation, and a more critical approach is needed. 6/10 ................
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