95507914-Report

Please download to get full document.

View again

of 17
10 views
PDF
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Document Description
Laboratory 1: Growth Kinetics Study of Microorganism in Shake Flask 1. Abstract This experiment is carried out to study the growth kinetics of microorganisms in shake flask. E.coli is grown in a LB broth medium and being fermented for 24 hours. Throughout the fermentation, the cell culture is taken out for every 3 hours and protein test, glucose test and cell dry weight are being performed. As for the optical density analysis, the absorbance reading from the spectrophotometer is taken while for
Document Share
Documents Related
Document Tags
Document Transcript
  1 Laboratory 1: Growth Kinetics Study of Microorganism in Shake Flask 1.   AbstractThis experiment is carried out to study the growth kinetics of microorganisms in shake flask. E.coli isgrown in a LB broth medium and being fermented for 24 hours. Throughout the fermentation, the cellculture is taken out for every 3 hours and protein test, glucose test and cell dry weight are beingperformed. As for the optical density analysis, the absorbance reading from the spectrophotometer istaken while for the glucose test, the reading of glucose level is taken from the YSI 2700 SelectBiochemical Analyzer or can also being performed by using DNS reagent and the absorbance value istaken. These absorbance values will then being compared with the standard curve to get the glucoseconcentration inside the shake flask at particular time. The cell dry weight, in the other hand, is takenafter the mass concentration is being dried overnight in the oven. The weight of the viral whichcontains the biomass before and after the drying process is recorded to get the dry cell weight.For the optical density of the cell, the absorbance value showed an increment which indicating that thecell was growing and number of cell is increased in the shake flask. The glucose concentration,however, cannot be determined as the absorbance values were increased and decreased unevenly andcomparison cannot be made with the standard curve as the data for the standard curve are notconsistent giving inaccurate curve. Therefore no conclusion can be made about the glucoseconcentration in the shake flask. Supposedly, as the number of cell increased, the glucoseconcentration would decrease as the glucose consumption by the cells is increased.The dry cell weight in the other hand can be seen that there is an increment from the beginning of thecultivation until the 6 th hour and showed unstable changes until the 24 th hour. Supposedly, as thenumber of cell increased inside the shake flask, the cell dry weight also should be increased.2.   IntroductionFermentation can be carried out as batch, continuous and fed-batch processes. In this experiment, theshake flask fermentation is being used. Shake flask fermentation is the example of batch fermentation.In shake flask, the culture flask usually Erlenmeyer flask is being used to place and growing themicroorganisms. It is the cheapest and easiest way to culture microorganism aerobically, in smallvolumes of nutrient broth.It is a small scale equipment which equivalent to stirred tank bioreactor.  2 In order to prevent any contamination to the culture, shake flask must be plugged. Different plug canbe made of cotton-wool, glass wool, polyurethane foam, gauze or synthetic fibrous material. The plughas to prevent airborne microorganism from getting into the medium while at the same time allowingfree flow of air into the flask.The cultures are incubated at certain temperature and shaking frequency in an incubator shaker toachieve a required growth rate. The shaking agitates the medium and the culture to keep the mixturerelatively homogeneous and also to ensure aeration, creating an aerobic condition. In batch culture,there is neither input supplied nor output generated throughout the fermentation. The medium cultureis initially inoculated with the microorganism. The growth keeps increasing until at certain extent, thegrowth is inhibited because of the decreasing substrate concentration and the presence of toxicmetabolites.3.   Aims  –    To study the growth kinetics of microorganism in shake flask experiment  –    To construct a growth curve including lag, log, stationary and death phases  –    To determine the Monod parameters4.   TheoryShake flask fermentation is one of the examples of batch fermentation. Batch culture is an example of a closed culture system which contains an initial, limited amount of nutrient. The inoculated culturewill pass through a number of phases. After an inoculation there is a period during which no growthappears to take place. This period is referred as the lag phase and may be considered as a time of adaptation. In a commercial process, the length of the lag phase should be reduced as much aspossible. Following a period during which the cell gradually increases, the cell grows at constant,maximum rate and this period is known as the log phase or exponential phase. The exponential phasemay be described by the equation below:  3  = µx -------------------1 where x is the concentration of microbial biomass t is the time, in hours µ is the specific growth rate, in hour -1  on integration, equation (1) gives   =       ------------------2 where   is the srcinal biomass concentration   is the biomass concentration after time interval, t hoursDuring the exponential phase, the organism is growing at its maximum specific growth rate,   forthe prevailing conditions.Equation 2 predicts that growth will continue indefinitely. However, growth results in theconsumption of nutrients and the excretion of microbial products. Thus after a certain time the cellgrowth rate will decrease until growth ceases. The cessation of growth may be due to the depletion of some essential nutrient in the medium when there is limitation in substrate.The decrease in growth rate and the cessation of growth due to the depletion of substrate may bedescribed by the relationship between µ and the residual growth-limiting substrate as follows: µ =         where     = maximum growth rate s = residual substrate concentration   = substrate utilization constant  4 Figure 4.1: The graph showing the relationship between the parameter of the Monod equation.The stationary phase in batch culture is the point where the growth rate has declined to zero. In theother word the growth rate is equivalent to the death rate. The cell death is might due to the nutrientlimitations due to their incorporation into cells during log-phase growth or a build-up of toxins due totheir release of fermentation products also during log-phase growth.The death phase is the result of the inability of the bacteria to carry out further reproduction ascondition in the medium become less and less supportive of cell division. The nutrient is extremelyinsufficient for the growth of the microorganism. Eventually, the number of viable bacterial cellsbegins to decline at an exponential rate. Industrial fermentation is usually interrupted at the end of theexponential growth phase or before the death phase begins.
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks