lab 6

ABSTRACK

This experiment is to study the growth kinetics of microorganism in shake flask. Erlenmeyer flask is used in this experiment to grow microorganisms. Escherichia coli (E.Coli) is grown in Terrific Broth (TB) medium at 350 rpm and 37°C. They were fermented for 24 hours. Throughout every 1hour for 4 hour then 2 hours for rest 20 hours, cell is taken out to obtain the value of absorbance by using spectrophotometer. Cell dry is then obtained after the mass concentration inside the flask is dried overnight. As for the optical density analysis, the absorbance reading from the spectrophotometer is taken. The cell dry weight, in the other hand, is taken after the mass concentration is being dried overnight in the oven. The weight of the tube which contains the biomass before and after the drying process is recorded to get the cell dry weight. Then, the obtained value can be turned into graph to observed the changing in growth kinetics of the cell . Graph of growth curve including lag, log, stationary and death phases. The others parameters that we studied includes cell concentration, absorbance reading, and cell dry weight. This experiment get a a few of human error when taking the reading for absorbance and mass of cell growth. But after we can observe the growth of microorganisms and construct the growth curve and determine the Monod parameters, the value of Monod parameter

of maximum growth gate µmax which is equal to the slope of the graph is 0.2443h-1, maximum net growth rate,µnet is 0.2747 h-1, specific growth rate, µnet is 0.7985 h-1 and mass doubling time, 𝜏𝑑 is 0.8681 h. This experiment was consider succeed .

1

INTRODUCTION

When microbial cells are inoculated into a batch reactor containing fresh culture medium and their increase in concentration is monitored, several distinct phases of growth can be observed. There is an initial lag phase, which is of variable duration. This is then followed by the exponential growth phase, where cell number (and dry weight) increases exponentially. This is also referred to as the logarithmic phase, the name arising from the common method of plotting the logarithm of cell number against time. Following this is a short phase of declining growth, and then the stationary phase. Here the cell numbers are highest. Finally the cell numbers decline during the death phase. Fermentation process such as batch, continuous and fed-batch processes. In this experiment, the shake flask fermentation was used. The shake flask fermentation is an example of batch fermentation. In shake flask, the culture flask usually Erlenmeyer flask is being used to place and growing the microorganisms. It is a small scale equipment which equivalent to stirred tank bioreactor. It is the cheapest and easiest way to culture microorganism aerobically, in small volumes of nutrient broth. The cultures are incubated at certain temperature 37°C and shaking frequency 350 rpm for 4hours in an incubator shaker to achieve a required growth rate. The shaking agitates the medium and the culture to keep the mixture relatively homogeneous and also to ensure aeration, creating an aerobic condition. In batch culture, it is closed environment that means there is neither input supplied nor output generated throughout the fermentation. The medium culture is initially inoculated with the microorganism. The growth keeps increasing until at certain extent, the grow this inhibited because of the decreasing substrate concentration and the presence of toxic metabolites. In order to prevent any contamination to the culture, shake flask must be plugged. The plug has to prevent airbone microorganism from getting into the medium while at same time allow free flow of air into the flask. Different plug can be made of cotton-wool, glass wool, polyurethane foam, gauze or synthethic fibrous material. The microorganism that we used to study in this experiment is E .coli . There are many specific media for growth of E. coli but in this experiment we used Terrific Broth (TB) media because it is the most commonly used medium in molecular biology for E. coli cell culture .The relationship between the specific growth rate (μ) of a microbial population and the substrate concentration (s), is an indispensable tool in all fields of microbiology, be it physiology, 2

genetics, ecology, or biotechnology, and therefore it is an important part of the basic teaching of microbiology (Kovárová-Kovar. K, et. al, 1998) .

OBJECTIVE

1. To study/observe the growth kinetics of microorganism in shake flask experiment. 2. To construct a growth curve including lag, log, stationary and death phases. 3. To determine the Monod parameters.

THEORY

Rate of microbial growth is known as 𝜇 net≅

1 𝑑𝑋 1

[ ]

𝑋 𝑑𝑡 ℎ

Yield coefficients ∆𝑋

𝑌𝑋/𝑆 = − ∆𝑆

[g cells/g substrate]

Mass doubling time (𝜏d) ln 2

𝜏𝑑 = 𝜇

[h]

𝑛𝑒𝑡

Monodequation 𝜇𝑔 =

𝜇𝑚 𝑆 𝐾𝑠 +𝑆

𝜇𝑔 = 𝜇 net when𝐾𝐷 =0 𝐾𝐷 = Endogenous metablosim 𝐾𝑠 = Saturation constant 𝐾𝑠 = 𝑆 𝑤ℎ𝑒𝑛 𝜇𝑔 = When𝜇𝑔 =

𝜇𝑚 𝑆 𝐾𝑠 +𝑆

1 𝜇 2 𝑚

, S