Manual Lab e Coli Lab 6

CHE506 Lab 6

Lab Manual

Edited Feb 2017

Laboratory 1: Growth Kinetics Study of Microorg anism in Shake Flask Flask

Objectives •

:

To study/observe the growth kinetics of microorganism microorgani sm in shake flask experiment

•

To construct a growth curve including lag, log, stationary and death phases.

•

To determine the Monod parameters of maximum growth rate ( max), yield of substrate (Yx/s), mass doubling time (t d), saturation constant (Ks), specific growth rate ( net).

1. Introduction In the shake flask fermentation, the culture flasks (usually Erlenmeyer) of 250 or 500 mL or larger are used for growing microorganisms. microorganis ms. Shake flask fermentation is the cheapest and simplest technique to grow bacteria or fungi, aerobically, in small volumes of nutrient broth. The broth is poured into Erlenmeyer Flasks equipped with cotton-wool stoppers, and autoclaved. After cooling, some microbes are "seeded" into the flask, and it is placed on a Shaker machine. The shaking agitates the content and so ensures aeration, so that the microbes could breathe. These flasks are shaken, generally, by an incubator shaker at a suitable agitation speed, which is usually in r.p.m. Shaken cultures are usually applied to aerobic processes. In general, filamentous microorganisms are grown for the production of secondary metabolites, which begins 1 to 3 days after inoculation and continues 3 to 4 days thereafter, for instance. In all such cases, the shaken cultures are used for strain improvement as well as for determination of the optimum conditions for the fermentation process. In many industrial processes, process es, it is also used for the initial stages of inoculum development. developm ent. Shaken cultures are a convenient method of growing microorganisms in submerged cultures under aerobic conditions created by shaking; it is a small scale equivalent of stirred tank bioreactor. Both the devices are extensively used with filamentous microorganisms and, often, with other types of microorganisms as well. Usually, complex media are used use d for shake flask cultures. However, to enhance the growing the synthetic medium is being devised for the fermentation process. Studies on inoculum size, temperature, agitation, nutrition are initially done using these cultures to monitor their influences on growth and product formation.

Page 1 of 9

CHE506 Lab 6

Lab Manual

Edited Feb 2017

Figure 1 Phases of a typical growth curve of E.coli in a batch culture

In a batch culture, 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 growth is inhibited because of the decreasing substrate concentration and the presence of toxic metabolites. Lag phase is the time between inoculation and reaching the maximum growth rate. There are two sub phases in the lag phase. In the first phase, there is no growth identified whereas in the second sub phase which is also known as acceleration phase, there is a constant growth begins. The second phase is exponential phase. The cells begin to proliferate with their maximum growth rate. The doubling time of E.coli   is 20 minutes. Exponential phase is important for determining the maximum growth rate, µ and doubling time, d since the growth at this time is the most constant and ideal. Retardation phase is the period between exponential and stationary phase, or in other words, the phase before the growth becomes stationary. Among the factors that inhibit the growth are reduced dissolved oxygen tension (DOT), substrate concentration, pH changes and presence of inhibiting metabolites. After retardation phase, the growth phase enters stationary phase where the growth becomes constant for a period of time before it declines. Finally, the growth declines from its stationary phase due to the cells lysation. This is indicated by the decrease of the viable cell number. There are many specific media for certain microorganisms like Luria Bertani (Lennox) and Terrific Broth media. Bacterial E.coli growth media: LB Miller broth/LB Lennox broth is the most commonly used medium in molecular biology for E.coli cell culture. LB broth contains the enzymatic digestion product of casein commonly known as peptone (some vendors term it Tryptone), yeast extract, and sodium chloride. Peptone is rich in amino acids and peptides. Its amino acid and peptide compositions reflect those of casein. In addition to amino acids and peptides, yeast extract also contains nucleic acids, lipids and other nutrients which are needed for bacterial growth. (LB Miller, Lennox)

Other media is Bacterial E. coli growth medium TB or Terrific Broth TB or Terrific broth is a phosphate buffered rich medium. In addition to 20% more peptone and 380% more yeast extract than LB broth, TB also has an added 0.4% glycerol as an extra carbon source. All these nutrients in TB can support E. coli growth to OD600 5 to 8 under normal shaking

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CHE506 Lab 6

Lab Manual

Edited Feb 2017

incubation conditions. TB is commonly used for protein expression and plasmid production in a laboratory scale. (TB broth)

2. Theories Rate of microbial growth

 net



1 dX

X dt

 

 net

 is characterized by specific growth rate:

[1/h]

Yield Coefficients ( Y X / s  ) are defined based on the amount of consumption of another material X

YX / s  

[g cells/g substrate]

S

Mass doubling time (  d ) is calculated based on cell numbers and the net specific rate of replication   d 

ln 2

 

[h]

 net

For substrate limited growth Monod equation is applicable in cellular system. Monod equation is as the following:  m

S

 

[1/h]

 g



 m

 = maximum specific growth rate when S >> K s

Ks



 g  =  net

Ks

S

when endogeneous metabolism is unimportant

= saturation constant or half-velocity constant

K s = S when

S>> K s , S