study of growth microorganism in shake flask
Short Description
study of growth microorganism in shake flask...
Description
INTRODUCTION
Fermentation can be carried out as batch, continuous and fed-batch processes. In this experiment, the shake 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 the microorganisms. It is the cheapest and easiest way to culture microorganism aerobically, in small volumes of nutrient broth. It is a small scale euipment which euivalent to stirred tank bioreactor.I n this experiment the microorganism used is E.coli.!he growth curve of E coli in a batch culture is been studies. !he graph below shows the phases of a typical growth curve of E coli in a batch culture.
Figure " #hases of a typical growth curve of E.coli in a batch culture
$ag phase is the time between inoculation and reaching the maximum growth rate. !here 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. !he second phase is exponential phase. !he cells begin to proliferate with their maximum growth rate. !he doubling time of E.coli is %& minutes. Exponential phase is important for determining the maximum growth rate, ' and doubling time, d since the growth at this time is the most constant and ideal. (etardation phase is the period between exponential and stationary phase, or in other words, the phase before the growth becomes stationary. )mong the factors that inhibit the
growth are reduced dissolved oxygen tension *+!, substrate concentration, p changes and presence of inhibiting metabolites. )fter 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. !his is indicated by the decrease of the viable cell number
OBJECTIVES •
!o study and observe the growth kinetics of microorganism in s hake flask
• •
experiment. !o construct a growth curve including lag, log, stationary and death phases. !o determine the /onod parameters of maximum growth rate *0 max, yield of substrate *1x2s, mass doubling time *td, saturation constant *3S and specific growth rate *0 net.
THEORIES
(ate of microbial growth 0
is characteri4ed by specific growth rate5
net
dX X dT *"2h 1
0 net 6
1ield 7oefficients *1x2s are defined based on the amount of consumption of another material
Δ X 1x2s 6 - Δ S
*g cells2g substrate
/ass doubling time *8 d is calculated based on cell numbers and the net specific rate of replication
¿2
8d 6 μ net
*h
For substrate limited growth /onod euation is applicable in cellular system. /onod euation is as the following5
μ
g6
μ ¿
S 2*3 S 9 S
m
*"2h
where 5 μ μ
m5
g ;
maximum specific growth rate when S ::3s μ
net
when endogeneous metabolism is unimportant
3 S 5 saturation constant or half-velocity constant 3 S 6 S when μ S ::3s , μ
g
S == 3s, μ
g6
g
6 < μ
6 μ μ ¿
m
m
S 2*3 S 9 S
m
APPARATUS AND MATERIALS
".
/icrobe 5 Escherichia 7oli
%.
Shake flask *%>&m$ flasks and "&&&m$ flasks
?.
Eppendorf tubes2falcon tube *".>m$
@.
7uvettes *spectrophotometer
>.
!hermostated rotary shaker2incubator shaker
A.
(efrigerated centrifuge
B.
/edia *for specific microbe
C.
Ethanol *B&D ethanol for swabbing for sterility
.
Spectrophotometer
"&.
unsen burner for sterility
"".
Graduated Flask for measuring media *"&&&m$, "&&m$, "&m$
"%.
$aminar Flow hood for sterility
"?.
iochemical )naly4er
"@.
#$7 for product measurement like ethanol
">.
7otton plugged
"A.
p meter
PROCEDURE i Preparation of media /edia is prepared according to the needs of microorganism used. a
ii
Terrific Brot preparation !he recipe is followed as stated at the bottle. !he media was autoclaved at "%"H7 for %& minutes.
Preparation of Ce!! C"!t"re
!he cell culture was maintained on an agar plate and liuid broth for the preparation of inoculum. ) suitable media were used to ensure the growth of microorganism. a
Seed c"!t"re preparation #inoc"!"m$ > loops of grown E 7oli were taken from the agar plates and were added to the
sterili4ed media of ">&m$ in "&&&m$ shake flask. Sterility were being sustained during the transfer. !he media were grown at ?&& rpm for > hours, assuming the exponential growth of E 7oli. !he + for seed culture was recorded by using the %
spectrophotometer. Main e&periment "&D of inoculum was transferred to the main experiment media by using aseptic techniue. Since "&D of inoculum was needed, thus only "> m$ of seed culture was needed if the total working volume was ">& m$. !he shake flask was capped by using the cotton plug and swabbed using B&D of ethanol. It was incubated in a thermostated rotary shaker at reuired rotational speed and temperature for %@ hours.
iii "
Samp!in' !he reuired amount of sample was transferred into the sampling tube with interval
time for every hour or every % or ? hours.
%
> m$ of the sample was taken every time sampling is done during the fermentation process to measure the optical density *+, glucose analysis and total cell number *biomass concentration 5 g2$
i( "
A%)or%ance Ana!*)i) #Optica! Den)it*$ #OD$ " m$ of the sample was transferred into a cuvette and the spectrophotometer was used
%
to measure the optical density with the wavelength set at A&&nm. " m$ of chosen media was used as the blank and the spectrophotometer was
?
calibrated to 4ero. !his method was used in order to measure the cell growth where high absorbance indicates high number of cells, which is caused by low transmittance and vice versa.
( " % ? @ > A B
Ce!! Dr* +ei't, #Bioma)) Concentration$ #-$ #'.L$ +ried centrifuge tubes were weighed and this were noted as the initial mass. " m$ of sample was added to the weighted centrifuge tube. !he sample was centrifuged at "&,&&& rpm and at ! 6 @H7 for %& minutes. !he supernatant was taken out from the mixture. !he centrifuge tube was dried in oven at C&H7 for overnight. !he dried centrifuged tube was left in the dessicator. !he centrifuged tubes were weighed and are noted as the final mass. Ce!! Dr* +ei't / 0ina! ma)) 1 Initia! Ma))
RESULT AND CALCULATION
a
Terrific Brot #TB$
Seed2inoculum !ime *h & >
(eal + *nm &.%"@ ".B>"
Time #$
A%)or%ance Optica! Den)it* #nm$
A%)or%ance Optica! Den)it* #nm$
#not di!"ted$
#Di!"ted$ 234
&
&.%%>
-&.&AB
"
-&.??B
-".&>C
%
&.A"?
-&."B?
?
&.AC
&."%%
@
&.A&&
-&.>?@
A
&.C"B
-&.C%%
C
&.BC
-&.B>
"&
&.BA%
-&."%>
"%
"."">
-&.?@B
"@
".&AA
-&."CB
"A
"."CA
&.%C&
"C
"."BA
&.">
%&
".%B>
&."BB
%%
".@@C
&.B&
%@
".@"B
&."@A
Optical density vs Time
2 1.5 1 0.5
Absorbance O ptical de nsity (nm) 0 0
5
10
15
20
25
30
-0.5 -1 -1.5
Time (hour)
Graph " 5 !he growth curve of E. coli plotted using absorbance optical density against time
7ell +ry eight *iomass 7oncentration
Time
Initia! 5ei't
0ina! 5ei't #'$
Ce!! dr*
Ce!! ma))
#$
#'$
#t"%e6)tic7er
5ei't #'$
concentration8
#t"%e6)tic7er
!a%e!6ce!!$8 m9
#m9 1 m2$
- #'.L$
!n -
!n -.-o
!a%e!$8 m2
&
".&BC
".&B
&.&&"
&.>
-&.A?
&.&&&
"
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&.&&%
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&
&.A?
%
".&A
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&.&&>
%.>
&."A
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?
".&B%
".&BA
&.&&@
%.&
&.A?
".?CA
@
".&>@
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&.&&>
%.>
&."A
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A
".&C"
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C
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A.&
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%.@C>
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%.?&?
In X vs Time 3 2.5
f(x) = 0.07x + 1.05
2 Ln X
1.5 1 0.5 0 0
5
10
15
20
25
30
Time (hr)
Graph 25 Graph of ln J against time which is plotted to construct the growth curve of the cell by using cell mass concentration, J *g2$ data. y referring Figure B.%, it shows that the exponential phase may occur at between ?rd and "%th hour. !hus, the data between that ranges of time will be used to find the value ' max.
In(X/Xo) vs Time 3 2.5
f(x) = 0.07x + 1.05
2
In(X/Xo)
1.5 1 0.5 0 0
5
10
15
20
25
30
Time (h our)
Graph ? 5Graph of ln J2J & against time is plotted to find the value of ' max From graph ?, the value of ' max is &.&AAA hr -" which find from the slope of graph ln J2J & against time. !hus the doubling time hr.
SAMPLE CALCULATIONS
!he value of cell dry weight can be determined by using the formula that will be shown below5 Ce!! dr* 5ei't / Initia! ma)) 1 0ina! ma))
!errific roth *! ". 7ell dry weight *g at time & th hour •
*Final weight of tube with cell K *Initial weight of tube ".&B K ".&BC 6 &.&&" g
%. 7ell mass concentration, J *g2$ at time & th hour • •
7ell dry weight L sample volume Molume of sample, % m$ 6 &.&&% $
6 &.&&" g L &.&&% $ 6 &.> g2$ ?. /aximum growth rate, ' max •
!he value is obtained from the slope of the graph of ln J2J & against time.
•
'max 6slope6 &.&AAA hr -"
@. +oubling time can be calculated by using formula of5 •
td 6 ln % L ' max
where 'max 6 &.&AAA •
ln % L &.&AAA
6 "&.@" hours
DISCUSSION
!his experiment is carried out to study the kinetic growth of microorganism. e choose E.coli as the cell and cultivated it inside a shake flask. !he growth of microorganism in shake flask is one of simple method called fermentation. !he media which contain carbon source is being supplied as the nutrients for the microorganism. !he flask was placed in incubator shaker to mix the cell and media for cultivation. It is to increase the homogeneity between media and cell and provide aeration for the cell. !he culture gone for fermentation for %@hours.ithin that period, the cell sample is taken out for every % hours to analyse the concentration of the cell *g2$ and the cell dry weight.
In order to determine the concentration of the cell inside the flask,t he absorbance reading of optical density is taken from spectrophotometer using A&&'m wavelength. !he higher the absorbance reading the higher the number of cell presence in the flask for particular time. From the graph " show the growth E.coli plotted based on absorbance value optical density *+ against time. !his graph somehow show the value of + is not same as theory. !he value of + did not show good pattern as it is increase and decrease at the particular time but at time %@hours,the cell has reached its deceleration phase where the growth of cell is started to slow down or in other word start to decay. !he optical density somehow give affect the result this might be during taking measurement of +, small air bubbles appeared without notice and it is counted as living cells. !he number of microscopic air bubbles, especially in dense cultures may be uite high. eside that the error might occur while conducting experiment is contamination while handling the culture that we are not aware of.
!he graph % shows the growth curve of the cell by using cell mass concentration.From the graph it can be observe the exponential phase is showed between ? rd to "%th hours.!he exponential phase or log phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission.!he rate of exponential growth of a bacterial cultured is expressed as generation time also the doubling time of the bacterial population.at time "%th to %%nd hours where the statationary phase occur.ased on the theory,the stationary phase is where the exponential growth cannot be continued forever in batch culture,it means that growth of bacteri remain contant and simply stopped growing,however the pattern graph
% shows at stationary phase there is still increment this is might be,if viable cells are being counted,it cannot be determined whether some cells are dying and an eual number of cells are dividing.)fter time %%hours and %@th hours it is call death phase .+uring the death phase,the number of viable cells decreases geometrically *exponentially,essential the reverse of growth during the log phase. Graph of ln J vs time is plotted in order to find the 0
.
max
From the graph, the exponential phase may be occur at time ? rd and "% th hour. !he value of 0max is obtained from graph of ln J2J & vs time. From the graph, the value of 0 max is &.&AAA hr "
. ence the doubling time is "&.@" hour. )s mentioned above, bacterial growth rates during
the phase of exponential growth, under standard nutritional conditions *culture medium, temperature, p, etc., define the bacteriumNs doubling time. +oubling time for bacteria vary from about "% minutes to %@ hours or more.
CONCLUSION
)t the end of this experiment,microorganism is suitable to be fermented inside a shake flask and it is simple methos to investigate the growth of microorganism.!he microorganism will go through several phases in their growth,several analyses on the cell need to be done to now the growth kinetics of the cell and the duration for each phase.!his include the cell concentration,glucose concentration and cell dry weight analyses. Growth kinetics deals with the rate of cell growth and how it is affected by various chemical and physical conditions. +uring the course of growth, the cells is continuously changing and adapting itself in the media environment, which can make physical and chemical condition of of cell growth change. In conclusion, the microbial culture in batch culture system *shake flask system goes through a lag phase, exponential growth phase, decelerating growth phase, stationary phase and sometimes the death phase depends on the end product desired
RECOMMENDATION
". efore start the bioreactor, it must be autoclaved to avoid contamination. !he bottles are plugged with cotton wool and sealed with aluminium foil. %. 7uvettes must be wiped with 3im ipe tissues to clean it and prevent any scratch so that the light from the spectrophotometer can pass through the cuvette and would not affect the spectrophotometer reading. ?. ear a glove while conduct experiment and spray on some ethanol before starting experiment this is to ensure no infections of bacteria. @. !he supernatant of cell concentration should be taken out carefully without any taking out of the biomass. >. !his experiment must be carried out under the laminar flow to prevent any contamination to the culture.
RE0ERENCES ttp3..555,%ioreactor),e".en.app!ication.optica!:den)it*:od:mea)"rement.
ttp3..555,mpi:%remen,de.Binarie).Binar*2;;,tm!
ttp)3..en,5i7ipedia,or'.5i7i.Bacteria!>'ro5t
RECOMMENDATION
A. efore start the bioreactor, it must be autoclaved to avoid contamination. !he bottles are plugged with cotton wool and sealed with aluminium foil. B. 7uvettes must be wiped with 3im ipe tissues to clean it and prevent any scratch so that the light from the spectrophotometer can pass through the cuvette and would not affect the spectrophotometer reading. C. ear a glove while conduct experiment and spray on some ethanol before starting experiment this is to ensure no infections of bacteria. . !he supernatant of cell concentration should be taken out carefully without any taking out of the biomass. "&. !his experiment must be carried out under the laminar flow to prevent any contamination to the culture.
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