lecture notes on safety and pollution controlFull description
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2 sample problems with solutions using Microsoft ExcelFull description
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biochemical testing
MicrobiologyFull description
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Ministry of Science and Technology Technology Department of Technical Technical and Vocational Education B.E (Chemical) 2! "inal E#amination 2$%&%2! '$am% &&$am ChE 05022 Biochemical Engineering ns*er any fi+e ,uestions &.
Deri+e Deri+e the rate e,uati e,uation on for the follo*i follo*ing ng en-yme en-yme reaction reaction using using the Briggs% Briggs% aldane approach. / & / $ S0E ES ES 10E / 2
2. su3st su3stanc ancee is con+er con+erted ted to a produc productt 3y the cataly catalytic tic action action of en-ym en-yme. e. ssume ssume that the Michaelis%Menten /inetic parameters for this en-yme reaction are 4 M 5 .$ mol6l7 r ma# ma# 5&$ mol6l%min (a) 8hat should should 3e the si-e of a steady%state steady%state CST9 to remo+e remo+e :; percent of incoming incoming su3strate ( Cso 5 & mol6l ) *ith a flo* rate of & l6hr< (3) 8hat should 3e the si-e of the reactor if you you employ a plug%flo* plug%flo* reactor instead of the CST9 in part (a)< $. 9oughly 9oughly s/etch a pro/aryo pro/aryotic tic cells7 la3el its parts7 and state a function function for each of these. =. 8hat are the 3asic procedures of recom3inant D> techni,ues< E#plain 3riefly these techni,ues *ith the help of flo* chart. ;. E#pl E#plain ain in deta detail il the the gro* gro*th th cycl cyclee of unice unicell llul ular ar micr micro%o o%org rgan anism ism for for 3atc 3atch h culti+ation. !. i3a et al.(&:!') reported the results of a chemostat study on the gro*th of a species strain of 3a/er?s yeast as sho*n in the follo*ing ta3le. The inlet stream of the chemostat did not contain any cells or products. Dilution ion @nlet Steady ady%st %state ate Stead eady%st %state 9ate Alucose Conc. Alucose Conc. Ethanol Conc. %& D7hr Csi 7g6l Cs7g6l C p 7g6l .'= 2&.; .;= .: .& &.: .: =. .&! 2&.2 .&$' '.; .&:' 2. .&'! '.== .2=2 &.' .22! =.;& (a) "ind the rate e,uation for cell gro*th. (3) "ind the rate e,uation for product (ethanol) formation.
.. pro/aryotic cells Typical The pro/aryotic cell is unit of structure7 t*o group micro3ial7 3acteria and 3lue green algae. 1ro/aryotic cell is simple and small. 1ro/aryotic cell is not compartmentali-ed 3y unit mem3rane. 1ro/aryotic cell ha+e t*o structurally distinguished t*o internal region cytoplasm and nuclear region. Cytoplasm contains graing dar/ spot due to the present of ri3osome. 9i3osome is the site of important 3iochemical reaction for protein synthesis. The nucleur contain deo#yri3ose nucleuic acid (D>) 7*hich contain genetic information that determine the production of protein and structure . The pro/aryotic cell is surrounded 3y cell *all and a cell mem3rane. The cell *all is considera3le thic/er than cell mem3rane7 and it protect cells from e#ternal influence. The cell mem3rane ser+e as the surface *hich other cells components attac/ and important cell function ta/e place. =. The 3asic procedures of recom3inant D> techni,ues ha+e t*o factor. They are (&) to oin the D> segment to the molecule and they can 3e replicated . (2) to pro+ide en+ironment that allo* the reproduction of oin D> molecules . The produce for the flo* chart of recom3inant D> techni,ues are
2
D> "oreign
Stric/y end
D> @lagse Tranaformation
Transformation cell "ig The procedure of recom3inant D> . "igure sho* the procedure of recom3inant D> techni,ues. The plasmid cut in definite si-e *ith restriction en-yme. The D> of the foreign clea+ed *ith restriction en-yme. Some of fragment ha+e interest gene. The plasmid and genome fragment mi#ed and oined 3y the D> Figases. The recom3inant plasmids are introduced into the 3acteria and coculati+ation of plasmid and 3acteria. s & .solution / & S0E
/ $ ES
ES
/ 2 The intermediate of reaction *ith respect to time is neglected. dCES 5 dt r CES 5 / &CsCE% / 2 CES% / $ CES 5 / &CsCE 5 / $ CES 0 / 2CES / &CsCE 5 (/ 2 0/ $ ) CES CE 5 (/ 2 0 / $ ) CES / &Cs rp 5 / $ CES
10E
(&)
(2)
By en-yme 3alance7 CEG 5 CE 0 CES 5 (/ 2 0 / $ ) CES / &Cs 5 ( / 2 0 / $ 0&) CES / &Cs CEG 5 (/ 2 0 / $ 0 / &Cs) CES / &Cs By di+iding 3y / & $
CES
5
CEGCS / 2 0 / $ 0Cs / &
By su3stituting of e, n (2) rp 5 / $ CES 5 / $ CEG CS / 2 0 / $ 0Cs / &
Comparing *ith r p 5 r ma# Cs / M 0CS / $ CEG CS 5 r ma# CS / 2 0 / $ 0Cs /M 0CS / &
∴ / 20/ $ ≅ / &
/ 2 / &
r ma# 5 / $CEG / M 5 / 20/ $ ≅ / 2 / & / &
2. Solution CS
Cs
/ M 5 .$ mol6F r ma# 5&$mol ∗ ! min 5 ' mol F min & hr F hr (a) + 5< #s 5:;H Cso 5& mol 6 F " 5 & F 6 hr CS5 CSG (&I #s) 5 & (& I .:;) 5 .; mol 6F
=
t the CST97 @nput I Gutput 0 general 5 "CSG I "CS 0r s+ 5 " (CSG ICS) 5 I r s+ " (CSG ICs) 5 r p+ " (CSG ICS) 5 r ma# Cs V 4 M 0CS
" V
5 r ma# Cs (4 M0CS)(CSG ICS)
" V
5 'J .; (.$0.;) (&%.;)
" V
5 $: .;$ J :.;
" V V
5 .=! hr %&
V V
5" .=!hr %& 5 & F6hr .=! 5 .&2: liter.
2 .(3)
CS
1lug "lo*
Cs
The plug flo* reactor 7 V 5 < IdCs 5 rma# Cs dt 4 M 0CS Cs I ∫ dcs 5 rma# Cs 5 Cs Cso dt 4M 0CS 4 M 0CS
t
∫ r ma#
;
Cso t ∫ 4 M 0CS dCs 5 ∫ r ma# t Cs Cs Cso Cso ∫ /M dCs 0 ∫ dCs 5 r ma# t Cs Cs / MlnCso 0 ( Cso ICs ) 5 r ma#t ( K plus 5 t 3atch ) / MlnCso 0 ( Cso ICs ) 5 r ma#K .$ ln & 0 (&%.;) 5 ' K .; .':: 0 :.; 5 'K :.:;':: 5 ' K K 5 .&2$hr K 5 V 6 " 5 .&2$hr V 5 .&2$ × & F6hr V 5 .&2$ Fiter
× hr
!. Soln (a) r # 5 < (3) r p 5 < L5
µ ma# Cs
ks + Cs
&
µ 5
y5
ks
&
µ ma# Cs
0
& µ ma#
m#0c
D 5 L hr %&
CS g6l
& C s
&
µ
.'= .& .&! .&:' .2=2
.;= .&! .&$ &'! .22!
&&.: & !.2; ;.; =.&$
&'.; &2.! .2; ;.$' =.=2
"rom graph7 C5
& µ ma#
5 &.!
!
&
Lma# 5
5 .!2;
&.!
ks
m5
µ ma#
5
.' &.2
5 .!!
/s 5 .=2 g6l !. (a) r # 5 < µ ma# Cs r # 5 k s + C s .!2;Cs
&.(a) The rates of product formation and su3stance of consumption are dC p dt dC S
5 / $CES
5 / &CsCE / 2CES → (&) dt ssume that the change of C ES *ith time7 d C ES6dt7 is negli3le compared to that of C 1 or CS . %
dC ES
5 / &CSCE / 2 CES / $ CES ≅ → (2) dt Su3stituting E, (&) into E, (2) confirms that the rate of product7 formation and that of the su3strate consumption are the same7 that is 7 dC p dC S r5 5% 5 / $ CES dt dt @f *e assume that the total en-yme contents are conser+ed7 CEG 5 CE 0 CES → ($) Su3stituting E, ($) into E, (2) for C E7 and rearranging for C ES CES 5
r5
C EoC S k 2
+
dC p dt
k $
+
k &C S
dC S
5%
dt
k $ C EO C S k 2
5
+
k $
k &
+
C S
5
r ma#C S K M
+
C S
8hile in the Briggs% aldane approach7 it is e,ual to ( / 2 0 / $ ) 6 / & . CS
Cs
2. 4 M 5 .$ mol6lit ma# 5 &$ mol6lit CSG 5 & mol6lit " 5 & F 6 hr
×
&hr & 5 Fit 6 min ! min !
(a) V 5< 7 CS 5 .; C SG 5 .; mol 6 lit (a) CST9 @nput output 0 generation 5 accumulation dC S "CSG I "CS 0 µS V 5V 5 dt " (CSGI CS) 5 I µS V F V F V
5 I 5
V5
µ S
C SO
−
C S
µ ma#C S
( K M
F ( K M
+
C S )(C SO
+
−
C S )(C SO
C S )
−
C S )
µ ma# C S
&
5 !
(.$ + .;)(& − .;) &$ × .;
'
5 .&2: Fit. (3) V 5<
F C
CS
SO
"or plug flo* reaction µ =
−
dC S dt
K M
− ∫ C C
S
SO S
K M C S
K M ln
µ ma# C S
K M
+ C S
C S
SO
∫ C C
=
C S C S
.$ ln
dC S
+
& .;
C S
dC S
+ ∫ C C
SO S
(C SO +
+
−
= µ ∫ t dt ma#
dC S
= µ
ma#
t
C S ) = µ ma#t
(& − .;)
= &$ ×
V & 6 !
V 5.&2$lit $. The pro/aryotic cell is the unit of structure in t*o micro3ial groups 3acteria and 3lue%green algae. The pro/aryotic is small and simple 7 as sho*n in fig 7 *hich is not compartmentali-ed 3y unit mem3rane systems .The cell has only t*o structurally distinguisha3le internal regions cytoplasm and nuclear region (or nucleoplasm ).The cytoplasm has grainy dar/ spots as a result of its content of ri3osomes7 *hich are composed of protein and ri3onucleic acid (9>) . The ri3osome is the site of important 3iochemical reactions for protein synthesis. The nuclear region is of irregular shape 7 sharply segregated e+en though it is not 3ounded 3y mem3rane .The nuclear region contains deo#yri3onucleic acid (D>) 7*hich contains genetic information that determines the production of proteins and other cellular su3stances and structures . The pro/aryotic cell is surrounded *ith a cell *all and a cell mem3rane. The cell *all7 considera3ly thic/er than the cell mem3rane7 protects the cell from e#ternal influences. The cell mem3rane (or cytoplasmic mem3rane) is a selecti+e 3arrier 3et*een the interior of the cell and the e#ternal en+ironment . The largest molecules /no*n to cross this mem3rane are D> fragments and lo*%molecular%*eight proteins. The cell mem3rane can 3e folded and e#tended into the cytoplasm or internal mem3ranes. The cell mem3rane ser+es as the surface onto *hich other cell su3stances attach and upon *hich many important cell functions ta/e place.
Transformed cell "ig7 Method for the production of recom3inant D> "ig sho*s the o+erall procedure for the production of recom3inant D> .The plasmid is cut at a num3er of defined sites *ith a restriction en-yme. The D> of a foreign genome is clea+ed *ith a restriction en-yme. Some of the resulting fragments may ha+e the gene of interest. 1lasmids and genome fragments are mi#ed and oined 3y D> ligase. The recom3inant plasmids are introduced into 3acteria 3y coculti+ation of plasmids and 3acteria. ;. Aro*th Cycle for Batch Culti+ation @f you inoculate unicellular microorganisms into a fresh sterili-ed medium and measure the cell num3er density *ith respect to time and plot it7 you may find that there are si# phases of gro*th and death7as sho*n in "ig. They are &. Fag phase period of time *hen the change of cell num3er is -ero. 2. ccelerated gro*th phase The cell num3er starts to increase and the di+ision rate increases to reach a ma#imum. $. E#ponential gro*th phase The cell num3er increases e#ponentially as the cells start to di+ide. The gro*th rate is increasing during this phase7 3ut the di+ision rate *hich is proportional to d ln Cno 6 dt7 is constant at its ma#imum +alue7 as illustrated in fig .
&
=. Decelerated Aro*th phas fter the gro*th rate reaches a ma#imum7 it is follo*ed 3y the deceleration of 3oth gro*th rate and the di+ision rate . ;. Stationary phase The cell population *ill reach a ma#imum +alue and *ill not increase any further. !. Death phase fter nutrients a+aila3le for the cells are depleted7 cells *ill start to die and the num3er of +ia3le cells *ill decrease. & Fag 1hase The lag phase (or initial stationary7 or latent) is an initial period of culti+ation during *hich the change of cell num3er is -ero or negligi3le. E+en though the cell num3er does not increase7 the cells may gro* in si-e during this period. The length of this lag period depends on many factors such as the type and age of the microorganisms7 the si-e of the inoculum7 and culture conditions. The lag usually occurs 3ecause the cells must adust to the ne* medium 3efore gro*th can 3egin. @f microorganisms are inoculated from a medium *ith a lo* nutrient concentration to a medium *ith a high concentration7 the length of the lag period is usually long. This is 3ecause the cells must produce the en-ymes necessary for the meta3oli-ation of the a+aila3le nutrients. @f they are mo+ed from a high to a lo* nutrient concentration7 there is usually no lag phase. nother important factor affection the length of the lag phase is the si-e of the inoculum. @f a small amount of cells are inoculated into a large +olume7 they *ill ha+e a long lag phase. "or large%scale operation of the cell culture7 it is our o3ecti+e to ma/e this lag phase as short as possi3le. Therefore7 to inoculate a large fermenter7 *e need to ha+e a series of progressi+ely larger seed tan/s to minimi-e the effect of the lag phase. B
C
D
E
"
&2 & '
t "ig Typical gro*th cur+e of unicellular organisms () lag phaseO (B) accelerated gro*th phaseO (C) e#ponential phaseO (D) decelerated gro*th phaseO (E) stationary phaseO (") death phase. t the end of the lag phase7 *hen gro*th 3egins7 the di+ision rate increases gradually and reaches a ma#imum +alue in the e#ponential gro*th period7 as sho*n 3y the rising inflection at B in fig. This transitional period is commonly called the accelerated gro*th phase and is often included as a part of the lat phase. @n unicellular organisms7 the progressi+e dou3ling of cell num3er results in a continually increasing rate of gro*n in the population. 3acterial culture undergoing 3alanced gro*th mimics a first%order autocatalytic chemical reaction. Therefore7 the rate of the num3er density (C n) of 3acteria present at that time r n
=
dC n dt
=
µ C n *here
the constant
µ is
/no*n as the specific gro*th rate
hr −& .The specific gro*th rate should not 3e confused *ith the gro*th rate7 *hich has
&&
different units and meaning. The gro*th rate is the change of the cell num3er density *ith time7 *hile the specific gro*th rate is & dC n
µ =
C n
dt
=
d ln C n dt
The gro*th of micro3ial populations is normally limited either 3y the e#haustion of a+aila3le nutrients or 3y the accumulation of to#ic products of meta3olism. s a conse,uence7 the rate of gro*th declines and gro*th e+entually stops. t this point a culture is said to 3e in the stationary phase. The transition 3et*een the e#ponential phase and the stationary phase in+ol+es a period of un3alanced gro*th during *hich the +arious cellular components are synthesi-ed at une,ual rates. Conse,uently7 cells in the stationary phase ha+e a chemical composition different from that of cells in the e#ponential phase. The stationary phase is usually follo*ed 3y a death phase in *hich the organisms in the population die. Death occurs either 3ecause of the depletion of the cellular reser+es of energy7 or the accumulation of to#ic products. Fi/e gro*th7 death is an e#ponential function. @n some cases7 the organisms not only die 3ut also disintegrate7 a process called lysis. !. (a) C#i5
