UNIVERSITY OF SAN CARLOS

Department of Chemical Engineering

Technological Center, Talamban, Cebu City, Philippines 6000
Telefax: (032) 3!6"#3
Chemical Engineering Laborator
To be Performe$ by: %u&yneil '( Cartel Course)*r( : +, Ch'!-
,uper&ising .aculty: /r( '&elyn 0u1ue!Taboa$a Target /ate to ,tart: +ay -2, 200#
3$&iser: 'ngr( 4amelito 3gapay Target /ate of Completion: +ay -5, 200#
E!periment " #$ A%%metric Re&'ction of ()O!oi%ophorone to Dih&ro!)()
o!oi%ophorone '%ing *a+er,% Yea%t -.art /$ U%ing *atch Sha+e)Fla%+0
I1 Ob2ecti3e% of the E!periment
This experiment aims to achie&e the follo6ing:
To in&estigate the ability of ba7er8s yeast to cataly9e the :a; re$uction of an
unsaturate$ 7etone (<=P) to a saturate$ 7etone (/<=P), an$ the :b; $egra$ation of
the pro$uct /<=P to actinol(
/escribe the rate of <=P re$uction by 0a7er8s yeast using the +ichaelis!+enten
7inetic mo$el(
II1 4aterial% an& E5'ipment%
61/ 4icro)organi%m
3cti&e $ry yeast (Saccharomyces cerevisiae) is pre!culture$ in 'xperiment >o( 6( This
6ill then be use$ in the asymmetric re$uction of )oxoisophorone to $ihy$roxy!!
oxoisophorone(

616 7ro8th Con&ition% of 4icroorgani%m 9/) (:
Ten grams ($ry 6eight basis) of ba7er8s yeast is incubate$ beforehan$ in 'xperiment
>o( 6 in a -!? 'rlenmeyer flas7 at 30
o
C, 220 rpm an$ 6ith 20 grams (anhy$rous) of glucose
as its substrate for 2 hours( @or7ing &olume of the culture is one liter an$ 6ill be $one 6ith
a $uplicate( /issol&e$ oxygen concentration an$ the pA 6ill be monitore$ e&ery after
sampling using <rion +o$el 2-0 3 pA meter an$ 3T= <rion +o$el #62 /< meter(
Con$itions Bustifie$ in 'xperiment >o( 6 6ill be a$opte$ on this experiment as 6ell as the
sampling inter&al(
III1 4etho&%
;1/ OI. Re&'ction 9/:
3$$ - gram of pure <=P in the bioreactor 6ith culture$ 0a7er8s yeast( Continue the
incubation for 2 hours 6ith the Bustifie$ con$itions in ,ection 2(2(
;16 Sample .reparation 9/< =:
Centrifuge the sample mixture in Programmable ='C Centra Centrifuge ('0#03) at
-0,000 rpm an$ CC for -0 minutes( ,eparate the supernatant li1ui$ from the settle$
biomass an$ transfer it to another &ial( ,tore the samples at 3
o
C (
-
;1; S'b%trate an& *ioma%% Anal%i% 9/< >:
.rom the centrifuge sample, filter the biomass 6ith @hatman filter or its e1ui&alent (0(2
m) paper an$ 6ash it 6ith $eioni9e$ 6ater( Then, transfer the biomass in a pre!$rie$ an$
pre!6eighe$ e&aporating $ish( Aeat the sample in the 3ll 3merican +icro6a&e <&en +o$el
3+@!2-0 ?+ at a po6er setting == for 20 minutes at a po6er setting ==( Then cool in the
$essicator to initial temperature an$ 6eigh again(
;1( 7l'co%e< Ethanol< an& Acetate Anal%i% 9> ) ?:
3naly9e the glucose consumption using the Dlucose (Trin$er) 3ssay +etho$, an$ the
ethanol an$ acetate using ,hima$9u DC!#3 as $escribe$ in 'xperiment >o( 6(
;1= OI.< DOI.< ACT< an& Enantiomeric E!ce%% Anal%i% 9(:
;1=1/ Calibration an& Stan&ar&i@ation
Prepare six stan$ar$s of <=P specifically 0(0- gE?, 0(02 gE?, 0(0"2 gE?, - gE?, -(2 gE?,
an$ 2 gE?( Det "20 ? ethyl acetate an$ a$$ to "20 ? of <=P stan$ar$( +ix an$ inBect 2
? of it to the DC( <bser&e an$ get the pea7( Plot the area of the pea7s against the
concentration of the stan$ar$s( Fse the e1uation of the line in $etermining the
concentration of <=P in the sample( /o the same for /<=P (64 an$ 6,) an$ actinol (4,
64 an$ ,, 64) analysis(
;1=16 Anal%i%
Fsing a micropipette, a$$ ethyl acetate "20 ? to "20 ? of the supernatant li1ui$ to
extract <=P, /<=P an$ actinol from the solution( 3ssay the organic layer in a DC!#3 gas
chromatograph using the follo6ing con$itions:
a() Column temperature: -20
o
C
b() =nBector temperature: -#0
o
C
c() /etector temperature: -#0
o
C
$() 4un time: -2(5 minutes
e() <=P retention time: ( minutes
f() 6R!/<=P retention time: 2(# minutes
g() 6S!/<=P retention time: 6(0 minutes
h() 4S, 6R!3ctinol retention time: -2(5 minutes
i() Carrier gas: Aelium
B() Column: A4 =nno6ax capillary column
7() Carrier gas flo6rate: -mlEmin
l.) =nBection &olume: 2 ?
+easure the enantiomeric excess (e(e() of 64!/<=P from the pea7 areas of the t6o
enantiomers obser&e$ bet6een the retention times of 2(# to 6(0 minutes( 'xpress the
enantiomeric excess as :4!,;E:4G,; in H(
;1> A'antification of E!perimental Re%'lt%
+easurements in the calculation of <=P, /<=P, an$ 3CT concentrations as 6ell as the
initial specific <=P shoul$ be correcte$ 6ith the actual &olume of the reactor an$ the
amounts ta7en out $uring the sampling(
Ialues of the initial cell $ry 6eight in the reactor 6ill be use$ in the calculation of
specific biomass pro$uction rate in grams per liter per hour(
2
Concentration of <=P, /<=P, an$ 3CT (in gE?) 6ill be plotte$ against the time (in hr)(
,pecific rate of <=P re$uction, /<=P pro$uction an$ its $egra$ation to actinol 6ill then be
calculate$ from the plot through the slope of each line( The <=P re$uction can also be
$escribe$ using the +ichaelis!+enten +o$el by $etermining the Js (gE?), $escribes the
affinity of the substrate to the catalyst 6hich is the 0a7er8s yeast) an$ r max (maximum rate
in gE?!h, 6hich $etermines the rate of <=P re$uction at 6hich it reaches the maximum)(
The final yiel$ 6ill be expresse$ in H as the ratio of the total /<=P pro$uce$ to the
total <=P fe$ 6hile the final /<=P selecti&ity 6ill be expresse$ as the ratio of the total /<=P
obtaine$ to the total /<=P an$ 3CT pro$uce$( Iolumetric pro$ucti&ity 6ill be expresse$ in
terms of total grams /<=P pro$uce$ per liter per hour(
;1# Calc'lation% 9(< B:
;1#1/ 7l'co%e Con%'mption in gram%
The actual glucose or substrate consumption, S in grams can be $etermine$ using
the follo6ing formula:
S C 9-CSo 1 Vo D CS 1 V:
6here C,o is the initial concentration of substrate in grams per liter an$ C, is the final
concentration of substrate in grams per liter in the bul7 li1ui$ 6hile Io is the initial
&olume in liter , an$ I is the final &olume in liter(
;1#16 Amo'nt *ioma%% .re%ent
The actual amount of biomass present $etermines the amount of yeast present
insi$e the sha7e flas7 after some time or in e&ery sampling( The follo6ing formula is
use$ in $etermining the amount of biomass present, E in grams:
E K 9-C!o 1 Vo D C! 1 V:
6here Cxo is the initial concentration of biomass in grams per liter an$ Cx is the final
concentration of biomass in grams per liter in the bul7 li1ui$ 6hile Io is the initial
&olume in liter , an$ I is the final &olume in liter(
;1#1; OI. Re&'ction
The amount of <=P re$uce$, OI. 6ithin the experimental run is expresse$ in grams
an$ is calculate$ using the follo6ing e1uation:
OI. C 9-COI.< initial 1 Vo D COI.< final 1 V:
6here COI.< initial is the initial concentration of <=P in grams per liter an$ COI.< final is the final
concentration of <=P in grams per liter in the bul7 li1ui$ 6hile Io is the initial &olume in
liter , an$ I is the final &olume in liter(
;1#1( DOI. .ro&'ction
The amount of /<=P pro$uce$, DOIP 6ithin the experimental run is expresse$ in
grams an$ is calculate$ using the follo6ing e1uation:
DOI. C 9-CDOI.< initial 1 Vo D CDOI.< final 1 V:
3
YDOI.FOI. (gEg) C GDOI.F GOI.
Selecti3it (gEg) C GDOI.F -GDOI. H GACT0
6here CDOI.< initial is the initial concentration of /<=P in grams per liter an$ CDOI.< final is the
final concentration of /<=P in grams per liter in the bul7 li1ui$ 6hile Io is the initial
&olume in liter , an$ I is the final &olume in liter(
;1#1= Actinol .ro&'ction
The amount of actinol pro$uce$, ACT 6ithin the experimental run is expresse$ in
grams an$ is calculate$ using the follo6ing e1uation:
ACT C 9-CACT< initial 1 Vo D CACT< final 1 V:
6here CACT initial is the initial concentration of actinol in grams per liter an$ CACT final is the
final concentration of actinol in grams per liter in the bul7 li1ui$ 6hile Io is the initial
&olume in liter , an$ I is the final &olume in liter(

;1#1> DOI. Yiel& Determination
/<=P yiel$ is the amount of /<=P pro$uce$ in grams per grams of <=P re$uce$( 3fter
the experimental run, /<=P yiel$ is calculate$ using the follo6ing formula:
;1#1# DOI. Selecti3it
/<=P yiel$ is the amount of /<=P pro$uce$ in grams per grams of total /<=P an$
actinol pro$uce$( 3fter the experimental run, /<=P selecti&ity is calculate$ using the
follo6ing formula:
IV1 Reference%$
:-; Castro et( al, ,creening 0a7er8s *east for its Capacity to Cataly9e the 4e$uction of !
<xoisophorone, Fni&ersity of ,an Carlos, Cebu City, 2003
:2; .abian et( al, 200( The 'ffect of >itrogen ,ource an$ Concentration on 0iomass *iel$ by
0a7er8s *east (,accharomyces Cere&isiae) in a 0atch ,ha7e .las7, Fni&ersity of ,an Carlos,
Cebu, City
:3; +'4CJ +icrobiology +anual, 2000(0erlin
:; Taboa$a, '( 0( 2002( In Situ Removal of Solid Products During Whole-Cell Biocatalysis,
TF/elft, 2626 0C /elft, The >etherlan$s
:2; Aer6it9, @( 2002( <fficial +etho$s of 3nalysis of the 3<3C =nternational, -"th e$(, Iol( = an$
==, 3<3C =nternational, Iirginia, F,3
[6] Trinder, P( -5"3.Determination of glucose in blood using glucose oxidase with an alternative oxygen
accetor.,!nnals in "linical #iochemistry, v.6, .$%&$6, '(6(
:"; Templeton, /( -55( Chemical nalysis and !esting !as", #a$oratory nalytical Procedure,
?3P!0--, /etermination of 'thanol Concentration in 0iomass to 'thanol .ermentation
,upernatants by Das Chromatography
:#; .oley, /( an$ Dagnon *( -55( >=<,A +anual of 3nalytical +etho$s (>+3+), 3C'T=C 3C=/:
+etho$ -603,
th
e$(, ,pringfiel$, I3
:5; Jargi .( an$ +ichael ,( 2002( 0ioprocess 'ngineering: 0asic Concepts, 2
n$
e$(, Prentice LAall
=nc(, >%

/ate ,ubmitte$: +ay -3, 200#

Chec7e$ an$ 3ppro&e$ by:
DR1 EVELYN *UAUE)TA*OADA
,uper&ising .aculty

>ote$:
EN7R1 RA4ELITO A7A.AY
3$&iser, Ch' -2 ?
Date$ IIIIIIIIIIIIII
2