54

CHAPTER 2

MATERIALS AND METHODS

2.1 CHEMICALS AND REAGENTS

The enzymes and reagents used in cloning experiments viz., NcoI,

XhoI, T4 DNA ligase, and Phusion™ Flash High-Fidelity PCR Master Mix,

M-MuLV reverse transcriptase, were purchased from New England Biolabs

(Manassas, USA). Taq Polymerase was procured from Fermentas (USA).

Plasmid miniprep spin kit, PCR purification kit, RNAprotect Bacteria

Reagent, RNeasy Mini Kit, RNase-Free DNase Set, random hexamers, dNTP

Mix (PCR Grade) and Proteinase K were procured from Qiagen (Germany).

Primers were procured from VBC-Biotech (Austria) and the inducer sakacin P

induction peptide (SppIP) was synthesized from GenScript (USA).

Chloramphenicol, ampicillin, kanamycin and lysozyme were bought from

Biobasic Inc (USA). Mutanolysin and 13C3- Glycerol were procured from

Sigma-Aldrich (USA). Culture media (LB and MRS) were purchased from

HiMedia Laboratories (Mumbai, India) and the individual media components

and other chemicals were obtained from either HiMedia Laboratories

(Mumbai, India) or from Merck (India). Since reuterin standard could not be

commercially procured, it was synthesized in our laboratory as described

under “Reuterin production by resting cells of L. reuteri ATCC 55730”.

2.2 CULTURE AND MAINTENANCE

The bacterial strains and plasmids used and modified in this study

are listed in Table 2.1.

55

Tab

le 2

.1 B

acte

rial

stra

ins a

nd p

lasm

id v

ecto

rs u

sed

in th

e st

udy

Stra

in o

r pl

asm

id

Des

crip

tion

Sour

ce o

r re

fere

nce

E. c

oli D

H5

Clo

ning

hos

t for

TA

ve

ctor

In

vitro

gen,

USA

E. c

oli E

C10

00

Clo

ning

hos

t for

pS

IP41

1 D

r Jan

Kok

, Uni

vers

ity

of G

roni

ngen

, N

ethe

rland

s

RB

C- T

A v

ecto

r TA

clo

ning

vec

tor

RB

C B

iosc

ienc

e C

orp.

, Ta

iwan

pSIP

411

E. c

oli-l

acto

baci

llus

shut

tle e

xpre

ssio

n ve

ctor

Sorv

ig e

t al (

2005

)

L. re

uter

i ATC

C 5

5730

H

ost

B

ioga

ia, S

wed

en

L. re

uter

i HR

2 L.

reut

eri w

ith y

qhD

Th

is st

udy

E. c

oli K

-12

MG

1655

So

urce

of y

qhD

gen

e Pr

of. T

akas

hi H

oriu

chi,

Nat

iona

l Ins

titut

e fo

r B

asic

Bio

logy

, Jap

an.

pHR

1TA

vec

tor w

ith y

qhD

Th

is st

udy

pHR

2pS

IP41

1 w

ith y

qhD

This

stud

y

L. r

eute

ri A

TCC

557

30 a

nd th

e E.

col

i stra

ins

wer

e gr

own

at 3

7 °C

in M

RS

brot

h (T

able

2.2

) an

d LB

(try

pton

e 1%

; yea

st e

xtra

ct 0

.5%

; sod

ium

chlo

ride

1%)

brot

h, r

espe

ctiv

ely.

A s

emi-d

efin

ed m

ediu

m a

s de

scrib

ed b

y

Lave

nder

et a

l., (

2001

) was

use

d fo

r met

abol

ic fl

ux a

naly

sis

with

out l

acto

se,

but w

ith g

luco

se (

27.7

5 m

M)

and

glyc

erol

(69

.38

mM

). C

asam

ino

acid

s in

the

med

ium

wer

e re

plac

ed w

ith c

asei

n en

zym

e hy

drol

ysat

e (ty

pe I

). In

addi

tion,

the

sem

idef

ined

med

ium

was

com

pose

d of

am

ino

acid

s, tra

ce

elem

ents

, vi

tam

ins,

nucl

eotid

es

and

twee

n 80

.Th

e re

com

bina

nts

wer

e

cultu

red

in m

edia

con

tain

ing

appr

opria

te a

ntib

iotic

s, am

pici

llin

(100

µg/

mL)

and

eryt

hrom

ycin

(200

µg/

mL

for E

. col

i and

5 µ

g/m

L fo

r L. r

eute

ri).

Gro

wth

was

mon

itore

d by

mea

surin

g th

e ab

sorb

ance

at

600

nm.

Cel

l dr

y w

eigh

t

56

(CD

W)

was

cal

cula

ted

from

a p

rede

term

ined

rel

atio

nshi

p be

twee

n L.

reu

teri

CD

W a

nd o

ptic

al d

ensi

ty (1

OD

600 c

orre

spon

ded

to 0

.33

g/l C

DW

).

For l

ong

term

stor

age,

E. c

oli a

nd L

. reu

teri

cel

ls w

ere

grow

n in

LB

med

ium

and

MR

S m

ediu

m re

spec

tivel

y, w

ith a

ppro

pria

te a

ntib

iotic

s at

37

ºC.

This

was

use

d to

inoc

ulat

e 10

ml o

f LB

or M

RS

and

the

cells

gro

wn

until

the

OD

600 r

each

ed 0

.8 to

1.0

(log

arith

mic

pha

se).

The

log

phas

e cu

lture

was

use

d

to m

ake

glyc

erol

(30%

) sto

cks a

nd st

ored

at -

80 ºC

.

Tab

le 2

.2 C

ompo

sitio

n of

MR

S m

ediu

m

Ingr

edie

nts

Con

cent

ratin

(g/l)

Y

east

ext

ract

5

Prot

eose

pep

tone

10

Bee

f ext

ract

10

Dip

otas

sium

pho

spha

te

2

Am

mon

ium

citr

ate

2

Sodi

um a

ceta

te

5

Mag

nesi

um su

lpha

te

0.1

Man

gane

se su

lpha

te

0.05

Poly

sorb

ate

80

1

Dex

trose

20

Fina

l pH

6.

5

57

Tab

le 2

.3 P

repa

ratio

n of

stoc

k so

lutio

ns o

f ant

ibio

tics

and

indu

cer

Rea

gent

/Che

mic

alSt

ock

Fina

l Con

cent

ratio

n

Am

pici

llin

100

mg/

ml i

n di

still

ed w

ater

10

0 µg

/ml

Eryt

hrom

ycin

10

0 m

g/m

l in

abso

lute

al

coho

l

10 m

g/m

l in

abso

lute

alc

ohol

200

µg/m

l (E.

col

i)

5µg/

ml (

L. re

uter

i)

Indu

cer (

SppI

P)

1 m

g/m

l in

dist

illed

wat

er

50 n

g/m

L

2.3

MO

LE

CU

LA

R B

IOL

OG

Y M

ET

HO

DS

2.3.

1 G

enom

ic D

NA

Isol

atio

n

The

geno

mic

DN

A o

f E.

col

i K

-12

MG

1655

stra

inw

as i

sola

ted

usin

g a

slig

htly

mod

ified

hig

h sa

lt, S

DS-

base

d m

etho

d (Z

hou

et a

l 199

6). 0

.5

ml o

f th

e ov

erni

ght c

ultu

re w

as p

elle

ted

in a

mic

roce

ntrif

uge

tube

at 1

0,00

0

rpm

for 1

0 m

in. T

he c

ell p

elle

t was

was

hed

in P

BS

buff

er (1

37 m

M N

aCl,

2.7

mM

KC

l, 2.

0 m

M K

H2P

O4,

10 m

M N

a 2H

PO4;

pH 7

.4)

and

susp

ende

d in

0.5

ml o

f TK

M I

buff

er (1

0 m

M T

ris, p

H 7

.6, 1

0 m

M K

Cl,

10 m

M M

gCl 2,

2 m

M

EDTA

). Th

e ce

lls w

ere

pelle

ted

agai

n an

d re

susp

ende

d in

TK

M II

buf

fer (

0.4

M N

aCl a

dded

to T

KM

I bu

ffer

) con

tain

ing

25

l of 1

0 m

g/m

l lys

ozym

e an

d

incu

bate

d at

37

°C f

or 1

5 m

in. T

o th

e su

spen

sion

, 50

l of

10%

SD

S w

as

adde

d, m

ixed

and

incu

bate

d at

55

°C f

or 1

0 m

in. T

hen

250

l of

5 M

NaC

l

was

add

ed t

o th

e su

spen

sion

and

mix

ed w

ell.

The

cells

wer

e pe

llete

d an

d

58

centrifugation as above. Finally the DNA was dried and resuspended in 50 l

TE buffer (10 mM Tris, 1 mM EDTA; pH 7.6).

2.3.2 Plasmid DNA Isolation

Plasmid DNA was isolated using Qiagen Mini prep kit using the

following protocol. 1.5 to 3 ml of E. coli culture was centrifuged at 8000 rpm

for 2min. Pellet was resuspended in 250 µl of buffer P1 by vortexing. 250 µl

of buffer P2 was added and mixed gently by inverting the tube 4-6 times until

the solution becomes clear. 350 µl of buffer N3 was added and mixed

immediately to form a white precipitate. The mixture was centrifuged at

13000 rpm for 10 min. Supernatant was carefully removed without any

precipitate and transferred to Qiaprep spin column. The column was

centrifuged at 13000 rpm for 60 s and the flow through was discarded. The

column was further washed with 500 µl of buffer PB and centrifuged for 60 s

at 13,000 rpm. 750 µl of buffer PE was added to the column and washed. The

column was transferred to a fresh 1.5 ml eppendorf and 50 µl elution buffer

was added, incubated for 2 to 5 min and then centrifuged to elute the plasmid

DNA.

For the isolation of plasmid from L. reuteri, Qiagen Mini prep kit

was used with the following modification: The cells in resuspension buffer,

were lysed with 30 mg/mL lysozyme (USB) and incubated at 37 ºC for

30 minutes. The rest of the procedure was done as described above.

2.3.3 RNA Isolation

RNA was isolated using RNeasy Mini Kit (Qiagen). The RNA in

the bacterium was stabilised by adding 500 µl (2 volumes) of RNA protect

bacteria reagent to 250 µl of bacterial culture. The mixture was vortexed for

5s and incubated at room temperature for 5 min. Further, it was centrifuged

59

for 10 min at 5000 x g and supernatant was decanted. The residual

supernatant was removed by gently dabbing onto paper towel. The pellet was

stored at -70 ºC for until use (upto 4 weeks). The following protocol was

adopted to isolate RNA from the processed culture.

L. reutrei cells were lysed in 100 l TE (10 mM Tris.Cl, 1mM

EDTA; pH 8.0)buffer containing 10 l proteinase K, 50 units mutanolysin

and 25 mg/ml lsyozyme and incubated at 37 ºC for 15 min. It was vortexed

for 10 s for every 2 min. 350 µl of buffer RLT was added and vortexed

vigorously. To this solution, 250 µl of absolute ethanol was added, mixed and

transferred to mini-spin column with collection tube. The sample was

centrifuged for 15 s at 8000 x g and the flow through was discarded. 350 µl of

RW1 buffer was added to the spin column and centrifuged for 15 s at 8000 x

g and flow through was discarded.

On-column DNase treatment was given, by adding DNaseI

incubation mix (10 µl of DNaseI stock solution + 140 µl of buffer RDD) to

the column and further incubating for 15 min at room temperature. 350 µl of

RW1 buffer was added to spin column and kept for 5min further centrifuged

for 15 s at 8000 x g. The collection tube was changed, and 500 µl of RPE

buffer was added to the spin column and centrifuged for 2 min at 8000 x g to

wash the spin column membrane. The mini spin column was placed in 1.5ml

tube and 50 µl of RNase free water was added to the spin column membrane.

The column was kept for 2 to 5 min and further centrifuged for 1 min at 8000

x g to elute the RNA. The isolated RNA was stored in -80 ºC until use.

2.3.4 Polymerase Chain Reaction (PCR)

PCR was performed using Taq polymerase and Phusion

polymerase. The PCR reaction mixture was made using the following

components (Table 2.4):

60

Table 2.4 components of PCR reaction

Components Final Concentration Forward Primer 500 nM

Reverse Primer 500 nM

dNTP 200 M

Template DNA 2 to 50 ng/ l

10X Polymerase buffer 1X

DNA Polymerase 1U

2.3.4.1 Cycling conditions for Taq polymerase

Initial denaturation 95 ºC 5 min

Denaturation 95 ºC 1 min

Annealing 55 - 65 ºC 1 min

Extension 72 ºC 1 min 20 sec (1 kb/min)

Final extension 72 ºC 10 min

29 cycles from step 2 to 4

2.3.4.2 Cycling conditions for Phusion polymerase

Initial denaturation 98 ºC 10 sec

Denaturation 98 ºC 3 sec

Annealing 65 ºC 5 sec

Extension 72 ºC 20 sec (1 kb/15 sec)

Final extension 72 ºC 1 min

24 cycles from step 2 to 4

61

2.3.

5 D

NA

Cle

anup

from

Aga

rose

Gel

The

band

cor

resp

ondi

ng t

o th

e de

sire

d D

NA

fra

gmen

t in

the

agar

ose

gel w

as r

apid

ly c

ut u

sing

a s

calp

el o

n a

UV

lam

p ta

ble.

The

siz

e of

the

gel s

lice

was

min

imiz

ed b

y re

mov

ing

the

extra

aga

rose

. DN

A fr

om th

e ge

l

was

pur

ified

usi

ng Q

ia q

uick

gel

ext

ract

ion

kit (

Qia

gen)

. Brie

fly th

e ag

aros

e

gel w

ith D

NA

was

sol

ubili

sed

with

buf

fer Q

G a

t 50

ºC a

nd tr

ansf

erre

d to

Qia

quic

k sp

in c

olum

n co

ntai

ning

sili

ca m

embr

ane,

whi

ch s

peci

fical

ly b

inds

to

DN

A w

hile

con

tam

inan

ts f

low

thr

ough

the

col

umn.

The

col

umn

is w

ashe

d

with

buf

fer

PE a

nd th

e pu

re D

NA

was

elu

ted

with

Tris

buf

fer

(10

mM

Tris

-

Cl,

pH8.

5) o

r wat

er.

2.3.

6 A

-tai

ling

of P

CR

Pro

duct

The

PCR

pr

oduc

t w

as

subj

ecte

d to

ge

l pu

rific

atio

n or

PC

R

purif

icat

ion

proc

ess

and

purif

ied

prod

uct w

as u

sed

for A

-taili

ng. B

riefly

in a

reac

tion

volu

me

of 1

0 µl

, 1 x

Taq

rea

ctio

n bu

ffer

, 3 µ

l (10

0 ng

) of

pur

ified

PCR

pro

duct

, 0.2

mM

dA

TP, 1

µl T

aq D

NA

pol

ymer

ase

was

add

ed a

nd th

e

mix

ture

was

incu

bate

d at

70

ºC fo

r 60

min

.

2.3.

7 PC

R P

urifi

catio

n us

ing

Spin

Col

umn

The

QIA

quic

k ge

l ext

ract

ion

kit (

Qia

gen)

bas

ed w

as u

sed

for

the

clea

nup

of D

NA

in P

CR

reac

tion

mix

ture

and

oth

er e

nzym

atic

reac

tions

. The

kit

uses

a s

pin

colu

mn

met

hod

whi

ch i

nvol

ves

the

spec

ific

bind

ing

of s

pin

colu

mn

mem

bran

e w

ith 1

00 b

p to

10

kb o

f D

NA

fra

gmen

ts, p

urify

ing

them

from

prim

ers,

poly

mer

ase,

sal

ts a

nd o

ther

com

pone

nts.

The

bind

ing

of D

NA

occu

rs a

t a p

H

7.5

and

elut

ion

is c

arrie

d ou

t hig

her p

H (p

H 8

.0).

Brie

fly, t

he

DN

A s

ampl

e is

mix

ed w

ith 5

vol

umes

of

buffe

r PB

and

tran

sfer

red

to s

pin

colu

mn

and

then

col

umn

was

was

hed

with

was

h bu

ffer

s and

the

purif

ied

DN

A

is e

lute

d us

ing

Tris

Cl (

pH 8

.0) b

uffe

r.

62

2.3.8 Restriction Digestion

Restriction reactions were carried out in a total reaction volume of

50 µl with 100 to 1000 ng of DNA, appropriate volume of restriction enzymes

and 1X reaction buffer. The reaction mixture was incubated at 37 ºC for 2 to 3

h. Restriction enzymes were inactivated by heating the mixture at 65 ºC for 20

min. For double digestion, compatible buffers were used for optimal activity

of both enzymes. The digested DNA was further purified using gel extraction

method or spin column method before ligation.

2.3.9 Ligation

Ligation was performed using T4 DNA ligase in reaction volume of

10 µl with appropriate ratios of insert and vector, 1 µl ligase and 2 µl 10X

ligation buffer. The vector/insert ratios were varied from 1:4 to 1:6 for

ligation. The reaction mixture was incubated at 16 ºC for 16 h.

2.3.10 Transformation

2.3.10.1 Transformation of E. coli Strains by Heat Shock

Chemically competent cells of E. coli strains were prepared using

the calcium chloride method (Sambrook et al 1989). Single colony was

inoculated into 4 ml LB broth for overnight, at 37 ºC with shaking at 180 rpm.

The overnight grown culture (250 µl) was used to inoculate 25 ml of LB broth

and grown until the OD600 reached 0.4 to 0.6. The culture was chilled on ice

for 10 min and then harvested by centrifuging at 7500 rpm for 10 min at 4 ºC.

The pellet was gently resuspended in 6 ml of ice cold 0.1M calcium chloride,

incubated in ice for 15 min and centrifuged at 3500 rpm for 20 min at 4 ºC.

The resultant pellet was resuspended in 12 ml of ice cold 0.1M calcium

choloride and kept on ice for 30 min. The cells were centrifuged at 3500 rpm

for 10 min at 4 ºC and the pellet was further resuspended in 500 µl of 0.1M

CaCl2 and stored at 4 ºC for one day as 100 µl aliquots.

63

To 100 µl of competent cells, 5 µl of ligation mixture or plasmid

DNA was added and gently mixed. The mixture was kept at 4 ºC for 30 min

and then subjected to heat shock at 42 ºC for 90 sec in a water bath. The cells

were then immediately chilled on ice for 5 min and 900 µl of LB media was

added. The cells were revived for 1 h at 37 ºC with shaking. The cells were

further plated onto LB agar containing appropriate antibiotics and incubated

at 37 ºC overnight for appearance of colonies.

2.3.10.2 Electrotransformation of L. reuteri

The electrocompetent cells were prepared as described by Berthier

et al (1996). In brief, 100 mL of MRS broth was inoculated with overnight

grown L. reuteri cells to an initial OD600 of 0.35 and incubated at 37 ºC (static

culture). At an OD600 of 0.6, the cells were chilled on ice and harvested,

washed once with ice-cold 10 mM MgCl2, and once with 10 mL of a solution

containing 0.5 M sucrose and 10% glycerol. Finally, cells were resuspended

in 1% of the same solution, aliquoted into eppendorfs and were either used

immediately or stored at -80 ºC upto one month. 5 µl of ligation mixture was

added to 40 µl of electrocompetent L. reuteri cells and transferred to a BTX

electroporator. 460 µL of MRS broth containing 80 mM MgCl2 was added to

the electroporated cells and transferred to an eppendorf and incubated at 37 ºC

for 2 h. The cells were plated on MRS agar containing the required antibiotic

and incubated for 24 - 36 h at 37 ºC until visible colonies were observed.

Electroporation conditions were:

Resistance 800 Ohm

Capacitance 25 µF

Voltage 1.5 kV

Time constant obtained was 11 – 13 milliseconds.

64

2.3.11 Screening of Recombinants by Lysate PCR

Individual transformed colonies were patched on LB agar plate

containing appropriate antibiotic and grown at 37 ºC overnight. A small

portion of patched colony was picked using sterile inoculation loop. The

picked cells were resuspended in 50 µl TrisCl buffer, and washed once.

Further lysis was performed by boiling the cells for 15 min in a water bath.

The lysed cells were centrifuged and 1.0 µl of supernatant was used as

template in 9 µl PCR mix containing forward, reverse primer and 5 µl of

2 x PCR master mixes. Polymerase chain reaction (PCR) was performed in

Eppendorf thermocycler.

2.4 ANALYTICAL METHODS

2.4.1 Agarose Gel Electrophoresis

An agarose gel of required strength (0.7 to 1.2%) was prepared

depending on the size of the DNA fragments to be separated. The gel is

created by suspending the desired quantity of dry agarose in 0.5X TBE buffer

(89 mM Tris, 89 mM Boric acid, 2 mM EDTA, pH 8.3), boiling until the

solution becomes clear, cooling it to about 55 ºC, adding ethidium bromide

(0.5 µg/ml) and then pouring it into a casting tray fitted with sample comb.

The gel was allowed to solidify at room temperature and then the comb was

removed. Further, the gel was placed horizontally in electrophoresis tank

containing TBE (0.5X) buffer. DNA samples were mixed with gel loading

buffer (0.21% Bromophenol Blue, 0.21% Xylene Cyanol FF, 0.2 M EDTA,

pH 8.0 and 50 % glycerol) and loaded onto wells. Bromophenol blue and

Xylene Cyanol dye fronts migrate through agarose gel along with double

stranded DNA fragments of 300 and 4000 bp respectively and thus were used

to mark the migration of DNA during electrophoresis. The voltage was set to

100 to 150 V and electrophoresis was performed at room temperature until

65

Xylene Cyanol reached bottom of the gel. The DNA bands were then

visualized under UV illumination using gel documentation system (Gel Doc

XR+ Imaging System, Biorad).

A similar procedure was adopted for preparing the agarose gel for

RNA analysis, by non-denaturing electrophoresis. 5 µl of RNA samples were

mixed with 5 µl of 2X RNA loading buffer (Qiagen) and heated at 70 ºC for

10 min and snap cooled on ice for 2 min to avoid secondary structure

formation. The sample was then loaded onto agarose gel and electrophoresis

performed at 100 V.

2.4.2 Quantitation of DNA and RNA

The concentration of DNA and RNA can be determined

spectrophotometrically by absorption in ultraviolet range. The readings were

taken at 260 nm and 280 nm. The OD at 260 nm allows calculation of the

concentration of nucleic acid in the sample. An OD260 of 1 corresponds to 50

g/ml for double stranded DNA and 40 g/ml for RNA. The ratio of

absorbance at 260 nm and 280 nm is used to assess the purity of DNA and

RNA. A ratio of ~1.8 is generally accepted as pure for DNA and a ratio of

~2.0 is generally accepted as pure for RNA. If the ratio is appreciably lower

in either case, it may indicate the presence of protein, phenol or other

contaminants that absorb strongly at or near 280 nm.

2.4.3 Cell Disruption by Enzymatic Method

L. reuteri cells were lysed enzymatically, using CelLytic B Plus Kit

(Sigma, USA), as per the manufacturer’s protocol. Briefly, cell pellet from

3 ml culture was resuspended in 1.5 ml of CelLytic B Plus working solution

(1 ml of cell lytic B, 80 l of lysozyme, 20 l of protease inhibitors and 100 U

of benzonase), vortexed briefly and incubated at 22 ºC for 30 min. The sample

66

turned transparent upon cell lysis and release of proteins. An aliquot of lysate

was directly mixed with appropriate volume of SDS sample buffer and

processed for protein analysis by SDS-PAGE. Another aliquot was

centrifuged at 13,000 rpm for 10 min at 4 ºC, to remove any insoluble

material. The soluble fraction was carefully removed from cell debris and

used for enzyme assays.

2.4.4 Total Protein Estimation

Protein concentration was determined with Bio-Rad protein micro

assay based on Bradford method. The dye-binding assay is based on

differential colour change of dye in response to various concentration of

protein (Bradford 1976). The absorbance maximum for an acidic solution of

Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding

to proteins. Each time the assay was performed, a standard curve was

prepared using BSA (Bovine Serum Albumin) as protein standard. 10 µl of

desired concentration of BSA was mixed with 190 µl of dye reagent and

mixed well. The protein concentrations were quantified in the microplate

format, as recommended by the assay kit and absorbance readings were

measured at 595 nm using ELISA reader (Labsystems Multiskan MS).

2.4.5 SDS-PAGE Analysis

Proteins present in sample were analysed by SDS-PAGE according

to the method of Laemmli (1970). The protein sample was mixed with sample

solubilisation buffer (SSB) and boiled to denature proteins for 10 min.

Subsequently, 5 to 15 µl (7 to 15 µg of protein) was loaded on to the gel. The

composition of the acrylamide gels (separating and stacking) is given in Table

2.5. The electrophoresis was started at 70 V until the bromophenol blue

reached the top of separating gel, then increased to 150 V. The electrophoretic

run was completed when the dye reached bottom of the gel.

67

Table 2.5 Components used in SDS-gel prepartion

Separating gel Stacking gel Tris/Hcl, pH 8.8 3.3 ml 3.4 ml

Tris/Hcl, pH 6.8 4.0 ml 830 µl

30 % Acrylamide/Bisacrylamide 2.5 ml 630 µl

10 % SDS 100 µl 50 µl

5 % TEMED 100 µl 50 µl

10 % APS (freshly prepared) 4 µl 5 µl

Acrylamide stock solution

Acrylamide - 29.2 g

Bisacrylamide - 0.8 g

Dissolve in distilled water to a final volume of 100ml

Electrode buffer (10X)

Tris Hcl - 0.25 M

Glycine - 1.92 M

SDS - 10 % (W/V)

Distilled water - upto 500 ml

Sample Solubilization Buffer (SSB, 4X)

Tris Hcl (pH 6.8) - 0.5 M - 1.0 ml

SDS (W/V) - 10 % - 0.8 ml

Glycerol - 40 % - 4.0 ml

- mercaptoethanol - 20 % - 2.0 ml

Bromophenol blue - 0.5 % - 50 µl

Distilled water - upto 10 ml

68

2.4.6 Coomassie Blue Staining

After electrophoresis, the gel was stained with coomassie brilliant

blue R-250 in a clean glass or a plastic container. Staining solution was added

approximately 5 times the gel volume and placed in a shaker for 4 to 14 h.

Stainer was saved for reuse and the gel was subsequently destained using

destainer.

Staining solution for Coomassie blue staining

Coomassie Blue R-250 0.3 g

Methanol 80 ml

Glacial Acetic acid 20 ml

Distilled water 100 ml

Destaining solution for Coomassie blue staining

Glacial Acetic acid 10 ml

Methanol 30 ml

Distilled water upto 100 ml

2.4.7 Biomass Analysis

Cell growth was monitored by measuring OD600 in UV-VIS

spectrophotometer (Hitachi U-1900). Cell dry weight (CDW) was calculated

from a predetermined relationship between CDW and optical density (1 OD600

corresponded to 0.33 g/l CDW and 0.3 g/L CDW for L. reuteri and E. coli

strains respectively).

69

2.4.8 HPLC Analysis

Concentrations of glucose, glycerol, 1,3-PD, ethanol, lactate, 3-

HPA and acetate in the culture broth were determined using a HPLC

(Shimadzu LC-10Avp) that was equipped with a refractive index detector

(RID) and an aminex HPX-87H column (300 x 78 mm, Bio-Rad, USA).

Resin-based HPLC columns like aminex, use multiple mechanisms of ion

exclusion, ion exchange, ligand exchange, size exclusion, reversed phase, and

normal phase partitioning to separate compounds.

Samples were filtered through 0.2µm PVDF (Polyvinylidene

fluoride) membranes before HPLC analysis and 20 µL of the filtered sample

was injected into sample injection port. The following conditions were

maintained during HPLC

Mobile Phase : acetonitrile and water in a ratio of 35:65

in 5 mM H2SO4,

Column temperature : 30 ºC

RID temperature : 50 ºC

Flow rate : 0.4 mL/min

Total time : 30 min

Standard graphs were made by plotting concentration versus area

obtained in the chromatograph. The standard graph equations were further

used to find out unknown concentrations of compounds in the samples.

3-HPA standard was synthesized in the lab using resting cells of L. reuteri

ATCC 55730 as explained in section 2.4.9.

70

2.4.9 Synthesis and Quantitation of 3-HPA

3-HPA was produced as described previously (Luthi-Peng et al

2002a, Spinler et al 2008). Briefly, the protocol for 3-HPA synthesis and

quantitation is as follows:

L. reuteri was cultured in 100 mL MRS broth, incubated

anaerobically at 37 °C for 24 h. The anaerobic condition was

maintained by sparging with nitrogen.

At the end of 24h, the culture was centrifuged and the pellet

washed with 50 mM sodium phosphate buffer (pH 7.4).

The cells were resuspended in 250 mM glycerol to a

concentration of ~1.5 x 1010 cells/mL and incubated

anaerobically at 37 °C for 2 h.

After the 2 h incubation, the culture was pelleted and the 3-

HPA-containing supernatant was collected and filter-sterilized

using a 0.22 µm filter and the filtrate used for HPLC analysis.

Quantitation of 3-HPA was done by HPLC, as described by Spinler

et al (2008). In short, the conversion of glycerol to 3-HPA occurs at a 1:1

ratio (Talarico et al 1988), and the concentration of 3-HPA in each sample

was determined by subtracting the remaining amount of glycerol from the

HPLC-determined concentration of the starting material.

2.4.10 Enzyme Assays

L. reuteri cells were grown on MRS medium containing 111 mM

glucose and 278 mM glycerol, in 15 ml static vials fitted with plastic screw

caps. Erythromycin (5 µg/ml) was added to the medium used for culturing the

71

recombinant strain. Cells were cultivated in batch mode at 37 ºC. Production

of carbon dioxide in the culture created and ensured aerobiosis. The

recombinant culture was induced with SppIP at an OD600 of 0.9 - 1.0. Samples

were taken at logarithmic phase to analyse enzyme activities.

1,3-propanediol oxidoreductase activity was measured

spectrophotometrically by following the initial rate of substrate-dependent

NADH formation at 340 nm ( = 6220 M-1cm-1) by a method adapted from the

work of Heyndrickx et al (1991). The assay mixture contained 100 mM 1,3-

PD, 10 mM NAD+, 30 mM ammonium sulfate, 100 mM Tris buffer (pH 9.0),

2 mM dithiothreitol and the whole cell lysate in a final volume of 1 ml.

The activity of NADP-dependent alcohol dehydrogenase (YqhD)

was also determined spectrophotometrically at 340 nm by the initial rate of

substrate-dependent NADPH increase ( = 6.22 mM-1cm-1). This method was

derived from the method described by Tang et al (2009). The assay mixture

contained 30 mM ammonium sulfate, 100 mM potassium carbonate (pH 9.0),

2 mM NADP, 100 mM 1,3-PD and the whole cell lysate in a final volume of

1 ml. One unit of enzyme activity is defined as the amount of enzyme that

catalyzes the conversion of 1 mol of substrate per min under the specified

conditions. Specific enzyme activity was expressed as units per mg protein.

All assays were performed in duplicates, and reported values are the averages

of two independent experiments.

2.4.11 Quantitative Realtime PCR Analysis

RNA integrity was examined by non-denaturating gel

electrophoresis. A260 and A260/A280 measurements using nanodrop

(Thermo Scientific, USA), were used to estimate the purity and concentration

of the RNA samples.

72

M-MuLV reverse transcriptase was used for cDNA synthesis. The

reaction mixture contained 1 g of RNA, 0.5 mM dNTPs, 0.5 g primers

(random hexamers), 100 U of M-MuLV, made up to a total volume of 60.0 l

with RNAse-free water. The reaction mixture was incubated at 37 °C for

60 min. The reverse transcriptase was then inactivated by heating the reaction

mixture at 65 °C for 10 min. The cDNA was diluted 3 times and stored at

-20 °C until use.

Quantitative real-time polymerase chain reaction (PCR) was

performed using Power SYBR Green PCR master mix (Applied Biosystems,

USA), in HT Fast Realtime PCR, model No 7900 (Applied Biosystems,

USA). The list of oligonucleotides used for qRT-PCR studies are listed in

Table 2.6. PCR was carried out using 96-well plate and the composition of

each reaction is as follows:

Forward primer 1 l (5 pmol)

Reverse primer 1 l (5 pmol)

cDNA 1 l (50 ng)

2X SYBR Green mix 10 l

Sterile water upto 20 l

The reaction mixture was incubated at 95 °C for 10 min, followed

by 40 cycles of denaturation (95 °C) for 15 s, annealing (50 °C) 2 min and

extension of 1 min at 60 °C. The reactions were performed in triplicates for

each gene.

The RQ manager software (Applied Biosystems, USA) was used to

calculate CT values for each gene amplification. The 16S rRNA gene that

codes for 16S ribosomal RNA was used as reference gene. It was used to

73

normalize the amount of cDNA used for each reaction. CT was calculated as

the difference between CT of the gene of interest and CT of the reference gene.

CT was calculated as difference between the delta CT of a gene in the

recombinant strain and CTof the same gene in the control strain at mid-log

phase. In the case of yqhD, CT was calculated for the induced recombinant

strain, with the uninduced strain as reference. The fold change was calculated

using the formula 2 CT (Livak & Schmittgen 2001).

Table 2.6 List of primers used for qRT-PCR analysis

Primer name Primer sequence 5’ to 3’

13pdor (Forward) CGATCCAATGTTGATGCTTGAT

13pdor (Reverse) CATTGACTATCAGTAATTGGGT

yqhD (Forward) GTGTGCTGACGCTGCCA

yqhD (Reverse) GGATCGAGCACGGCAAATAC

pgl (Forward) TCCTCAGAACAGAGGCTTGG

pgl (Reverse) GCAATGGAATTCTCCAAAGCA

fba (Forward) GGTGACTATGATGCAGCGA

fba (Reverse) CAATCTCTTTGGTTTTGGCA

aad (Forward) GCCATCGCTGATATGTGT

aad (Reverse) GTGAAGTGGCCTTATACATTG

ackA (Forward) TGTTGGTCACCGTATCTCTC

ackA (Reverse) AGCCTTTATTCCCGCTAAACCA

ackA2 (Forward) GCATTGTACAGTGTGCCTTA

ackA2 (Reverse) GCACCTGCTCCAATATGCAT

16S rRNA Hufner et al (2008)

74

2.5 STRATEGY FOR CLONING OF YQHD GENE IN

LACTOBACILLUS REUTERI

In order to obtain controlled, high-level expression of yqhD in

L. reuteri, the E. coli – lactobacillus shuttle expression vectors, pSIP 409 and

pSIP 411, were chosen. Both the vectors are characterized by the presence of

certain common features including:

A strong and sakacin-inducible promoter (PorfX)

Gene for erythromycin (ermL ) selection marker that functions

in both E. coli and lactic acid bacteria and

A broad host range and high copy number (pSH71) (de Vos,

1987) as in pSIP 411 or a narrow-host-range Lactobacillus

replicon from plasmid p256 (Sørvig et al 2005) as in pSIP 409.

The 1.163 kb yqhD gene fragment (GenBank accession number

NC010498), was amplified from the chromosomal DNA of E. coli K-12

MG1655 using the primers yqhDF and yqhDR (Table 2.7). PCR conditions

employed were:

1. Initial denaturation 98 ºC 10 sec

2. Denaturation 98 ºC 3 sec

3. Annealing 65 ºC 5 sec

4. Extension 72 ºC 20 sec (1 kb/15 sec)

5. Final extension 72 ºC 1 min

24 cycles from step 2 to 4

75

The amplicon was cloned into TA vector and transformed into

E. coli DH5 to generate the recombinant plasmid pHR1. Further, the yqhD

gene was sub-cloned from pHR1 into NcoI/XhoI site of pSIP411 and pSIP

409, with E. coli EC1000 as the cloning host, to yield constructs pHR2 and

pHR3 respectively. However, while pHR2 could be easily electroporated into

L. reuteri ATCC 55730, to generate the recombinant strain L. reuteri HR2,

the pHR3 plasmid could not be transformed due to the incompatibility of its

256rep replicon with the host. The clones were screened by lysate PCR using

the primer pair PorfXF and yqhDR (Table 2.7). The strategy used for

generating L. reuteri HR2 is depicted in Figure 2.1.

Figure 2.1 Strategy adopted for cloning yqhD gene in L. reuteri ATCC

55730

76

Table 2.7 Primers and peptide sequences used in the study

Primer name Primer sequencea

yqhDF (Forward) 5’-CATG CCATGGACAACAACTTTAATCTGCACACC-3'

yqhDR (Reverse) 5‘-CCG CTCGAG TTAGCGGGCGGCTTC-3’

PorfXF (Forward) SppIP

5’-TGAAAATTGATATTAGCG-3’ MAGNSSNFIHKIKQIFTHR

a The restriction sites in the primers NcoI (forward) and XhoI (reverse) have been underlined

2.6 METABOLIC FLUX ANALYSIS

In order to perform flux analysis, L. reuteri strains were grown on

semi-defined medium in 250 ml static flasks fitted with plastic screw caps.

They were inoculated with early log phase culture to obtain an initial OD600 of

0.2-0.3. They were cultivated in batch mode at 37 ºC for 7 h with an initial pH

of 6.5. Production of carbon dioxide in the culture created and ensured

anaerobiosis. The recombinant culture was grown in two different conditions

– uninduced and induced with SppIP at an OD600 of 0.6. Aliquots were

removed periodically to track growth and analyse metabolite profiles.

Experiments were carried out in duplicate and the mean values were estimated

together with the standard deviations for OD600, specific growth rate and

fermentation products.

Metabolic flux analysis is based on the pseudo-steady-state

assumption for intracellular metabolites, which implies that there is no

accumulation of any intermediates. Metabolic flux analysis for glucose and

glycerol – grown cells consisted of the following fluxes and unknown

intermediates: the glycolytic pathway consisted of twelve fluxes (v1 to v12)

and nine intermediates (Figure 2.2); the glycerol pathway comprised of three

fluxes (v13 to v15) and one intermediate (Figure 2.2). The degrees of freedom

77

for the glucose and glycerol pathways are 3 and 2 respectively. The system

becomes classically over determined as number of measured fluxes include

four in glycolytic and three in glycerol dissimilation pathways. These fluxes

were calculated from substrate utilization and product formation data using a

stoichiometric model of glucose and glycerol metabolism (Figure 2.2). The

metabolic fluxes through phosphoketolase (PKP), Embden-Meyerhof (EMP)

and glycerol dissimilation pathways were estimated for the period of

maximum growth rate as described by Årsköld et al. (2008). The fluxes in the

stoichiometric model, were derived from the relationship Qx = max * Yx,

where Qx is flux of the specific metabolite x (expressed as mol of x / mol

biomass-h), max represents maximum specific growth rate (h-1) and Yx

represents the specific metabolite production (mol of x / mol biomass). The

carbon balances were determined for control, uninduced and induced

recombinant L. reuteri strains, based on the total amount of carbon-containing

products and biomass formed (in C-mol) and the amount of glucose and

glycerol consumed (in C-mol). Carbon balances for glucose estimated during

the exponential growth phase in control and uninduced cultures were found to

close on C-mol basis at ~103%. However, the fluxes could not be analyzed

for the induced recombinant strain since carbon balance closure could not be

attained (data not shown). Carbon dioxide production was calculated from the

stoichiometric relationship with acetate and ethanol production (Arskold et al

2008).

To understand the influence of the heterologous alcohol

dehydrogenase on carbon flux, flux partitioning at key branch points, was

examined as previously described (Sridhar & Eiteman 2001). To determine

the flux-partitioning at a specific node, the carbon flux entering that node was

scaled to 100. Each scaled flux exiting the specific node therefore represents

the fraction of the incoming carbon flux exiting by that route.

78

Figure 2.2 The glycolytic and glycerol dissimilation pathways and

corresponding in vivo fluxes in L. reuteri ATCC 55730 The fluxes in glycoltic pathways are represented by v1 to v12 and fluxes in glycerol pathway by v13 to v15. Abbreviations: G6P glucose-6-phosphate, 6PG 6-phosphogluconate, R5P ribulose-5-phosphate, X5P xylulose-5-phosphate, AcP acetyl phosphate, AcCoA acetyl-CoA, F6P fructose-6-phosphate, FBP, fructose-1,6-bisphosphate, DHAP dihydroxyacetone phosphate, GAP glyceraldehyde-3-phosphate, Pyr pyruvate, 3-HPA 3-hydroxypropionaldehyde, GDHtglycerol dehydratase, 1,3-PDOR 1,3-propanediol oxidoreductase, YqhD E. coli alcohol dehydrogenase

79

2.6.1 Analysis of Glycerol Metabolism in L. reuteri ATCC 55730

using 13C- NMR Studies

For the labeling experiment, the following protocol was followed:

i. Inoculated 2 ml MRS broth with 10% of overnight grown L.

reuteri culture. Incubated at 37 °C for 1-2 h, until the culture

entered the log phase (OD600 of 0.9 to 1.0 obtained)

ii. 500 µl of the log phase culture was used to inoculate 5 ml of

MRS broth containing 13C3-labeled glycerol (130 mg).

Incubated the culture at 37 °C for 10 - 12 h, until the culture

reached late stationary phase (OD600 of 4.5 reached).

iii. Culture harvested and centrifuged to separate the fermentation

broth containing extracellular metabolites.

iv. The supernatant was concentrated by lyophilisation,

redissolved in 500 l of deuterated water (Sigma-Aldrich, St.

Louis, MO) and used for the identification of extracellular

metabolites.

13C-NMR spectra were recorded at 125.77 MHz on a Bruker AV III

500 MHz FT spectrometer with a broadband gradient probe head (5mm) at

room temperature. Signals for glycerol and 1,3-propanediol were assigned by

comparison with previously published chemical shifts (Spectral Database for

Organic Compounds, Japan) (A 1.2 and 1.3).

2.7 BIOREACTOR STUDIES

2.7.1 Batch Fermentation

To study the effect of glucose and glycerol concentration on growth

and metabolite formation in L. reuteri ATCC 55730, co-fermentation was

80

performed in batch mode with two different ratios of glucose and glycerol, 0.4

and 0.5, in MRS medium. The inoculum for the batch reactor was grown in

150 mL MRS broth at 37 ºC until an OD600 of 0.8 – 1.0 was reached. The seed

was then inoculated into a 2 L fermentor (KLF 2000 – Bioengineering AG,

Switzerland) filled with 1.2 L MRS medium containing erythromycin and

glycerol (278 mM or 217 mM). Fermentation was carried out at 37 ºC and

250 rpm, in an anaerobic condition. The pH was maintained at 5.5 by the

addition of 1.5 M NaOH or 1.5 M H3PO4 (El-Ziney et al. 1998). The

anaerobic condition was established by flushing with sterile nitrogen.

Similar batch bioreactor studies involving the recombinant L.

reuteri strain were carried out in MRS medium with erythromycin. A glucose

to glycerol ratio of 0.4 was used. At 0.8 OD600, the recombinant culture was

induced with 50 ng/mL of sakacin P induction peptide (SppIP). Samples were

removed periodically for determining OD600. The culture pellet and

supernatant were stored at – 20 ºC, to be used later for protein and metabolite

analyses respectively.

Cultures for qRT-PCR studies, were grown in a 7.5 L fermentor

(New Brunswick Scientific, BIOFLO 415 Fermentor, USA) filled with 3 L

MRS medium containing glycerol (278 mM). Rest of the procedure was

similar to that of the batch fermentation described earlier. The samples were

removed at mid-log and late-log phase for qRT-PCR analysis.

2.7.2 Fed-batch Fermentation

Fed-batch fermentation was performed with L. reuteri ATCC

55730 strain, in a 7-L fermentor (BioFlo 415-New Brunswick Scientific,

USA) containing 3-L MRS medium at 37 ºC, 250 rpm and pH 5.5 (controlled

by adding 1.5 M NaOH or 1.5 M H3PO4) supplemented with 1.84 g/L

(20 mM) glycerol, under non-aerated conditions. The pre-culture was grown

81

in 300ml MRS broth at 37 ºC until an OD600 of 0.8-1.0 was reached and was

inoculated into fermenter. Two kinds of feed were given – Feed solution I

contained only glucose and glycerol, whereas feed solution II contained

limiting nutrients (Table 2.8). Two types of feed solution I (A and B) were

used in this study. Feed Solution I (A) containing low concentration of

glycerol, was used to primarily improve growth and prevent the accumulation

of toxic 3-HPA. Since 1,3-PD is growth-associated, feed Solution I (B)

containing higher glycerol concentrations, was given when an OD600 of ~9.5

was reached, to improve cell density as well as 1,3-PD formation.

Table 2.8 Composition of feed solutions used in fed-batch study

Type of feed Composition

Feed solution I (A) Glucose - 720 g/l (4 M)

Glycerol - 73.6 g/l (0.8 M)

Feed solution I (B) Glucose - 362.25 g/l (2.0 M)

Glycerol - 287.5g/l (3.12 M)

Feed solution II Beef extract - 200 g/l

peptone - 20 g/l

Yeast extract - 20 g/l

At glucose concentrations below 5 g/L (27.7 mM), 42 g of Feed

Solution I (A) and 60g of Feed solution II were added to the reactor medium.

For second phase feed, 114.78 g of Feed Solution I (B) and 60g of Feed

solution II were added simultaneously into the reactor. Samples were

collected at regular intervals for monitoring OD600. The samples were also

centrifuged and the supernatant stored at – 20 ºC, to be used later for

metabolite analyses.

82

2.8 1,3-PROPANEDIOL TOLERANCE STUDIES IN L. REUTERI

The inhibitory effect of 1,3-propanediol on growth of L. reuteri

was studied under batch conditions in 50 ml bottles fitted with plastic screw

caps. Different concentrations of 1,3-PD – 0, 50,100 and 150 g/L, were added

to the growth medium (MRS broth), either before inoculation or when the

culture reached early exponential phase (OD600 1.0) and incubated at 37 °C

for 24 h. Growth was monitored by measuring OD600 at regular intervals. The

absorbance values were then converted to Cell dry weight (CDW) values as

explained in 2.4.7.

2.9 BY-PRODUCT INHIBITION ANALYSIS

Formation of by-products, lactate and ethanol, was curtailed using

inhibitors. Log phase cultures of L. reuteri grown in MRS were exposed to

varying concentrations of lactate inhibitors, sodium iodoacetate and oxalic

acid for up to 4 h and their growth and metabolite production analysed by

HPLC. To inhibit ethanol formation, cultures of native and engineered strains

of L. reuteri in latent (before inoculation) and log phase in MRS, were treated

with 15mM benzaldehyde, an ethanol inhibitor (Shone et al 1981) and

incubated at 37 °C for 24 h. Growth was monitored by measuring OD600. The

concentration of acetate, lactate, ethanol and 1,3-PD in the culture supernatant

were determined by HPLC.

10% of the overnight grown L. reuteri cultures were transferred to

15 ml vials containing MRS with 111mM glucose and 278mM glycerol and

was incubated at 37 °C till early log phase of 0.8 -1.0 was reached. Varying

concentration of lactate and ethanol inhibitors were added to the culture at log

phase and samples were collected at different time points to monitor cell

growth and metabolite concentrations.

83

2.10 BIOINFORMATIC ANALYSIS

To determine if the sequence of the redox-sensing global regulatory

protein Rex and its DNA-binding sites, are present in the genome of

Lactobacillus reuteri ATCC 55730, bioinformatic analysis was adopted.

However, genome of L. reuteri ATCC 55730 strain is not manually curated

for the presence of Rex repressor and its binding sites. Therefore relevant

information from another strain, L. reuteri JCM 1112, that is available at

RegPrecise, an online database of manually curated inferences of regulons

(Novichkov et al 2010), was exploited. The amino acid sequence of Rex

protein from L. reuteri JCM 1112 strain (UniProt id: B2G5X5) was blasted

against the whole genome of L. reuteri ATCC 55730 to identify the

corresponding putative Rex sequence. In the rex regulon of JCM 1112 strain,

Rex protein was found to bind upstream of the following five genes, namely,

rex (Redox-sensitive transcriptional regulator Rex), gldA (Glycerol

dehydrogenase), dhaT (1,3-propanediol dehydrogenase), noxE (NADH

oxidase), adhE (Alcohol dehydrogenase, EC 1.1.1.1; Acetaldehyde

dehydrogenase, EC 1.2.1.10). The Rex-binding sites upstream of the 5 genes

in JCM 1112 strain were used as queries and blasted against the whole

genome of ATCC 55730 strain, to identify the presence of similar putative

Rex-binding sites.

2.11 SIMPLE UNSTRUCTURED KINETIC MODEL

A modified version of the kinetic model proposed by Tobajas et al

(2008) has been adapted to describe the evolution of biomass, consumption of

substrates and formation of products at various time points, in glucose-

glycerol co-ferementation in L. reuteri. The kinetic model was used by the

authors to describe the behaviour of L. reuteri PRO 137 strain during

co-fermentation in batch mode. In the present study, the modified model has

been utilized for describing the behaviour of native and recombinant

84

(L. reuteri HR2) strains of L. reuteri ATCC 55730. The simple unstructured

model developed by my colleague, Ms. Geethalakshmi, considers the mass

balances for eight state variables such as biomass (X), glucose (S), glycerol

(G), acetate (A), lactate (L), ethanol (E), 3-HPA (R) and 1,3-propanediol (P)

(Equations 2.1 – 2.8) with eight parameters , viz., k1, k2, k3, k4, YXS, YLX, YEX and

YAX. Essentially all the mass balance equations have been adopted from the

model proposed by Tobajas et al (2009) except the ones describing the

production rate of 1,3-PD and glycerol, which have been slightly altered.

The equation used to describe the growth of L. reuteri not only

considers biomass evolution and substrate concentrations, but also the

inhibitory influence of 3-HPA accumulation. This is given by:

RkXSk

dtdX

2

1 (2.1)

where 1k represents the growth constant and 2k represents the saturation

constant for 3-HPA.

Since glucose is the limiting substrate for the growth and biomass

formation in L. reuteri, the rate of glucose consumption could be linked to the

yield of biomass on glucose as follows:

/( )XS

dS dX dtdt Y

(2.2)

where YXS denontes the yield of biomass on glucose.

The equations describing the production rate of 1,3-PD and

glycerol are based on the conversion of glycerol to reuterin and its subsequent

reduction to 1,3-PD, as is evident from experimental data in literature (Luthi-

Peng et al 2002a, Talarico et al 1990). The additional direct glycerol

85

bioconversion assumed in the earlier model, was omitted in our study.

Accordingly the equations describing the glycerol consumption rate and 1,3-

PD formation rate have been modified. The rate of glycerol consumption

depends on biomass and reuterin formation as given by the expression:

GkdtdG X3 (2.3)

where 3k is the Kinetic constant for 3-HPA production.

The rate at which reuterin is produced is related to its production by

glycerol dehydration as well as its conversion to 1,3-PD by reduction. Both of

these processes are reflected in the equation:

XRkXGkdtdR

43 - (2.4)

where 4k is the Kinetic constant for 1,3-propanediol production

The equation describing the formation rate of 1,3-PD could be

directly linked to the reduction of reuterin as:

XRkdtdP

4 (2.5)

The rate of production of by-products such as lactate, acetate and

ethanol, could be related to cell growth as expressed by the following rate

equations:

dXdtdL YLX / dt (2.6)

where LXY is the yield of lactate on biomass.

86

dXdtdE YEX / dt (2.7)

where EXY is the yield of ethanol on biomass.

dXdtdA YAX / dt (2.8)

where AXY is the yield of acetate on biomass.

2.11.1 Parameter Estimation

Parameters of the unstructured model have been estimated using

genetic algorithm (GA), an effective stochastic global search algorithm (Park

et al 1997, Roubos et al 1999, Sarkar and Modak 2004), along with a Runge-

Kutta integration method. Experimental data of the batch experiments have

been interpolated for accurate parameter estimation. The parameters have

been estimated by minimizing the objective function as given in Equation

(2.9), which is the normalized squared error between the estimated and

measured state variables in MATLAB 7.7. The optimization tool box with

Genetic algorithm as a solver has been used to estimate the parameters. The

objective function is defined as,

m

j

n

iij ijeij XXX

1 1

2max

2functionObjective (2.9)

where n is the number of observations (interpolated data)

m is the number of state variables

Xeij represents the estimated state variables

Xij represents the measured state variables and

Xij max is the maximum value of the state variable

87

In an attempt to make the model more realistic, the estimated

parameters were constrained by upper and lower boundary values that were

based on batch data and relevant literature (De Vries et al 1970, Pancheniak

et al 2012, Teusink et al 2006, Tobajas et al 2009). The estimated parameters

have been used to simulate the entire profile of the state variables for the two

batch experiments, by solving the above nonlinear differential equations in

MATLAB.