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THE PRCDUCTTOI'J Q"t;' LACTIC ACID FROJ.! WP.EY BY COT':'l'H:UOUS

CULTURE AS A POSSIBLE l\�ANS OF VfA:STE DISPOSAL

A thesis presented in partial fulfilment of the

requirements for the degree of Doctor of Philosoplw

i.n Biotechnology at J,:assey University.

KEVT!'!. l!.A"R.SHALL

1_972

ABSTRACT THE FR.ODU�TIC'�r C"l" L4.CTJC ACID Fll0rf W:fl':'f BY crr�CITI:LJO TS

CUI.TlJ't� AS A FC3STRL::: !:'E/:NS OF '.'.'AST"'3: DIS9CSAL.

A study wa s made of the fermentation of lactose in lactic casein

whey to lactic acid u s ing a strain of LactobaciJ.lus bulgaricus.

Both batch and continuous culture were useu .• A culture ves sel capable of be ing operated under controll ed conditions \

was de signed and built for thi s s tudy. Temperature, pH, gas �tmospher�,

degree of agitation and medium flow rate could be altered and controlled.

A meter was developed far the continuou s measurement of lactic acid

production. The meter used a capacitance probe to measure the volume

of alkali addsd to the culture to maintain a constant pH. The kinetic s of lactic acid production in a batch culture of whey

were characterized by :

dP �� + �N) p p m dt

= K + p p p m The kinetic s of bacterial cell growth were cons i stent with the normally

accepted Monod equation but no direct verifi cation of this was made .

A notable feature of the production of lactic acid in a batch culture

was the considerable amount of lactic acid formed by non-dividing

bacterial cells . More than 50 p ercent of the acid produced during a

batch culture was synthe sised while the cell population was in a

s tati onary growth phase.

The maximum cell number was not l imited by the concentration of

lactose . Supplementation with tryptophan , casamino acids and a number

of vi tamins increased the cell popul ation and the rate of acid production

and de creased the batch time . S odium caseinate was a good source, of

es sential and stimulatory nutrients.

The optimum heat treatment of the whey involved heating to 69°C. In unsupplemented whey the removal of suspended material by centri­

fuging and filtration prevented the formation of acid. To maintain

maximum acid formation rate s the impeller Reynolds number had. to be

greater than 10,000.

The presence of oxygen prevented the growth of the bacterial cell

popul ation, but once the maximum c ell p opulati on had be en reached

oxygen did not effe c t the acid synthe si s .

In a sir.gle stage continuous culture reactor the concentration of

lactic acid was eiven by :

p p m K + p P

p m

The constants were deter·mined from batch culture data.

A single s tage continuous culture is not suitable for the conversion

of all the lactose in the whey to lactic acid. If lactic aci d production

by continuous culture is t o be considered as a means of waste dispo sal

it will be neces sary to u se feed back of cells to a single - stage

reactor or mul ti-stage s tirred taru�s.

I n continuous culture studies it was shovm that the optimum

temp eratu!'e fo r the fermentation of lactic casein whey was 4-6°C. A

pH in the rage 5.4- - 6.0 was best. Outside this range, p roductivity

and yield were decreas ed.

I t can b e conclude d that though continuous production of' lactic

acid from whey is fea sibl e , multi-stage continuous reaction systems

an�or cell feedback are necessa�y to reduce the lactose concentration

t o an acceptable level. The whey should be supplemented with a source

of amino acids.

ACKN OVH;EDGEr,;ENT S

I wish to thank my supervisory committee and my colleagues

in the New Zealand Dai� Research Institute for helpful and

stimulatin g discussions, advice and assistance.

iv

1

2

2.1 2.1 .1 2.1 .2 2.1 .3 2.2 2.2 .1 2.2.2 2.2.3 2.2.4-2.2.5 2.2.6 2.2.7 2.3

3

3.1 3.1 .1 3.1 .2 3.1 .3 3 .1 .4-3.1 .5 3.1 .6 3.1. 7 3.1 .8 3.1 .9 3.2 3.2 .1 3.2.2 3.2.3

TABLE 01<' COl'TTENTS

ACKJIJOWLEDG 3f.:Erns.

TABL}; OF COI\'T�NTS.

FIGURSS.

TABLES. INTRODUCTION.

A REVIJ.t.'W OF THE RELEVAN'£ LITERATURE.

Microbial Kinetics.

Models of cell growth.

Models of metabolit e production.

Continuous culture.

Lactic acid.

Whey medium.

Organisms.

CroVith factors.

Temperature.

Heat treatment of whey.

pH.

Continuous culture.

Agitation.

EXPERIMENTAL.

Equipment.

Culture vessel.

Agitation.

Continuous operation.

Gas supply.

Temp era tu re • pH.

Alkali volume meter.

Cost of the equipment.

Flask cultures.

Materials.

Organism.

Whey.

Chemicals.

iv

V

viii

xiii

1

5

5 5 8

10 13 13 14-14-15 15 16 17 17

20

20 20 20 28 28 28 28 29 33 34-34-34-35 36

3.2.3.1

3.2.3.2

3.3

3.3 .1

3.3.2

3.3.3

3.4

3.4 .1

3.1 .... 2

3.4.3

3�5

4

4.1

4.2

4.2 .1

4-. 2.2

4.2 .3

4. 2 ·'+ 4.2.5

4.2.6

4.2.7

4. 2.8

4.3

4.3 .1

4.3.2

4.3.3

5

5.1

5.1 .1

5.1 .2

5.1 .3

5·.1 .4

5.1 .5

5.1 .6

5.2

5.3

�upplementary nutrient solutions.

Sodium case inate.

�'easurements.

Cell concentrati on.

Lactose concentration .

Lactate concentrat ion .

Methods.

Batch culture .

Contin11ou s culture.

Flask culture .

Method or expres s ing re sults .

RESULTS. Selection or Variables .

Batch culture .

Typ ical batch culture .

Whey concentration.

Supplementary nutrients .

Ste rile ril tration .

Cas ein as a suppl ementary nutrient .

Controlled atmosphere .

Agitation .

·cell recycle .

Ccntinuous culture .

Opt imum value s or pH , temperature and

dilution rate ror a sinele stage continuous

culture us ing steriJized whey.

S terilized whey with added s odium caseinate .

Pasteurized whey.

KINETICS OF LACTIC ACID PRODUCTION FROM WHEY.

Batch culture .

vi

Comparison with the kinetic equation derived by Pirt.

Exp onential 5rowth rate phase .

Declining growth rate phase.

S tationary growth rate phas e .

Enzyme kinetics .

Batch culture k inetics .

Continuous culture .

Prediction of continuou s op eration from batch data.

36

45

ll-5

45

52

53

60

60

67

73

83

85

85

95

95

99

99

102

.1 02

103

106

106

108

111

112

6.

7.

1 .• 2. 3.

5.

6.

DISCUSSION.

CONCLlJSIOJ\'S.

l\'OJ.�NCLATURE.

REFERENCES.

APPENDICES.

Lactic casein whey.

Classification of LBR.

Sample calculation.

Yield of lactic acid from lactose.

Analysis of a three variable central

composite rotatable design.

vii

114-

131

132

131+

14-0

1�-0

14-1 1414-

14-6

14-7 Experimental designs and selection of variables.150

3 .1

3.2

3. 3

3 . 4

3 . 5

3 . 6

3 . 7

3 .8

3.9

Schematic outline of the culture vessel�

The culture vessel and controls.

Details of the ports in the culture ve�sel and

the coupling used to conr.ect flexible hoses.

Detail of the top plate of the culture vessel.

Detail of the bottom plate of the culture vessel.

The impellers used for ag1tating the culture medium.

The imp eller �uift seal.

The calibrat1on curve for the alkali burette.

A typical recorder trace obtained from the

capacitance probe in the alkali burette.

Time IJeasured in the vertical dirc;ction and

volume of alkali in the horizontal. The stepped trace is due to the on-off operation of the

pF! controller.

3.1 0 A typical lactose calibration. Cptical d.ensity at

4.2

370 nm of the lactosozone solution forrEed by

reaction of lactose with phenylhydrazine as a

functi on of lactose concentration.

A typi cal batch culture. B9 . 1 .5. Acid concentrat­

ion as a function of time. '::hey 35 g/1, pH - 6.0,

A typical batch culture; B9 . 1 .5; ln cell number

concentration and ln aci1 concentration as

viii

21

22

23

24

25

26

27

30

31

41

functions of time. Whey 3 5 g/1, pH 6 . 0, 46°G. 47

A typical batch culture; B9 .1 .5; the rate of acid

formation as a function of time.

\Yhey 35 g/1, pH 6 . o, 46°c.

Acid and lactose concentrations during the batch

culture of vrhey. Whey 75 g/1 with supple­

mentary nutrients, pH- 5.5 , 46°C .

48

49

4.5 Ln deor<Jribonucleic acid (DNA) concentrabon

4-.6.a

4.6.b

as a function of time during the batch culture of whey. Whey 35 g/1, pH 6.0, 46°c.

Acid concentration as a function of time during the

batch culture of pasteurized sterile-filtered whey

with supplementary nutrients. a - salts,

b - vitamins, c - amino acids, d - nucleic acid

compounds. Whey 35 g/1, pH 6.0, 46°C.

Acid concentration as a function of time during the

batch culture of' sterile-filtered whey '?fi th supple­

menta� nutrients. a - salts, b - vitamins,

c - amino acids, d - nucleic acid compounds.

Whey 35 g/1, pH 6.0,l1-6°c;.

4.7 The reduction of acid synthesis caused by centri-

4.8

fuging or sterile filtering and pasteurizing

whey. Whey 35 g/1, pH 6.0, 4-6°C.

The effect on the rate of acid synthesis of adding

5 g/1 sodium caseinate (AD 74) to sterile

fi1 tered whey (B9 .3 .12) cf fig .4. 7.

Whey 35 g/1, pH 6.0, 4-6°c.

4.9 Different batches of sodium caseinate added to

4.10

sterile filtered whey. 10 g/1 Na Cas.

Whey 35 g/1, pH 6.o, 46°c.

Acid concentration during batch culture of whey as

a function of sodium caseinate (HF 145)

concentration. Whey 35 g/1, pH 6.0, 46°C.

4.11 Acid synthesis rate during the phase of stationa�

cell population as a function of the concen­

tration of sodium caseinate (134-136)

Whey 35 g/1, pH 6.o, 46°C.

4.12 The effect of shaking various whey cultures in

conical flasks. Acid concentration after

16 h incubation at 4-6°C without pH control.

S - shaken; NS - not shaken. Whey 75 g/1.

ix

50

55

61

63

65

66

68

4. 1 5

4. 16

4. 17

4.1 8

Acid concEntration after 7 h incubation at 0 46 � of ·whey cul tu;res shaken and unsh:1.ken

with different gases in the head space.

'.'.'hey 75 t;/1. The effect of air on acid productjon at pH G.o, 46°C.

A - air sparged at 1 20 1/h from the tirr:e of inoculation, pasteurized whey, 35 &'1.

B - air sparged at 1 20 1/h for 19 h after inoculation; air flow stoppe c1 and culture

reinoculated with a fresh inoculum of LB�. Whey 35 g/1.

C - air sparge a� 1 20 1/h coreruenced after 8 h

incubation . Whey 70 g/1.

Acid production in the bacterial exponential growth

phase as a function of impeller speed and dia­

meter. (ln x ln). Pasteurized whey 70 g/1,

pH 6.o, 46°C.

Acid production rate in the bacterial stationa�

growth phase as a function of iiL.peller speed

and diameter. (ln x ln) . Pasteurized whey 70 g/1,

pH 6.o, 46°C .

Acid production in the b acterial exponential growth

phase as a function of the impeller Reynolds

Number. (ln x lu). D.- 25 lL.ffi paddle,

o - 51 mrr: paddle, o- 1 02 mm paddle.

Pasteurized whey 70 g/1, pH 6 .0 , 46°C.

Acid production rate in the bacterial stationa�

growth phase as a function of th8 impeller

Reynolds Number. (ln x l n) . D. - 25 mm paddle,

paddle.

0 - 51 mm pa:ldle,

Pasteurized whey 70 g/1,

o-1 02 mm 0

pH 6 .0, 46 C.

X

6 9

72

77

7 8

7 9

80

4 .• 19

4.20

4.21

4.22

4.23

4.24-

Acid production in the bacterial exponential

phase as a function cf P f' ( = 1'.' l 3 2 -

(d.- v1) n cl ) , the power d.issip3ted

by the po.c1::J_c. ( ln x J.n). !:l - 25 mr;1

padd.le, o- 51 mm paddle, 0 - 1 02 lllill

paddle. Pasteurized whey 70 g/1, pH 6.0,

46°c.

Acid production rate in the bacterial stationary

growth phase as a function of P� 3 2 J.

( = wl (d.- w) n d. ) , the power

dissipated by the paddle.(ln x ln). Ll - 25 m:rt paddle, 0 - 51 mm paddle,

0 - 102 mm paddle. Pasteurized whey 70 g/1, pH 6.0, 46°C.

Increase in the acid production rate by adding

stationary phase cells of LB� to a batch

culture at 7 h. o - control .6. -cells

ad.ded at time shovm by the arrow.

Vihey 35 g/1, pH 6.0, L;..6°C.

The productivity of lactic acid (��) as a

funct:lon of temperature, pH and dilution

rate in a single stage continuous culture.

Sterilized whey 75 g,ll. Acid concentration (P) as a function of temper­

ature,pH and dilution rate in a single stage

xi

81

82

84

91

continuous culture. Sterilized whey 75 g/1. 92

Yield of lactic acid from lactose (Y) as a

function of temperature, pH and dilution

rate in a single stage continuous culture.

Sterilized whey 75 g/1. The rate of acid production ���) as a function of

pH and temperature at a dilution rate of

0.2 h-1 • Sterilized whey 75 g/1.

The steady state value of the productivity of

lactic acid from a single stage continuous

culture of whey. -1 D - o.o6·J h ,

Pasteurized whey 35 g/1,

pH 6.0, 4-6°c.

93

94

98

5.1

5.2

5.3

5.5

5.6

6.1

A typical batch culture (139.12.13). Ln cell

number concentration, acid concentration

and rate of acid synthesis a:> functions

of time. FasteurizecJ. whey 70 €/1,

pH 6.o,

A typical batch culture nith supplewentary

amino acids and salts alded (B9. 2 .1).

Rate of acid synthesis and specific rate

of cell growth as functions of time.

Sterile-filterea. vrhey, 35 g/1, pH 6.0, �-6°C.

The rate of acid synthesis as a function of

cell number concentration in the exponential

growth phase. 0 - B9 .1 2.13.

0 - B 9. 2 • ·1 • � - B 9 • 1 • 5 •

The specifi c r ate of acid production is a linear

function of the specific rate of cell gro11th

in the bacterial declining gro?;th phase.

0-B9.12.13. D-B9.2.1.

xii

100

101

104-

�- B9.1 .5. 105

The reciprocal of the rate of acid synthesis as a

function of the reciprocal of the lactose

concentration, as a test of equation 5.4

(B8.2.6.)

The reciprocal of the rate of acid synthesis as a

function of the reciprocal of (Pm -P) ·where

Pm is the maximum potential acid concentratjon

as a test of equation 5.6 (B9.12.13)

The factor p N

K + p - p p m F-m - p

as a function of the reciprocal of the

dilution rate as a test of equation 5.10.

Sterilized whey 70 g/1. Graphical estimation of the operation of a multi­

stage continuous culture. (Ricica,1969b)

Data f ror..: 39.3 .13 . -.-:hey 3 5 g/1, !1 " 1 0 - 11 H 6 o 46°c. .a ._,as, 01 , p _ • ,

107

109

1 13

123

TABLES 3.1 �easured values of the capacitance at di£ferent

burette volumes for the level meter •.

3 .2 Analysi s of lactic casein whey powder.

SupplementaFJ nutrients.

3 .4- Lactose determination by the methods of Wahba and Lane & Eynon on three whey solutionso

4-.1 A t ypical batch culture (B9.1 .5).

4-.2 Changes in the acid and lactose concentrations in

4-.3a

a typical bat ch culture (B8.2.6).

The e£fect of nut rients on the rate of acid pro­

duction in batch £e1�entation of whey;results.

4-.3b The e£fect of nutrients on the rate of acid pro-

4-.4-

duction in batch fermentation cf whey;

analysis of variance.

Nutrients add.ed to whey.

1�.5 The effect of supplementary nutrients on cell con-

centration and pH a£ter 7 h i ncubation at 4-6°C.

ll-•6 Batch fermentation of whey with add.i tional sodium caseinate.

4-.7 Batch fe1�cntation cf whey in conical flasks; the

4-.8

4-.9

4-.10

4-.11

4-.12

effect o£ shaking.

Head-space gas mixtures.

The effect of headspace gas on acid production in

shaken and not shaken cultures of wh ey.

Paddle impeller diameters and speeds used to deter­

mine the effect of agitation intensity on the

rate of acid production.

Typical result for a batch fe�entation of whey;

effect of agitation (B9.12.13).

The effect of agitation intensi� on the rate of

acid production.

xiii

32

35

37

4-0

51

52

56

57

58

59

62

67

70

70

73

75

76

4-.1 3

4-. 14-

4. 15

4. 1 6

4-.17

4-.1 8

4-. 1 9

5. 1

5.2

5.3

5.5

A 1 . 1

A 1.2

A2. 1

A2.2

A2.3

A2.4

"xperimental con:iitions in continuous culture

experiments to determine the effect of pH, temperature and dilution rate on product­

ivity, acid concentration and y ield .

The effect of pH, temperature and dilution rate

on lactic acid production.

Regression coefficient ( "t standard ei-roi':)

for the equations -relatint, produc.tiv� ty,

�cid- concentra ti6ri and yield '-wi tb pH,

temperaturE: and. dilution rate .

Analysis of variance for the equg_tions re1at-

ing productivi�;, acid concentration and

yield wi th pH, temperature and di1ution rate.

Continuous culture of whey with added sodium

caseinate.

Continuous culture with pasteurized whey.

�eproducibility i n continuous culture experiments

usir.g pasteurized whey.

xiv

8 6

87

89

89

95

9 6

Maximum specific growth rate for whey batch cultures. 103

Batch culture kinetics; value of constants in equation 5. 1.

Data from B8.2. 6. as a test of equation 5.4-.

Values of V and K for equation 5. 6. m z

The rate of acid production from experimer.tal re-

103

10 6

108

sults (B9. 12. 13) and calculated from equation 5.7. 1 1 1

Typical composition of whole milk and lactic

casein whey.

Some phYsical properties of lactic casein whey.

Lactobacilli classification - test cultures.

Growth in 1':RS broth. Optical density after 4-8 h.

Microscopic appearance of lactobacilli cultures.

Acid production in sterile reconstituted skim milk.

140

140

14 1

142

14-2

142

A5. 1

A5. 2

A5.3

A6.1

A6 .2

Sugar fermentation in modified JmS broth

after 4-8 h.

Central composite rotatable design for three

variables.

Regression coefficients for the equation show­

ing the effect of pH, ten:perature and

dilution rate on acid concentration .

Analys i s of variance for the equation showing

the effect 0f' p H, temperature and dilution

rate on acid concentration.

Matrix for 1 6 runs for a Plackett and Burman

design.

Levels of the indep endent variables in the

Plackett and Burman screening experiments.

Screening of variables; experimental results .

Screening of variables, increase in acid.

cor1c:entra tion .

::)creening experiments, productivity .

Screening experimen�s, yield.

Primary and two-factor confounding in a 1 6 exp eriment Plackett and Burman design.

XV

14-3

14-8

14-9

1 53

1 55

1 56

156

157