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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 TSCUI.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 m pdt = K p + p m p
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 m p K p + p m P
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 lacti c 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 sourceof 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 stimulating discussions, advice and assistance.
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 impeller �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
typical batch culture. B9 . 1 .5. Acid concentration 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
21
22
23
24 25 26
27
30
31
41
functions of time. Whey 3 5
g/1,
pH 6 . 0, 46°G. 47A 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 supplementary 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
46 � 0 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 1 201/h
from the tirr:e ofinoc ulation, pasteurized whey, 35
&'1.
B
-
air sparged at 1 20 1/h for 19 h after inoculation; air flow stoppe1 and c culture reinoculated with a fresh inoculum of LB�.Whey 35
g/1.
C
-
air sparge a� 1 20 1/h co
reruenced after 8 hincubation
.
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 bacterial 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 02mm
paddle.Pasteurized whey 70 g/1, pH 6 .0, 46°C.
Acid pr
o
duction
rate in the bacterial stationa�growth phase as a function of th8 impeller Reynolds Number. (ln x l n
)
. D.-
25mm
paddle, paddle.
0
-
51mm
pa:ldle,Pasteurized whey 70 g/1,
o-1
02mm
pH 6 .0, 46 C. 0
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'.' l3 2 -
(d.- v1)
n cl)
, the power d.issip3ted by the po.c1::J_c. ( ln xJ.n).
!:l - 25 mr;1padd.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 xln).
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 b atch 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 afunct: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 temperature,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 afunction of temp erature, 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 75g/1.
The steady state value of the productivity of lactic acid from a single stage continuous culture of whey.
D - o.o6·J h , -1
Pasteurized whey 35 g/1, pH 6.0, 4-6°c.
93
9 4
9 8
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 rate 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.
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)
·wherePm 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 " .a ._,as, 1 0 01 -11 , p H _ 6 • o , 46°c.
107
109
113
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 n
u
trients o n the rate of acid production 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 int ensi� 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-.13
4-. 14-
4. 15
4. 1 6
4-.17
4-.1 8 4-. 19
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 yield.
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 with 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.
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 m K z for equation 5. 6.
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 fe rmentation in modified JmS broth after 4-8 h.
Central composite rotatable design for three variables.
Regression coefficients for
the
equation showing the effect of p H, ten:perature and
dilution rate on acid concentration .
Analys i s of variance for the equation showing the effect 0f' p H, temp e rature and dilution rate on acid concentration.
Matrix for 1 6 runs for a Plackett and Burman des ign.
Levels of the indep endent variables in the Plackett and Burman screening exp eriments.
Screening of variables; exp erimental result s.
Scree ning of variable s, increase in acid.
cor1c:entra tion .
::)creening experiments, pro ductivity . Screening exp erimen�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
