whe ther the open fermentation vats rep o rte d. as bcir.g used in the coJL.mercial
production of lac tic acid are desirable ( C ampbelJ , 1 953 ; O etiker, 1 96 0 ;
and O l ive , 1 93 6 ) .
Ano ther reascr. f o r not us i ng op en vats i s the inhibit ory effect of
oxygen en the growth of LDR. Air in the headspace a.bove the w!:.ey, part
icularly if the cul ture s u rfa c e was not quie s cent , caus ed the groi7 th of
the LBR culture to cease due to the
adverse
effect of oxyge n . Then the air was replaced with ni trogen or carbcn d i oxide cel l growth proc eede dnormally . Once a culture of LBR had entere d the s tationary growth phase
air had no effe ct on acid p roluction, even when bubbled through the culture medium. Acid was b e ing produced by non-dividing cell s and
oxyg en appeared to be toxic only tc cel l s whi ch were grow ing and dividing .
The advers e effect of oxygen on cell growth is c onsi stent ·;;i th the
des crip tion of as an anaerobic or microaerophilic organi sm
(Bre ed e t 1 957 ) . One p o s s ibl e cause o'f the toxic effe ct of oxygen is the lack of the enzyme p e roxida s e in LBR . This enzyme reduc es hydrogen
peroxi de formed by cell metabol ism. If the hydrogen p eroxide is not re moved from the mediuo it cau s e s death of the cell . LBR was in fact k illed when air was sparged into the medium. The organi sm was incap abl e of re
producing after air sp arging ceased. However no p ermanent toxi c c omp ound accumulated as was shown by normal grovtth after reinoculation
(4.. 2.6�
Catalase ad.ded to the medium would have no effec t because thi s er:.zyme isinactivated at 4.5°C .
Experiments on the effect of agitation s how an in crease in acid p ro duction rate as the level o f ag itation i s increased to a parti cul a r level ,
but above that level further increas e s in agitation offer no advantag es .
The decrease in acid product ion at high imp eller sp eecls ob served by Keen
( 1 972)
was not obs e rved.For each imp eller there was a dis tinct increase in a cid produc t i o n
rat e with incre a s e d speed of rotation ( ?i g .4. . 1 5 , 4 . 1 6 ) . The average irnp eller shear rate is a function of the imp eller sp eed and is imp ortant if coalescence of p article s in p arts of the vessel remote from the im
p eller caus e s reaction rates to be l imited. As dis cus sed la ter, at l ow
imp eller sp eeds the agitati o n was insuffi cient to susp end all solid
p arti cles and thi s is re.f1ected in Figs
.
4 . 1 5 and 1+ . 1 6 .The maximum imp eller shear rate i s a function of tip speed and woul d
b e imp ortant if the bacterial sy stem was sensitive to shearing forces.
1 28
c reased. This is al s o refle c t ed by the small changes in the average
chain length as agita t i o n was increased. I t can the refore be concluded that thi s ba cterial sys tem is not s ensitive tc shear forc e s .
The correlation of Reynolds number wi th acid. production rate
( Fi g . 4- . 1 7 , L� . 1 8 ) is similar i n sr..B.p e to that ob ta ined by Kneule ( 1 956)
in a n inves tigation of the effect o f ag itati o n intens i ty on ma ss- transfer co efficients fo r s olid p articles i n agitated l i quids. Kneul� correlated
the mass-transfer co e f:ficient with the imp ell e r Reynolds l':umber. He showe d that two di stinc t regions exist and c oncluded tha t up to a
p articular agitatio n intens i ty the particl e s w e re inc ompletely susp e nded . Cnce the particles were comp l etely suspended, increased mixing resulted in only a small incre a s e in the Mas s -transfer rate.
For the system of Lac tobac illus fermenting l a c t o s e in whey to lacti c ac i d a similar explanation i s likely . B elow a R.eynolds !':umb e r of 1 0 , 000 s olids i n t h e medium are p robably inc ompl etely suspended.
I n fac t , in runs 1 and 8 ( Re < 5 , 000 table 4 . 1 2 ) a layer of s ettled s olids was ob served o n the b a s e o f the fermenter. 1 t i s unl ikely the
lack of susp ension of bacterial cells i s the cause of reduced a cid
p roduc tion rate. Bacterial c el l s have a spe c ifi c gravi t-y of approximately 1 . 03 and hence \7ill b e rea dily susp ended eve n at low levels of agitatio n .
Howeve r there are o ther s ol ids i n the whey which consist o f casein :fines , b acte J'ial debris and ins olub l e , denatured whey proteins. Y!hen
whey wa s centrifuged at 1 0 , 000 x g to remove s ome of th e susp ended. s o l ids , total acid p rodu ctio n and the a c id ::;ynthe s i s rate were redu ced cortpared
with whey at the s.:1me lactose concentra tion. ','/hen the whey vra s s terile
filtered, thu s removing e s s enti e�ly al l the su sp ended s ol id s , c el l gro\'rth
and acid proCJ.uction were reduc ed almost to z ero . ( Fig .l+ .7 ) .
l'�o toxic component was b e i ng introdu ced by the pro cedure b ecause a
supply of amino acids allowed cell growth and acid productio!1 to p ro ce e d normally . The amino acid s c oul d b e added i n a defined form ( tryp tophan and c a s amin o acids, f ig .4 . 6 ) or as s odium cas e inate ( fi g .l+ . 8 ) . Heating to
69°C
aft er sterile -filtration did no t increase the acid production rate .Henc e , when agitation o f the whey culture medium is insuffici ent to fully su sp end the ins oluble o r c olloidal material in the whey the redu ced availab i l ity of some e s s ential nutrient c aus e s the acid p ro duction rate to b e c onsiderably redu ced. As shown by Kneule ( 1 956) an increase of the imp el l e r Reynold s �!umb er up to 1
O,COO
cau s e s an increa s e in the mass transfer rate from the susp ende d � olids . Once the solids are c ompl e te ly1 29 suspended there i s only a minor increase in the rate of convers i on of lac tos e to l ac ti c acid.
Kerr,pe and ·:
.
est(1 959)
and K een(1 972)
clain1ed a possible increase inthe rate of rr:ass transfer between the bact erial cell wall and the medium as the agitation intenni ty was increased. If tl-;.e rate of' reaction \';as limited by the rat e of L1as s -trans:fer then it 7tould be exp ec t ed :rro!n th e empirical correlation derived by C: alderbank
( 1 967 )
that the rate of acid.formation v;ould increase as the 0 . 25 power of the pow·er input . The value of 0 .032 ( 0.065 if a single s traight l ine i s d.ravrn through all
exp erimental points fig .1f . 20
)
ob tained with whey i s significantly le s s than 0 . 25 , s.howing tha t mass-transfer b e tween s olid and liquid i s not the rate limiting step in the reaction sequence.Kempe and '.':est
( 1 959)
ob tained a value of 0.08 as the exponent for agitator speed as a function of acid production rate. Their exp eriments were performed with a single turbine and if it is assumed that power is prop ortional to n3 then their results can be exp res sed as :dP
a.t = aP
0 . 027 0
whi ch is very close t o the exp ress ion obtained in the work reported here .
Keen ( 1 972) reported a de crease in the rate of change of pH and hence
p rc swrably acid synth es i s ra te as the l ev el of agitation 11as increased in a culture of lac tis gro-;Ying in skim-milk . '
,
'iith LBR g rowingin pas teuri zed whey the rate of acid synthesis is increase d as the
agitation level i s increased.
From the results obta ined in this v.rork it can b e conclud.ed that the main requirement of agitation in the whey-LB� system is to maintain in
s oluble or collo idal material in suspension ensuring max imum availability of nutrients for the bacterial cell s , allowing the maximum rate of cell growth and acid p roduction. The imp ell er 'teynolds �;umber should be great er than 10,000.
The production of lactic acid from lactic cas ein whey is obviously
technically feasible. The economi c viability will depend on a number of factors including the cost of the fermentation, the c o s t of recovery and purification of the lactic acid from the fermented whey and the available markets. The latter aspects are outs ide the scop e of this study.
Successful operation in continuous culture is po ssible with product
ivities improved compared with batch culture. Eowever yields are redu ced unless multi-stage cultures are used.
1 30
In batch culture the bes t overall productivity obtained was
2.0
g/1 h in whey sup p l emented w ith amino ac i :ls , salts ar.d. vitamins( abed
fig.\.6b).In a typi cal batch cul ture the p roduc tivity ·,·;a;; 0 . 3
g/1
h . "'hi s incll;.:'l.e s only the fe ri!'enta tion tiL:.e and not the time t o p repare t h e ves s el and the medium. In continuous culture,prod.u c tivities varied with the iilution !'ate.The ;uaximum attained with 35
g/1
w�ey s o l ids was i . 8g/1
h but the yield was only0.23 .
At lower d.ilution ra tes so that the produc tivity approached0 . 8 g/1
h ( 0 . 061 h-1 with p a s t euri zed vth3y) the yield i'ias incre a s ed t o7 0 p er cent c ompared. vrith 9 5 p ercent i n the batch culture . The e s t imated productivity from the three stag e continuous uni t would bo 1 .8 g/1 h but
the yield would be increased to 95 p ercent. Pro ductivity i s p rop o rtional
to th e concentra t ion of whey s ol ids .
I t i s interes ting t o note tha t with the L3� organism, the opt imum c ond.i tions o f fermentation and the addition of' s o dium caseinate to the whey, the time to completion of a b atch culture of whey v;-as ab out 1 5 h c ompared with the literature rep o rted values o f from one to s ix days (Whittier and Webb , 1 950) .
The production o f lac tic ac id from lacti c casein Ylhey as a me an s of
was te di spo s al w ill be successful only if mul ti- s tage c ontinuous culture
with cell fee d-back is used. A s ingl e - s tag e c o ntinuous c ul ture vr ill not
1 31
Though the lactose in lae tic cas ein whey can be fermented t o lactic acid with yields in batch culture of 90-95 p ercent , in a s i ngle stae; e
continuous cul ture fermentat ion the y i eld of lac ti c acid and the util
ization of lac t o s e are unac cep tably lov: . If p ollution abatement is the main aim the l a c t o s e in the effluent from the fermenter mus t b e redu ced
to a s low a level as p o s s ible .
I n continuous cul ture the ac i d concentration and decrease in l acto s e
c oncentration are l imited by the low level o f e s s ential nutrients in whey for the op timum g rowth of LBR , a s t rain of The cell
con centrat ion and the rat e of acid formation can be increased by the
addi tion of supplementary nutrients in the form o:' tryp tophan and casamino
a cids , or s odium cas einate.
The rate of acid synthes i s is al s o increased by the use o f p a s t
euri z ed rather than s terile whey, by op erating at the opt imum temp era ture
o f 46 °C and pH 5 .4 - 6 . 0 , providing s ufficient agitation to fully
susp end insoluble o r collo idal comp oun1s in the whey and restri cting the
a ccess of oxygen to the g rowing cell s .
Kinetic equations derived from batch culture data can b e u sed t o p redict the op eration of variou s type s of continuous culture app aratus . These equations show that the use of a multi-stage c ontinuou s cul ture system with feedback of' cells will b e ne ce s sary to reduce the lactose c oncentration in the effluent whey low
enough to g ive an accep tably low BOD . The u s e of such a system
i s p o s sible b ecau s e non-dividing cell s of LBR are capable of c onverting lactose to lactic acid, ac ting as a sourc e of gly colytic enzyme s to catalyse the rea c t i on .
The two b i o ch emical problems, p roduction o f lactic acid by non-dividing o rganisms and the increa sed rate of acid synthes i s after the culture has b een exposed t o a l ow pH, are worthy o f further study .
An economic asses sment of the c ost of the multi-stage
continuous culture op eration compare d with batch cul ture op eration will need to b e carri ed out before a firm de c i s i on can be made as
to the bes t system for c ommercial op eration. Such an asses sment
1.<.;·�
woul d be part of an overall study the re cove �J o f the lactic acid from the fermented vrhey, an aspect not studied
a A b c d D g i K K