INDUSTRIAL PROCESSES

There are t hree bas i c proces s e s used for c ommerc i a l product ion of c it r i c a c i d ( Lockwood , 1 9 7 5; Rohr et a Z . ,

1 9 8 3 ) : -

1 . The Ko j i ferme n t at ion proc e s s

'2 . The l i quid cult ure s hallow p a n process 3 . The s ubmerged ferme n t at ion proce s s

The pre c ise te chn ical de t a i ls o f t he s e proce s s e s remain s ecret , but examinat ion of t he re levant patents i s s ued to t he companies concerned has g iven s ome i n format i o n a s t o t he proces s e s used ( Lockwood , 19 7 5 ) .

The Fermentat ion Pr oces s

This i s a s imple ferme n t at ion proces s , deve l oped i n

Japan . C oo ked , s o l id vege table res idues , u s u a l l y s weet

potato or wheat bran , are spread in t rays and i n o cu lated with a s e le c t ed s t rain of A . n i g e r . Dur i n g incubat i o n , t he

amylase produced b y A . n i g e r s ac char i f i e s t he s t a r c h and

much of t he hydrolysed s ugar is t he n converted t o c it r i c acid . The temperature of t he s o l id mas s i s ma i n t a i ned at 2 8 0 C and t he pH drops to 1 . 8 to ·2 . 0 as c i t r i c

accumulates . The mas s is extracted with wat er

acid in percolators after 5 to 8 days incubat ion and t he c i t r i c acid is pur if ied . The e s t imated annual product ion by t h i s met hod is only 2 5 0 0 tons ( L ockwood , 1 9 7 5 ; Rohr e t a Z . , 1 9 8 3 ) .

The C ulture Shal low Pan Proc e s s

--.----

More commonly re ferred to as t he s ur f a c e c u lt ure

Europe . I t it e s t imated t hat in a l ar ge c it r i c a c i d produc t i o n p lant , approx imat e ly 3 0 acres o f s ha l l ow p a ns are requ ired . To prevent met a l ion cont am i nat ion o f t he s ub s t ra t e , t he pans are manufactured f rom h i gh -pur i t y s t a inle s s s teel or a lumi n i um . The mos t c ommon s ubs t r at e i s beet mo l a s ses , but raw s ugar o r gluc o s e s yrups c a n a ls o be us ed . T r a ces of iron are removed by treat ing t he m o l as s es with f e r r o cyan ide and f i lter ing o f f the r e s u l t i ng pre c ip i t a t e . The med ium is then adj us ted t o pH 2 . 5 t o 4 . 0 u s i n g s u lphur ic ac i d . I nocu lat ion i s w i t h spores of A .

niger b lown over t he s t er i l e s o lut ion i n t he pans . The s pores rap idly germinate and cover t he s o lu t ion w i t h a t h in white pe l l i c le of myce l i um . The temperat ure i s mai n t a in e d a t 3 0°C and s teri le humid i f ied a ir is b l own s lowly over t he s ur face o f the solut ion for about 5 to 6 days . Af t e r 8 t o 1 0 days o f incubat ion , the s ugar con t e n t has b e e n reduced from 2 0 0 g / l to 10 g / l and the maximum c it r i c a c i d concentrat ion i s achieved . The yie ld o f c it r i c a c i d from this proce s s is approx imate ly 8 5 % on t he bas i s o f s ugar used .

The Ferment at ion Proces s

A s chema t i c diagr am of this proc e s s is shown i n F i gure 2 . 1 ( Lockwood , 1 9 7 5 ) . The raw mater i a l s used are gener a l l y beet mo las s es , glucos e s yrups and h i gh - t e s t cane s yr up . A pe l letal form of a se lected s train o f A . niger i s u s ed t o inoculate t he fermenter . The culture i s a g i t at e d and aerated throughout the ent ire fermen t at i o n . The durat i o n o f the fermentat ion depends o n the init ial s ugar c o n c e n tr a t i o n ,

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s ubsequent s u gar addit ions and t he amount o f growt h . I t usually ranges from 6 t o 14 days , but for a given s e t o f cond i tions , i t is a con s t ant period . T he c i t r i c ac i d y ield from t his process is about 95% on t he basis of s ugar ut i l i sat ion ( Lockwood and Bat t i , 1965 ) . The s ubmerged fermentat ion proces s , be cause of t he f o l lowing advant ages:

- higher y ield of c it r ic a c id based on s ugar u s e d - improved proces s control

- reduced ferme n t at ion per iod

- reduced requirement for f loor space - reduced manual handl ing

- lower i nves tment cos t

is t he preferred proce s s f or curre nt commercial c it r i c ac id product ion ( Sodeck e t a l . , 1982 ) .

I n s ummary , approx imat e l y 80% o f t he c i t r ic a c i d requ ired in t he wes tern wor ld i s produced b y t he s ubmerged fermentat ion proces s , us ing mo las s e s as t he s ub s t rate

( Sodeck et a l . , 1982, ) .

2 . 5 B IOCHEMISTRY OF C ITRIC JlCID JlCC UMULATION BY A. NICER

Many theories have been put f orward t o e xp la i n t he accumulat ion of c i t r ic acid by A . niger ( e . g . Per lman and S ih , 1960; Meyrath , 1967; Kubi ce k et a l . , 1980; Hos s a i n et

a l . , 1984 ) . The s it uat ion , however , is s t i l l t hat n o s in g le hypot hes is fully explains t he opt imum phys i o l o g ical condit ions required to obtain h i gh yie lds . I t i s ge n e r a l l y accepted t hat t he final s tep i n t he s yn t hes is of c it r i c ac id is t he condens at ion of ace t yl C oA and oxaloacet a t e ( F i gure 2:2- ) , and t hat this condens at i o n is t he ma j or r o u t e o f

c itric acid s yn t he s i s ( Kapoor e t a l . , 1 9 82 ) . Two p r o b lems ,

t hen , need t o be addre s s ed . F irs t , t he reason why c it r i c a c i d is accumulated r a t her t han met abol i sed . S e c o nd , t he s ource o f oxaloace t ic ac id , s in ce accumu lat ion o f c i t r i c a c i d impl ies some dis t urban ce i n t he normal operat i o n o f t he TeA- cyc le , which cons eque n t ly prevents t he produ c t i o n o f oxaloacet i c a c i d b y t h i s ro ute .

The mec han ism o f c i t r i c ac id product ion by A . n i g e r was s t udied by Shu e t a l .

glucose - 1 -C 1 4 as t he

( 1 9 5 4 ) , us ing a med i um c o n t a i n in g s o le carbon s ource . F o l lowing mathema t i cal analys i s of t he ir dat a , they conc luded t ha t 3 7 t o 4 0 % of t he t o t a l c it r ic a c i d was formed f r o m C4 - d i c arboxyl i c acid , w h i c h had b e e n p r o d u c e d v i a t h e TCA ­ c y c l e . I n contras t , Boms te in and Johnson ( 1 9 5 2 ) a n d C le land and Johnson ( 1 9 5 4 ) demo n s trated t hat ver y l i t t l e C4

d i c arboxylic acid was produced via t he TeA cycle whe n t he

fermentat ion condit ions were s uc h t o give 5 0 t o 7 0 % y i e lds of c itr ic acid . U s ing t he radio labe l le d g luc o s e a s s ubstrate , C le land and John s on ( 1 9 5 4 ) conc luded t ha t g luco s e was f irst sp l it i n t o two C3 - f ragments ( pyruv i c a c i d )

f o llowed by the format ion o f a C2 -fragme n t ( acetyl C o A ) by decarboxylat i on and a C4 - fragment ( oxaloacet i c a c i d ) b y c arboxylat ion .

c itric acid .

The s e two fragme n t s the n conde n s e d t o form

S ince t hen , t he enzymes phosphoe n o lpyruvat e

c arboxykinase and pyruvate c arboxylase have been demons trated to be act ive pur ing c i tr i c ac i d p r o du c t i o n

( Woron ick and Johns on , 1 9 6 0 ; Bloom and Johns on , 1 962 - ) . I t

lyas e and oxaloacetate hydro lase are act ive in A . n i g e r

dur ing oxalic acid and c i t r i c a c i d product ion ( Ve r ho f f and Sprad l i n , 1 9 7 6 ) . Hence, t here i s ev i dence t ha t a maj or s ource of oxaloacet ic acid dur ing c it r i c acid a c c umu l a t i on i s v i a carboxylat ion of a

C3

There a number of repor t s in t he l iterature c on c er n i ng t he invo lvement of s ome TeA-c ycle e n z ymes in c it r i c a c i d accumulat ion . Three key e n z yme s t hat have been e x am i n e d are aconitase and both NAD - l inked and NAD P - l inked i s o c i t r a t e dehydrogenases . Ramkr ishnan e t a t . ( 1 9 5 5 ) rep o r t e d t hat c it r i c a c id accumu lated due to the d i s appe arance of aconitase and i s o c itrate dehydrogenase at the end of t he i n i t ial growt h phase . I t was not s t at e d , however , whi ch i s o c itrate dehydrogenase was examine d . They f ur t her demons trated that dur ing c it r i c a c id accumula t i o n , t he act iv i t y o f the condens ing e n z yme i n cre ased t e n f o l d a n d t hat the accumulated c it r ic acid inhibited t he a ct iv i t y of i s oc i trate dehydro genas e . However , t he p r e s e n c e of a conitase and both NAD - l i n ked and NAD P- li n ke d i s o c i t rate dehydrogenases dur ing c i t r i c acid accumu l at ion was demon s trated by La Nau z e ( 1 9 6 6 ) , a l t hough i n lower a c t i v it y dur ing the citric ac id product ion phase t han d ur in g t he

i n i t ial growt h phas e of t he fungus . There is a maj or d e f e c t in the report o f Ramkr ishnan e t a t . ( 1 9 5 5 ) , i n t hat t h e y f a i led t o give det a ils of the me t hod us e d for t he preparat ion of the c e l l-free extract , and more impor t a n t ly , f a i led t o give de t a i l s of the prec aut ionary meas ures t aken t o avo id enzyme inact ivat ion dur ing t h i s prepara t i o n

procedure . Thus , the obs erved dis appearance o f t he s e

e n z ymes may have been t he res u lt o f inact ivat ion d ur i n g t he preparat i o n procedure , par t i cu l arly s i nce many s ub s e que n t inve s t igat ors have demons trated t he s e e n z yme s t o be a c t ive dur i n g c it r ic ac id a ccumulat ion . Abmed e t a Z . ( 1 9 7 2' ) re­ e xamined t he role of t he TeA-c yc le . dur ing c i t r i c ac i d accumu lat ion , in part icular s t udyi n g m i t o chondr i a l f u n c t i o n , TeA-cyc le e n z ymes and interme d i a t e s of t he TeA-cyc le . They demons trated t hat certain TC A- cyc le e n z yme act ivi t ie s , e . g . t he c it r i c ac i d conde n s i n g e n z yme , acon itase and bot h NAD­ l i n ked and NADP- l in ked isoc i trate dehyd rogenas es , w e r e a s h i g h dur i n g t he product ion phas e a s dur ing t he i n it i a l growth phas e . The pres ence o f TC A- c yc le intermed i a t e s , a s demons trated b y Ahmed ( 1 9 7 0 ) , gives n o s uppor t t o t he

concept o f a complete blockage of t he TeA- c yc le dur i n g

c it r i c ac id accumulat ion .

S z c z odrak ( 1 9 8 1 ) s tudied t he act ivities o f a c o n i t as e , both NAD - l inked and NAD P - l inked i s o c it r at e dehydr o g e n as e s and c itrate s ynthase ( condens i n g enz yme ) i n ce l l - f r e e extracts of A . n i g e r dur i n g c it r ic ac i d product io n . He reported t hat during the product ion phas e , t he act iv i t i e s o f aco n i t as e and bot h NAD - l inked and NAD P - l inked i s o c i t ra t e dehydrogenases de creased s ign i f icant ly c ompare d w i t h t he ir act iv i t i e s dur ing t he growt h phas e ( but d i d not d i s appe ar complete l y ) and t hat c itrate s ynt hase act iv i t y was