Hnh 1: Cu to ca acid citric C6H8O7

1. GII THIU V LCH S PHT TRIN: Forter m u quyn sch Hot ng ha hc ca nm bng mt tuyn b: C l cc nghin cu tp trung vo qu trnh ln men acid citric cn nhiu hn c nhng nghin cu v cc chc nng khc trong qu trnh bin dng nm mc, tuy vy, nhng kin thc c bn c quan tm n c l t hn c nhng qui trnh c miu t trong quyn sch ny tuy nhin nu xt trong lch s nghin cu, ln men acid citric l qui trnh v nm u tin c tim nng trong cng nghip c khm ph. Thc vy, t khi iu ny c cng b th s lng cc bi bo v ch ny tng ln ng k v hi vng rng n s gp phn xng ng vo khi kin thc v cng ngh v sinh ha vi sinh vt. Nm 1891- 1893, ln u tin s to thnh acid citric c quan st thy bi Wehmer, n c xem nh l mt sn phm ph trong qu trnh sn xut acid oxalic bng cch nui cy Penicillium glaucum trn mi trng c ng. Nm 1893, ng phn lp c hai chng mi c kh nng tch ly acid citric v cho rng chng l mt chi mi. Cytromyces c xem nh l chng Penicillia n vng (Wehmer, 1903). Nhng th nghim ca Fabrique de Produit Chimiques de Thann et the Mulhouse nhm sn xut to acid citric trn quy m cng nghip b tht bi. Nguyn nhn dn n tht bi l do qu trnh ln men phi c tin hnh trong vi tun th mi thu c mt sn lng ng k v vy rt d b tp nhim. c bit l v Wehmer tin vo nhng tuyn b mang tnh thuyt phc ti thi im rng to ra v duy tr sn phm acid th acid to thnh phi c trung ha.

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Nm 1913, Zanhorsky c cp patent (bng sng ch c bng quyn) cho quy trnh sn xut acid citric khi s dng Sterigmatocytis nigra mt tn gi khc ca cho Aspergillus niger. Tuy nhin chnh kt qu nghin cu ca Currie mi m ra con ng cho nhng thnh cng sau ny ca vic sn xut acid citric quy m cng nghip. Nm 1916, J.N. Currie vi vai tr l cng s ca tin s Ch. Thom - ngi ng u phng th nghim vi sinh Cc Ha Hc ti thnh ph Washington.DC, ln u tin quan st thy mt s lng ln cc chng Aspergillus niger c trong phng th nghim c kh nng sn xut mt lng ng k acid citric trong nhng iu kin thun li. Pht hin c cho l quan trng bc nht chnh l Aspergillus niger pht trin rt tt khi gi tr pH nm trong khong t 2,5 3,5 m acid citric li c to ra nhiu khi gi tr pH ca mi trng mc thp. Hn th na, im ni bt ca pht hin ny chnh l vi gi tr pH thp nh vy th sau t 1-2 tun c th thu c gn 60% sn lng trn mi trng ti thiu vi kh nng b tp nhim l rt thp. Cho n lc ny, tng lng dung dch acid citric c to ra t nc p tri cy (ch yu l t chanh) khong 10.000 t/a, trong hm lng acid citric nguyn cht chim 7-9% v tn ti di dng mui calcium. sn xut ra 1 tn acid citric cn khong 20 - 30 tn chanh (tng ng vi din tch khong 2,7 ha). Lc ny, Italy l nc cung cp hn 80% nhu cu v acid lactic ca th gii. Nhiu cng ty cc nc khc nhau tin hnh nhp t mui calcium citrate n acid citric ch yu l cung cp cho ngnh cng nghip sn xut thc ung. Sau khi kt thc chin tranh th gii th nht c nc xut khu l Italy ln cc nc nhp khu u ri vo hon cnh ti chnh kinh t b hn ch, v vy hnh thc sn xut c khng cn ph hp, ngi ta tin hnh tm kim nhng phng thc sn xut acid citric mi. Chnh lc ny, nhng pht hin trc y ca Currie mi tr thnh nhng cng c quan trng nht cho s pht trin ca ngnh cng nghip sn xut acid citric trn quy m ln bng hnh thc ln men. Vic Currie pht hin ra rng Aspergillus niger c th pht trin v tch ly acid citric ti pH < 2 cho ngha l khi qu trnh ln men c bt u nng pH ny th kh nng ca s xm nhim c th c loi b hon ton. Hn na, ng tm ra rng nng ng cao l iu kin thun li nht cho t l sn phm cao v ng quan st thy rng sn lng ti u ch c th t c di iu kin gii hn pht trin. Nm 1919, nhng n lc khc nhm a hnh thc ln men acid citric vo trong sn xut thng mi b tht bi (Societ des Produits Oganiques de Tirlemont, B), nhng n nm 1923, Chas. Pfizer & cng ty ti New York, thnh cng khi p dng phng php ca Currie v h tr thnh nhng ngi sn xut acid citric ln nht th gii. Sau , ti Anh, J v E. Sturge tin hnh sn xut calci citrate theo quy trnh theo quy trnh tng t da trn bn quyn sng ch ca Fernbach v cs. (1927- 1932). Nm 1928, mt nh my sn xut acid citric da trn ngun carbon r tin l mt ng c ci c xy dng ti Kaznjov gn Plzen, Cng ha Czech (Montan - und Industrialwerke, vorm. J.D. Stark, 1924; Szucs, 1925). Kh khn khi s dng loi vt liu ny chnh l s hin din ca cc ion kim loi, trong ng vai tr quyt nh l cc ion kim loi chuyn i. Theo Leopold v Valtr (1964) th cc nh my ny cn s dng hexacyanoferrate (HCF) trong bc u tin ta hoc to phc hp vi cc kim loi khng mong mun trong mi trng. iu ny c ct gi b mt v c cng b kh tr vo nm 1938 vi bng sng ch c cp cho Mezzadroli (1938).

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Nhiu nh my c xy dng ngay sau nhiu nc nh l Lin bang Soviet, c (Joh. A. Benekiser GmbH), B (La Citrique Belge) v mt vi nc khc. K t nm 1933 theo Perquin (1938) tng lng acid citric trn th gii l 10.400 tn vi ch 1.800 tn c sn xut t Italy, phn cn li c sn xut vi quy m ln bng cng ngh ln men ( Chu u: 5.100 tn, M: 3.500 tn). Rt nhiu tc gi tham gia nghin cu gip lm sng t c ch sinh ha ca qu trnh sn xut acid citric nh l Bernhauer (1940), Foster (1949), Prescott v Dunn (1959) hay Johnson (1954). Mt cu hi kh th v c t ra: c bao nhiu gi thuyt c a ra cho ti khi con ng sinh tng hp acid citric t con ng ph v carbohydrate thng thng c cng b v phn ng nn tng ca chu trnh tricarboxylic acid (TCA) s tch t acetyl CoA v oxaloacetate, c hiu mt cch r rng. Tuy nhin, c ch iu ha dn n s tng hp acid citric vi hm lng cao v s bi tit nhanh chng ch mi c bt u nghin cu t thp nin 1970 v n tip tc c nhiu nhm nghin cu quan tm. Ban u vic ln men acid citric c tin hnh theo hnh thc nui cy trn b mt. Sau ny hnh thc ln men chm c ngh tuy nhin hnh thc ny cn ny sinh nhiu vn ch yu l do iu kin ca h thng ln men v c bit chng sn xut nhy cm vi nhiu yu t m cho n nay vn cha c nhn thc r. Tc ng ca cc ion kim loi chuyn i c cng b nhiu hn v iu l cn thit la chn cc loi vt liu th ph hp. Trong sut thp nin 1950 t l ca cc phc cht c bn trong qu trnh ln men c tuyn b. Ch vi hnh thc ln men ny m c rt nhiu chin lc c bit c pht trin tng ng vi cc loi vt liu th. Nm 1944, Szucs a quy trnh hot ng chia thnh 2 giai on chnh: giai on u kch thch s pht trin ca h nm si, giai on sau gii hn s pht trin to acid citric. t c iu ny cn cung cp mt lng phospho (~ 70mg/l) vo trong mi trng giai on pht trin v chun b mt lng phospho t do trong mi trng giai on sn xut sn phm. Nm 1948 - 1949, Shu v Johnson ci tin mi trng ln men c th tin hnh c hai giai on trong cng mt h thng. Cc tc gi ny ch ra rng iu kin ti u ca qu trnh ln men i hi phi c s cn bng gia mangan, km, phospho. Trong ln men thng mi vic b sung cc nhn t cn thit l rt kh khn v quy trnh ny s dng cc vt liu th c thnh phn khng c xc nh r rng (v d mt r ng). Rt nhiu cng trnh nghin cu c thc hin tm kim iu kin ti u khi s dng cc loi c cht r tin nh l mt r ng (Shu, 1947; Karow v Waskman, 1947; Woodward v cs., 1949; Moyer, 1953), tinh bt (Moyer, 1953a, b), tinh bt c thy phn (Schweiger v Snell, 1949). C rt nhiu quy trnh x l v tinh sch mt ng c bit l vic loi b cc kim loi vt. Hu ht chng u c x l bng HCF u tin, hin nay ngi ta ngh vic s dng cc ht trao i ion (Perlman v cs.,1945 Woodward v cs.,1949 .). Tuy nhin hu ht cc th nghim ch ra rng HCF va ng vai tr l cht loi b ion, va ng vai tr l cht ng c ch s pht trin c th tch t c nhiu acid . Mt bc t ph chnh t c ti M bi nhng ngi ngi nghin cu ca tp on Miles Laboratories. Trong qu trnh pht trin k thut cc tc gi gii thiu phng php s dng tinh bt c thy phn nh l mt loi c cht r tin. Khi nghin cu vi nhng vt liu th tinh khit hn, h pht hin ra rng hot ng bt li ca cc kim loi vt, c th b bt hot nu thm vo mt lng va ion ng

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(Schweiger 1959,1961). Hn th na, tng t HCF, nu ion ng c thm vo vt qu mt lng nh th s rt thun li cho vic sn xut acid vi sn lng cao. Hon cnh hin ti: nhiu cng ty s dung nhng thnh tu k thut ca ln men chm v sn xut khong 80% lng acid citric trn th gii. Ch mt vi cng ty vn cn tip tc s dng phng php nui cy b mt truyn thng .

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Hnh 2: Con ng chuyn ho n gin trong sn xut acid citric t carbohydrate v n-alkan

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Vo thp nin 1960 mt quy trnh mi c ra trong khi nghin cu m rng trn vic s dng n-alkan nh l mt ngun carbon cho qu trnh sn xut sinh khi vi sinh vt (protein n bo, SCP) v nhng sn phm khc nh acid amino, vitamin, acid hu c .v.v. Cc nh nghin cu ti Nht Bn l nhng ngi u tin s dng cc chng vi khun Corynephom t bin thay cho Corinebaterium, Arthobater v Brevibacterium trong cng ngh ln men ngay sau tr thnh mt cng ngh c thit lp vi nhng c tnh tiu biu ca nhng thnh tu tr tu. Nm 1966 - 1967 mt vi chng t bin c to ra sn xut acid citric t n-parafins (C9-C30 )v cc c cht lin quan. Mc d n c ng k bn quyn (Tanaka v cs., 1968), tuy nhin n khng c khai thc trn quy m cng nghip. Sau ngi ta pht hin ra rng nm men, chng Candida v cc dng lin quan s dng n-alkan lm ngun carbon cho ngun sn xut sinh khi ng thi n cng c th sn xut ra mt lng ln acid citric t nparafins sn phm c trn ln (isocitric acid) (Fukui v Tanaka,1980). Nhng n lc c lp acid citric nh l mt sn phm ph trong qu trnh sn xut SCP sm c thay th bng vic u t nghin cu sn xut acid citric nh l mt sn phm chnh. iu ny cng do vic sn xut SCP t ngun hydrocarbon khng c chp nhn v nhng nh hng ca n n sc khe vt nui. Mc d sn lng acid citric to ra t cc alkan t C9 C20 trong phng th nghim t hiu sut 100% nhng vic a vo sn xut cng nghip vn b hn ch bi mt vi l do: vic sn xut acid citric t c cht ny lun km theo mt lng ln isocitric acid m s c ca cht ny khng c chp nhn trong nhiu ng dng, v d trong sn xut thc n cng nghip; vic chn dng v xc nh cc thng s ti u ca qu trnh cn c xem xt. Trong thi gian ch i th r rng n khng phi l mt c cht r to ra sn phm cui cng l acid citric. Cho d nh th no th vic nghin cu li ng dng ca nm men hoc vi khun cng em li nhiu hiu qu th v. c bit trong thi k ca vic sn xut acid citric nh l mt sn phm ph ca sn xut SCP. Vic ny cn thit thc y cc quy trnh c lp dung dch acid citric. Phng php li trch bng dung mi c pht trin v ngy nay c ng dng ph bin trong hot ng ln men truyn thng. Mt khc, ngi ta cng quan st thy rng c nhng chng nm men c th sn xut c acid citric khi pht trin trn mi trng c cha glucose hay cc ngun carbon tng t (Tabuchi v cs., 1969; Abe v cs., 1970). Nhng kt qu tm ra c nghin cu m rng v pht trin xa hn trong nc CHDC c. Nhng lin kt vi ngi c c l hn ch cc n lc tin xa hn a cc k thut mi vo sn xut trn quy m ln. 2.C S SINH HA S IU HO TCH LU ACID CITRIC 2.1 Loi v nng ng khi ng con ng tch ly acid citric khng thng qua s iu ha Glycolytic C nhiu thng s nh hng n hiu qu tch ly acid citric khi s dng Asp. niger. Trong s loi v nng ng l yu t nh hng nhiu nht. Trong khi nng ti u ca cc kim loi vt phosphate v nitrogen c mi tng quan vi nhau th nng v loi ng c xem nh l mt thng s c lp. S thch hp ca cc ngun carbon khc nhau trong sn xut acid citric bng Asp. niger c a ra tho lun bi Xu v cs (1989b): ch nhng ngun carbon nh glucose v sucrose mi c

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nm chuyn ha nhanh chng cho kt qu sn lng v t l acid c tch ly cao. Nn tng sinh ha ca tc ng ny khi khng thng qua s iu ha glycolytic: c t nht 3 enzyme trong con ng chuyn ha hexose biphosphate ca Aspergilli kim sot phn ln qu trnh l hexokinase (EC 2.7.7.1), 6-phosphofructokinase (EC 2.7.7.11; PFK 1), v pyruvate kinase (EC 2.7.1.40) (Smitt v NG, 1972; Kubicek, 1988b). Cn ch rng c ch iu ha bin dng tt l nguyn nhn ca s iu ha glycolytic tc l khi gia tng hot tnh ca mt vi enzyme glycolytic th khng dn n s gia tng lng acid citric to ra. S khuch i gen v d pki A (gen m ha pyruvate kinase) khng dn n s ci tin ca chng sn xut (l. De Graaff, J.Visser, S. Choojun, M. Roehr v C. P. Kubicek, kt qu ny cha c cng b). Tng t, s iu ha glycolytic nm men khng b tc ng bi s khuch i cc gen chn lc (Heinisch, 1986).

Hnh 3: Quy trnh chuyn ho ca acid citric c im ca PFK1 trong A. niger c hiu r hon ton v rt nhy cm trong kim sot nhiu con ng bin ng khc nhau nh l c hot ha bi Fru2,6-P2 AMP, ion NH4+ v b c ch bi phosphoenol Pyruvate, citrat v ATP (Habison v cs., 1983; Arts v cs., 1987). c nhng bng chng gin tip v s iu ha protein 8

kinase da vo c-AMP (Legisa v Bencina, 1994). S hot ha bi Fru-2,6-P 2 kt ni cc PFK1 vi nhau v iu ha 6-phosphofructose-2-kinase (EC 2.7.1.105; PFK2). N kch thch s t m rng enzyme sau iu ha km trong A.niger. V kt qu ch ra rng hot ng ca enzyme ny c bin i chnh do s gn Fru-6-P ln b mt (Harmsen v cs., 1992). V vy s chuyn i ca Fru-6-P thnh Fru-1,6-P c xem nh l mt im quan trng trong s iu ha dng chy glycolytic. Theo mt bo co cho rng s tch ly acid citric ch c th xut hin di iu kin PFK1 khng th kim sot dng chy glycolytic mt cch hiu qu. S trung ha vai tr ca NH+4 - tch ly di iu kin Mn (Kubicek v cs., 1979a) - s c ch citrate ca PFK1 l mt thng s lin quan trong qu trnh ny (Habison v cs., 1979,1983). Tuy nhin s tch ly acid bi nng ng cao li ln quan ti mt con ng bin dng khc: nng ng cao lm gia tng nng Fru-2,6-P 2 ni bo (Kubicek Pranz v cs., 1990). y ch l mt quan st vi ngun carbon cho sn lng acid citric cao. V nng Fru-6-P ni bo ch cao di mt vi iu kin, nghin cu tm thi cho thy rng n ph thuc loi ngun carbon v nng acid citric tch ly v s khc nhau ca chng nh hng n nng Fru-6-P trng thi bn m iu nh hng n s thnh lp cc nhn t Fru-2,6-P2 hot ha glycolytic. Tuy nhin, ngi ta ra mt gi thuyt m nu ng n s gi ra cc bc bin dng trc khi to thnh Fru-6-P ngha l hexokinase (phosphoglucose isomerase xc tc phn ng cn bng nhanh chng v khng nm trong nhng cht iu ha quan trng) c l tham gia vo trong vic iu ha phn ng thy phn glucose v tch ly acid citric di nhng iu kin ph hp. Bng vic s dng phng php phn tch nhy ca trng thi bn, Torres (1994a,b) cho rng hoc c s chuyn i hoc phosphoryl ha glucose y mnh qu trnh tch ly acid citric. c im sinh ha ca qu trnh phosphoryl ha hexose trong Asp. niger c nghin cu k (Steinbock v cs., 1993): khng ging nh S. cerevisiae (Lobo v Mattra 1977) nhng n li tng thch vi chng Khuyveromyces lactis (Prior v cs., 1993), Asp. niger ch cha mt loi hexokinase. Enzyme ny c to ra khi nng sucrose hay glucose mc cao. c im ng hc ca n gn ging nh enzyme ca ng vt, ngha l n khng b c ch bi glucose-6-phosphate. Vic s dng chng A.niger t bin khng 2-deoxyglucose trong sn xut acid citric c loi b (Fledurek v cs., 1988; Kirimura v cs., 1992) v n kh hot tnh ca hexokinase. th hot ng ca hexokinase chng li s tch ly citrate trong chng t bin c ngh rng hexokinase khng b gii hn trong dng bin dng. Pht hin ny cho rng vic kh hot tnh ca hexokinase c th l h qu ca dng chy carbon t l thp trong chng t bin khng desoxyglucose. Nh vy hu nh cc nhn t kim sot hiu qu cc bc chuyn ha sm ca glucose c khm ph 2.2 Pyruvate carboxylase v vai tr ca s phn ngn chuyn ho c lp vi qu trnh iu ha sn phm cui cng ca qu trnh glyco gii hiu kh l pyruvate. Trong t bo bnh thng cc sn phm ca qu trnh d ha pyruvate i vo trong ti th v c chuyn ha thnh acetyl-coA. Asp. niger v mt vi loi nm khc c cha pyruvate v carboxylase c kh nng bin i pyruvate v CO2 to thnh oxaloacetat (Osmani v Schutton, 1983; Bercovitz v cs., 1990; Jaklitsch v cs., 1991). Enzyme ny c cm ng khi nng carbon hydrat hin din cao (Feir v Suzuki 1969; Hossain v cs., 1984). S lin quan ca pyruvate carboxylase trong qu trnh hnh

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thnh acid citric c chng minh bng cc th nghim vi cht c nh du (Cleland v Johnson, 1954) v vic o lng lng kh thot ra t qu trnh ln men (Kubicek v cs., 1979b). im khc bit ln nht gia pyruvate carboxylase ca nm vi hu ht cc sinh vt c nhn chnh l v tr ca n trong t bo: hu ht cc enzyme ca sinh vt c nhn c cha trong ty th (Osmani v Scrutton, 1983; Turohtt v Ratledge, 1988) cn trong Arpergillus spp. n xut hin ti cc v tr chuyn bit trong cc phn cytosolic (Jaklitsch v cs., 1991) y chnh l im thun li c bn nht trong qu trnh tch ly acid: oxaloacetat khng cn chuyn ra t ti th v vy n s khng b kh thnh malate. V cht mang tricarboxylate s dng dicarboxylic acid nh mt ng c cht. Kt qu c trong s to thnh citrate v malate. Hot ng ca citosolic pyruvate carboxylase kt ni qu trnh glyco gii n s to thnh citrat t ti th. 2.3.Hot ng ca chu trnh Tricarboxilic Acid (TCA) v s tch ly acid citric Hu ht nhng nghin cu trc y v s tch ly acid citric u tp trung vo s c ch ca mt bc trong chu trnh TCA v cha nhng im mu thun vi nhau. Hu ht cc nh nghin cu u cho rng nht thit phi c mt bc c ch trong chu trnh tch ly acid hu c, trong khi mt s khc - s dng cc ti th c lp - li cung cp bng chng cho thy s hin din ca mt chu trnh bnh thng ( xem xt li xem Roehr v cs., 1992; Kubicek v Roehr, 1986; Kubicek v cs., 1994). Khng ai dm chc rng c hay khng s gia tng nng citrate l cn thit cho s tch ly acid citric nhng n c th xut hin nh l mt sn phm ca c tnh iu ha cc enzyme trong chu trnh TCA. Trong khi c nhng bng chng cho thy rng aconitase khng b c ch khi lng citrate to ra qu nhiu (Rubicek v Roehr, 1985), iu ny khng th c loi tr i vi mt vi enzyme khc trong chu trnh TCA. V citrate c ch NADP - c hiu cho isocitrate dehydrogenase ( Mattey, 1977; Legisa v Mattey, 1986), t l kh in t trong bin dng [NADH/(NAD + NADH] c ch NAD - c hiu cho isocitrate dehydrogenase (Kubicek, 1988a), oxaloacetate, cisaconitate, NADH c ch 2oxoglutarate dehydrogenase (Meixner Monori v cs., 1985). Tuy nhin iu quan trng i vi s c ch chnh nu chng xut hin trong in vivo th cha c hiu r: v dng ra ca citrate ph thuc vo dng vo malate, nguyn nhn c ch ny c vch ra bi s tch ly citrate l c lp bi s gia tng vn chuyn trong t bo cht. Tht khng may, c tnh ca cht mang tricarboxylase ca Asp. niger, ti th vn cha c nghin cu. Ngi ta vn tip tc quan tm cao n iu ny bi v n gip gii thch ti sao isocitrate v cis aconitate mc d c s cn bng ca aconitase vn khng c tch ly bi Asp. niger. Cn phi ch rng phng php phn tch nhy cm ca trng thi bn cho thy s quan trng ca s tun tro citrate trong ti th i vi t l tch ly citrate (Torres, 1994b). 2.4 S iu ha qu trnh hnh thnh sn phm trong cc iu kin mi trng khc nhau: Bn cnh cc yu t nh nng v loi ng nh hng n s tch ly citrate ( c ni phn trn) th cc thng s mi trng khc cng nh hng n s hnh thnh acid citric i km vi s hnh thnh mt s sn phm ph khc nh l gluconic acid v oxalic acid.

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iu kin mi trng ti u cho s tch ly acid citric l: - Nng ng cao: 120 250 g/l - S thiu ht cc kim loi vt c bit l Mn2+ v Fe3+. - p sut oxy ha tan cao (> 140 mbar) - pH: 1.6-2.2 - Nng phosphate c iu chnh mc ti thiu cho yu cu pht trin. - Lm tng nng NH4+ C s sinh ho v s tc ng ca chng s c cp phn di. 2.4.1 Nhng ion kim loi vt: Rt nhiu kim loi vt c gi tr c bit l Mn2+, tuy nhin trong s tch ly acid bi A. niger th ch Fe3+ v Zn2+ ng vai tr quan trng, s thiu ht Mn2+ l bt buc thu c sn lng cao. C nhiu cch gii thch c ngh quan tm n c im sinh ha trong hot ng ca kim loi vt (Roehr v cs., 1992). Vai tr ca s thiu ht mangan c l tp trung lm gim s tng hp cc i phn t (Kubicek v cs., 1979a; Hockertz v cs., 1989) do cm ng gia tng protein bin tnh (Kubicek v cs., 1979a; Ma v cs., 1985). S gia tng nng NH4+ tch ly trong si nm hot ha PFK1, m s c ch ca n c trung ha bi citrate nh thc y s glyco gii (Kubicek v cs., 1979a). Gi thuyt c a ra cho chng A. niger t bin khi PFK1 nhy cm vi nng citrate ng thi lm gim s nhy cm ca s tch ly acid citric bi Mn2+ (Schreferl v cs., 1986). S gim st trong qu trnh tng hp cc i phn t c cho l s c ch ca ribonucleotide reductase, dn n s gim st trong vic tng hp DNA do thiu tin cht monomeric ca s nhn i DNA (Hockert v cs., 1988, 1989). Mt vi tc ng khc ca s thiu ht DNA nh l nh hng ln sinh tng hp vch t bo v hnh thi hc hyphal, mi lin h ca s ri lon trong nhn i DNA cha c lm r (Kubicek v Roehr, 1986). Tc ng sau cng l yu t ni bt nht v c th c s dng nh l tiu chun d on s thnh cng ca qu trnh ln men acid citric: s thiu ht mangan trn si nm pht trin c khng bo mnh, phn nhnh cao, vch t bo c lm c, xut hin nhng ch phnh. Hin tng ny c th lin quan n s nh hng pht trin ngn bi s mt nh hng vn chuyn cc ti. Kh nng ni bt ca Cu2+ l trung ha nhng tc ng c hi ca s xut hin Mn2+ bi v n c kh nng c ch Mn2+ bng cch hp thu, s xut hin ca n l c hiu trong h thng vn chuyn c i lc cao (Seehaus v cs., 1990). nh hng ca cc kim loi khc trong s tch ly acid citric hu c bi Aspergilli hin nay vn cha c r rng: mt vi nh nghin cu cho rng Fe3+ c nh hng to ln nhng quan im ny b bc b bi nhiu ngi khc (Kubicek v Roehr, 1986). Trong quan im nng cao ca Fe3+ cn thit tch ly citrate, cc tc gi ny cng nhn rng tp cht mangan c th tht s nh hng n kt qu quan st c (Kubicek v Roehr, 1986). 2.4.2 p lc oxy ha tan v s thng kh: S hnh thnh tt c cc acid hu c u tng di iu kin thng kh mnh. S hp thu oxy vi hm lng cao l cn thit cn bng chuyn ha gia s thnh lp citrate vi vic s dng hm lng ng cao. iu ny xut hin khi kh nng chui h hp ca A. niger khng to thnh NADH vi t l cao. Hn th na, s gn kt ca phc hp 1 t chui h hp ( NADH: ubiquinone reductase) tr nn ri lon trong iu kin

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tch ly acid citric (Wallrath v cs., 1991; Schmidt v cs., 1992). Trng thi ny c b p bi s hot ha mt con ng h hp khc m trong s ti oxy ha NADH th khng c kt hp to ATP (Zehentgruber v cs., 1987). Cc thnh phn khng xc nh ca con ng phi ph thuc vo s duy tr p lc cao ca oxy v mt s gin on ngn c th lm gim hot ng ca n ( Kubicek v cs., 1980). Nhng n lc nghin cu c ch quan trng ny gip trnh khi s d tha ATP m khng th s dng trong phn ng sinh tng hp. 2.4.3 pH: S tch ly acid citric bi Asp. niger chu nh hng ca pH tc l acid citric ch xut hin khi gi tr pH